
==== Front
Mol Cancer
Mol Cancer
Molecular Cancer
1476-4598
BioMed Central London

39245737
2105
10.1186/s12943-024-02105-9
Review
Advances on immunotherapy for osteosarcoma
Yu Shengnan 1
Yao Xudong yaoxudong@hospital.cqmu.edu.cn

2
1 https://ror.org/033vnzz93 grid.452206.7 0000 0004 1758 417X Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
2 https://ror.org/00r67fz39 grid.412461.4 Center for Joint Surgery, Department of Orthopedic Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
9 9 2024
9 9 2024
2024
23 19220 7 2024
30 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Osteosarcoma is the most common primary bone cancer in children and young adults. Limited progress has been made in improving the survival outcomes in patients with osteosarcoma over the past four decades. Especially in metastatic or recurrent osteosarcoma, the survival rate is extremely unsatisfactory. The treatment of osteosarcoma urgently needs breakthroughs. In recent years, immunotherapy has achieved good therapeutic effects in various solid tumors. Due to the low immunogenicity and immunosuppressive microenvironment of osteosarcoma, immunotherapy has not yet been approved in osteosarcoma patients. However, immune-based therapies, including immune checkpoint inhibitors, chimeric antigen receptor T cells, and bispecfic antibodies are in active clinical development. In addition, other immunotherapy strategies including modified-NK cells/macrophages, DC vaccines, and cytokines are still in the early stages of research, but they will be hot topics for future study. In this review, we showed the functions of cell components including tumor-promoting and tumor-suppressing cells in the tumor microenvironment of osteosarcoma, and summarized the preclinical and clinical research results of various immunotherapy strategies in osteosarcoma, hoping to provide new ideas for future research in this field.

Keywords

Osteosarcoma
Immune microenvironment
Immunotherapy
http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 82203007 http://dx.doi.org/10.13039/501100002858 China Postdoctoral Science Foundation 2023MD744162 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
==== Body
pmcIntroduction

Osteosarcoma is the most common primary malignant bone tumor that mostly occurs in children and young adolescents [1]. It originates from mesenchymal stem cells, characterized by osteoid product [2]. Osteosarcoma exhibits a predilection to occur in the metaphysis of long extremity bones, such as distal femur, proximal tibia, proximal femur, and proximal humerus [3]. It was reported that approximately 60% of patients are between 10 and 20 years old and osteosarcoma is considered as the second leading cause of death in this age group [1]. The incidence of osteosarcoma is 3–5 per million per year [4]. Despite their rare incidence, osteosarcoma has a high disability and mortality rate. The current standard treatment for osteosarcoma is neoadjuvant chemotherapy-surgery-consolidation chemotherapy treatment mode, which achieves great clinical success for patients with localized osteosarcoma [5]. The 5-year survival rate has reached a plateau in osteosarcoma patients with localized disease ranging from 60 to 70% since the introduction of systematic chemotherapy [6]. Unfortunately, osteosarcoma is a tumor with a high tendency for metastasis, and 15-20% of newly diagnosed osteosarcoma are detected with metastasis [7]. The most common metastatic site is lung. And the paitients with metastatic disease either at diagnosis or at the time of recurrence portends a dismal prognosis with 5-year survival of only 20-30% [8].

Limited progress has been made in improving the survival outcomes in patients with osteosarcoma over the past 40 years. Especially, the prognosis of metastatic or recurrent osteosarcoma is still unsatisfactory. Given these bottlenecks to conventional treatment regimens, there is an urgent need to exploit novel therapeutic strategies to improve the treatment situation of patients with osteosarcoma. In addition to traditional treatment methods including surgery, chemotherapy, and radiotherapy, new therapeutic approaches, such as targeted therapy and immunotherapy, are also under intense research for osteosarcoma. Currently, research on targeted therapy for osteosarcoma mainly revolves around anti-angiogenic small molecule inhibitors, with representative drugs being regorafenib and sorafenib. These anti-angiogenic drugs, do have certain effects on metastatic osteosarcoma, but they have short progression-free survival (PFS) time and insufficient objective response rate (ORR) [9].

In recent years, immunotherapies involving the application of immune checkpoint inhibitors (ICIs), genetically modified T cells, tumor vaccines, immune modulators and cytokines, have received considerable attention. Importantly, due to their impressive efficacy, ICIs are recommended for the treatment of several human malignancies, including melanoma [10], non-small-cell lung cancer [11], head and neck cancers [12] and so on. Chimeric antigen receptor T (CAR-T) cells as an adoptive cell therapy (ACT) have showed remarkable clinical outcome in the treatment of malignant hematological tumors, such as acute lymphoblastic leukemia [13], chronic lymphocytic leukemia [14], and non-hodgkin lymphoma [15]. Immunotherapy can more accurately target tumors and activate the host’s anti-tumor immune response. Compared to traditional chemotherapy, immunotherapy has fewer side effects and possesses long-lasting anti-tumor activity. The microenvironment of osteosarcoma has complex heterogeneity and immunosuppressive properties, which makes immunotherapy not yet approved for indications in osteosarcoma. However, it is undeniable that immunotherapy is becoming an increasingly attractive therapy strategy for osteosarcoma. In this review, we will present on the tumor microenvironment characteristics of osteosarcoma, including immune-suppressive related cells and tumor-suppressive related cells, as well as their roles in the osteosarcoma microenvironment. We will also summarize the results of preclinical and clinical research on immunotherapy, especially ICIs and CAR-modified immune cell therapies, as well as the immuno-combination strategies, with the aim of further understanding the immune characteristics of osteosarcoma from multiple perspectives and providing new directions for the application of immunotherapy for patients with osteosarcoma.

Immune microenvironment of osteosarcoma

The tumor microenvironment (TME) is a complex ecosystem with high heterogeneity, which includes tumor cells and a large number of non-tumor cells, embedded in altered extracellular matrix. In the same way, the TME of osteosarcoma also contains many components which are closely related to the escape of tumor cells from immune surveillance, uncontrolled proliferation, and metastasis. The immune cells in the TME not only play a crucial role in the occurrence and development of tumors, but also are important targets of anti-tumor immunotherapy [16]. There are various types of immune cells in osteosarcoma, mainly including tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), T cells, B cells, natural killer cells (NK cells), and dendritic cells (DCs) and so on. In addition, molecular pathways, especially immune related molecules in the microenvironment of osteosarcoma, are closely related to the development of tumors and the prognosis of patients.

Characteristics of cell composition in the TME of osteosarcoma

The TME of osteosarcoma is distinctly suppressive, with immune cells exhibiting complex and diverse functions. Understanding the roles and mechanisms of different immune cells within it, and developing corresponding therapeutic strategies targeting specific elements, can help improve the prognosis for patients with osteosarcoma.

Tumor-promoting cells in TME

Tumor cells

Tumor cells, as the key components of TME, which can actively interact with the immune system and promote the formation of inhibitory immune networks [17]. In recent years, circulating tumor cells (CTCs) have been frequently mentioned. Under the impact of local and systemic treatment, some tumor cells can still survive in small amounts and escape from the primary lesion into the circulatory system, causing distant metastasis or recurrence of osteosarcoma [18]. Therefore, research on these tumor cells and the development of corresponding treatment methods will be benefit to reduce the metastasis and recurrence of osteosarcoma.

TAMs

TAMs are the most abundant immune cells in the immune microenvironment of osteosarcoma and account for approximately 50% of the immune cells [19]. The polarization state of TAMs has a significant impact on tumor cells. Generally, TAMs polarize into two types, M1 and M2, primarily due to the influence of different cytokines and signaling pathways in the tumor microenvironment [20]. In the TME of osteosarcoma, M2-type TAMs are the main proportion.and play a complex and crucial role. M2-type TAMs are typically associated with anti-inflammatory and tumor-promoting characteristics, and they participate in processes such as tumor angiogenesis, extracellular matrix remodeling, and immune defense and regulation [21]. The polarization state of M2-type TAMs can promote tumor progression by releasing specific cytokines and chemokines, such as producing anti-inflammatory cytokines like IL-10 and transforming growth factor-beta (TGF-β), which are involved in anti-inflammatory responses, and participate in tumor angiogenesis and extracellular matrix remodeling [22]. M2-type TAMs in the TME of osteosarcoma may also affect tumor immune evasion and drug resistance through interactions with other immune cells. For example, they may interact with cancer stem cells, supporting the function of cancer stem cells [23]. In addition, the presence and functional state of M2-type TAMs are closely related to the prognosis and therapeutic responsiveness of osteosarcoma. Studies have shown that the degree of infiltration of M2-type TAMs can serve as one of the biomarkers for judging the prognosis of osteosarcoma patients [21]. Various immune checkpoint proteins expressed on the surface of TAMs, such as PD-1 and CD47 receptor [19]. Therefore, therapeutic strategies targeting M2-type TAMs, such as promoting the polarization shift of M2-type TAMs to M1-type or targeting the immune checkpoints of TAMs, may provide new directions for targeted therapy of osteosarcoma [21]. In addition, the M3-TAMs, actually the FABP4+ TAMs previously identified as the alveolar macrophages in the lung, were dominant in the lung metastatic osteosarcoma lesion [24]. High expression of FABP4 provides fatty acids for tumor cells to promote the occurrence and development of cancer [25]. There has been some research on the mechanism of FABP4 action in tumors, but the specific mechanism is not yet fully understood. M3-TAMs contribute to the metastasis and colonization of osteosarcoma cells in the lungs [26], exacerbating the malignant progression of the tumor. Developing targeted therapeutic strategies against M3-TAMs may potentially suppress the pulmonary metastasis of osteosarcoma.

MDSCs

MDSCs are significant compotent in the TME of osteosarcoma, MDSCs attenuate anti-tumor immunity particularly the activity of T cells, by secreting factors such as arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS), thereby facilitating tumor cells to evade immune surveillance [27]. MDSCs also contribute to tumor angiogenesis through the secretion of factors like vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9) [28]. Moreover, MDSCs interact with other cells in the tumor microenvironment, such as TAMs and Tregs, to jointly promote immune evasion and tumor progression [29]. MDSCs establish pre-metastatic niches in distant organs, creating favorable conditions for the spread and colonization of tumor cells [30]. And they also secrete TGF-β and hepatocyte growth factor (HGF), which contribute to epithelial-mesenchymal transition (EMT), thereby promoting the invasion and metastasis of osteosarcoma [31]. Recent study has discovered that CD300ld, a surface molecule on MDSCs, plays a significant role in the immune-suppressive function of MDSCs and may potentially serve as a novel therapeutic target [32]. Given the critical role of MDSCs in immune suppression and tumor progression, they have become potential targets for immunotherapy. Treatment strategies targeting MDSCs may help to lift immune suppression and enhance the efficiency of the anti-tumor immune response. However, that need more exploration before clinical application [33].

Treg cells

In the osteosarcoma microenvironment, Treg cells exert their effects through various mechanisms. They secrete immunosuppressive cytokines such as IL-10, IL-35, and TGF-β to inhibit the activity of effector T cells, and inhibit the formation of osteoclasts through direct cell contact-dependent manners [34]. Interaction of CD4+ Tregs and osteoclasts significantly alters TME and is connected to poor prognosis of osteosarcoma [34]. And the interaction between T cell immunoglobulin and mucin-domain containing 3 (TIM-3)+ T cells and monocytes, naive CD4+ T cells, and Gal9-expressing CD4+ CD25+ Tregs could resulting in progressive suppression of Th1 responses [35]. Moreover, Zhou et al. Report that infiltration of T cell immunoreceptor with Ig and ITIM domains (TIGIT) expressing Treg cells in osteosarcoma tissue, providing a new target for the immunotherapy of osteosarcoma [4]. Treg cells not only help tumor cells evade immune surveillance but also participate in promoting tumor angiogenesis [36]. Furthermore, the heterogeneity of Treg cells in the osteosarcoma microenvironment and their proliferation and activation status are closely related to tumor development, potentially serving as targets for immunotherapy.

TANs

TANs are a heterogeneous and functionally diverse subset of neutrophils that infiltrate the tumor microenvironment. TANs could also be polarized to the anti-tumor N1 phenotype and pro-tumor N2 phenotype. Infiltrating neutrophils combat tumor cells by orchestrating the recruitment of immune cells and also by facilitating antibody-dependent cellular cytotoxicity [37]. Yang et al. identified that neutrophils are more abundant at the primary tumor site than those at metastatic sites by detecting the specific marker CD11b [38]. It is reported that TANs significantly decresed in the highly hypoxic tumor microenvironment. Hypoxia may downregulate immune cells, thereby facilitating tumor immune evasion and metastasis [39]. Web-like chromatin structures known as neutrophil extracellular traps (NETs) plays important roles in immune protection, inflammatory and autoimmune diseases and cancer [40]. In osteosarcoma, a signature derived from NETs is associated with tumor recurrence and metastasis, and can predict patient prognosis [41, 42]. Currently, research on TANs in osteosarcoma is in its infancy, and there is a long road ahead in developing anti-tumor therapeutic strategies associated with TANs.

Cancer stem cells (CSCs)

CSCs are a subset of tumor cells that play a pivotal role in various stages of cancer development, including tumorigenesis, drug resistance, recurrence and metastasis [43, 44]. Extensive research suggests that osteosarcoma may arise from mesenchymal stem cells (MSCs), which contribute to modulate immune responses, facilitate cell fusion and differentiation [45]. MSCs are capable of secreting anti-inflammatory factors while suppressing pro-inflammatory agents, thereby aiding osteosarcoma cells in evading immune surveillance. This process is mediated by extracellular vesicles (EVs), particularly exosomes, which can be released through autocrine or paracrine mechanisms. MSC-EVs inhibit T cell proliferation and the immunological effects of B cells [46, 47]. TGF-β and interferon-γ (IFN-γ) associated with MSC-EVs can facilitate the transformation of mononuclear cells into Tregs [48]. Furthermore, MSCs can promote the polarization of TAMs towards the M2 phenotype by secreting IL-6 [49].

Tumor-associated fibroblasts (TAFs)

TAFs comprise a large proportion of cells in the microenvironment and produce components of the extracellular matrix that facilitate communication between cells, promote cell adhesion, and stimulate proliferation [50]. TAFs can be reprogrammed in lung under the influence of TGF-β1 in osteosarcoma-derived EVs, which promotes metastatic progression of osteosarcoma [51]. The interaction of osteosarcoma cells expressing CXCL14 and fibroblasts expressing integrin α11β1 promotes metastasis and demonstrate that targeting the CXCL14-integrin α11β1 axis is a potential strategy to inhibit osteosarcoma lung metastasis [52].

Tumor-suppressing cells in TME

T cells

T cells, particularly cytotoxic T cells, play a significant role in the immune microenvironment of osteosarcoma. T cell infiltration is the first step in its anti-tumor effectiveness. Analyses of TME of osteosarcoma demonstrate an immune cell infiltrate consisting of both macrophages and T cells [53, 54]. TIM-3(+)PD-1(+) T cells-specific immunosuppression that was amplified by M2-type tumor-associated macrophages. depletion of CD163(+) macrophages significantly improved T cell proliferation and proinflammatory cytokine production [55]. In osteosarcoma, the number of T cells in metastatic lesions is significantly higher than that in primary and recurrent lesions in situ [56]. An increased number of tumour-infiltrating T cells and PD-L1 expression in metastases compared with primary tumours, suggesting accessibility for T cells, could imply that osteosarcoma patients with metastatic disease may benefit from T-cell-based immunotherapy [56]. Tumor infiltrating lymphocytes (TILs) are mainly distributed in the region expressing human leukocyte antigen Class I, whereas CD4+ and CD8+ T cells concentrated at the pulmonary metastases interface [33]. The CD8+ T cells in TME of osteosarcoma are functionally inactive with increased expression of PD-1 and TIM-3. TIM-3 blockade restored the T cell alloreactive function of the CD8+ T cells in vitro and in vivo [57]. A study claimed that CD8+ T cells have a positive association with prognosis, whereas γδT cells are associated with poor prognosis [58]. As the main killer of tumor cells in the microenvironment, T cells have become the most important research object for developing immunotherapy.

DCs

DCs, as professional antigen-presenting cells (APCs), act crucial roles between innate and adaptive immunity [59]. DCs detect tumor antigens and present them to helper cells and cytotoxic T cells, thereby triggering the anti-tumor immune response. During this process, DCs also transform from an immature state to a mature state. During the initial phases, DCs engage in robust proliferation and maturation, which is essential for the activation of both helper and cytotoxic T cells. However, as the tumor progresses, osteosarcoma cells evolve to generate variants that are resistant to the actions of DCs and phagocytes. This resistance results in diminished DC activation, ultimately allowing the tumor to evade the immune surveillance [60]. A study found that in group with high immune scores, the quantity of resting DCs was significantly higher than that in the low immune score group, and the degree of DC activation was positively related with anti-tumor therapy response [61]. The role of DCs in the immune microenvironment is complex. A study reported that DCs drive the pathogenesis of osteosarcoma through oncogenes and the tumor suppressor glutamate metabotropic receptor 4 (GRM4) [62]. Compared with wild-type DCs, GRM4-knockout DCs secrete more IL-23 and IL-12, leading to rapid tumor growth and accelerated progression in vivo [63]. The proportion of CD1c+ DCs is larger in lung metastatic lesions than that in primary and recurrent lesions [4]. At present, the most popular therapeutic strategy for DCs is vaccination. In osteosarcoma, researchers tested the efficacy of DC vaccines in preclinical studies and demonstrated that DCs vaccines induced tumor suppression [64]. However, in clinical trials, DC vaccines showed limited effect for the treatment of osteosarcoma [65, 66].

NK cells

NK cells play key roles in the killing of tumor cells by releasing perforin, granzyme, and tumor necrosis factor-α (TNF-α) and expressing FasL [67]. But the anti-tumor effects of NK cells are also affected by the PD-1/PD-L1 interactions [68] In the osteosarcoma microenvironment, NK cells were suppressed, but TGF-β expression increased. TGF-β plays a key role in blunting the NKG2D-mediated tumor surveillance [69]. TIGIT also is expressed on NK cells. TIGIT inhibits human NK cytotoxicity against the tumor by interacting with the ligands, including CD155 and CD112 [70]. Therefore, targeting immune checkpoints and/or TGF-β pathway maybe can reinvigorate tumor elimination efficiency of NK cells. Additionally, stimulating the inherent tumor-killing function of NK cells is also a promising strategy. Adoptive NK cells showed initial success in the treatment of osteosarcoma [71].

B cells

B cells are not only the protagonists of humoral immunity by producing antibodies but are also a type of antigen-presenting cells involved in immunoregulation. Regulatory B cells are a type of B cells with immunosuppressive effects. Regulatory B cells inhibit CD4+ T cells, cytotoxic T lymphocytes (CTLs), macrophages, and DCs by secreting inhibitory cytokines such as IL-10, TGF-β, IL-35, and expressing membrane surface regulatory molecules such as FasL and CD1d, and promote the transformation of T cells into Tregs, thus weakening anti-tumor immune responses [72]. Osteosarcoma patients with high infiltration of B cells had a better prognosis and activated B cells were positively correlated with survival [73]. Research on B cells in osteosarcoma is still very limited, and new therapies based on B cells lack satisfactory results.

M1-type TAMs and C1Q + TAMs

TAMs are typed into M1 and M2. Generally, M1 macrophages participate in anti-tumor effects, while M2 macrophages promote carcinogenesis [20]. Although M2-TAMs are predominant in the osteosarcoma microenvironment, M1 macrophages also play their respective roles. M1 macrophages release pro-inflammatory mediators such as IL-1, IL-12, IL-18, and TNF-α, which have anti-tumor effects. In addition, a analysis of single-cell RNA datasets and bulk RNA datasets demonstrate that complete component 1q (C1Q)-positive TAMs have anti-tumor effect and predict a better prognosis for patients with osteosarcoma [74].

The cellular components infiltrating the tumor microenvironment are diverse (Fig. 1), and their functions are not singular but also varied. Immune cells in different states may primarily promote tumor progression, or they may exert anti-tumor effects when the surrounding environment changes. Therefore, it is necessary to view the cells in the microenvironment dialectically and develop appropriate anti-tumor treatment strategies based on their characteristics.

Fig. 1 Schematic illustration of the cellular components infiltrated in the osteosarcoma microenvironment. The TME of osteosarcoma contains tumor cells and a large number of non-tumor cells, embedded in altered extracellular matrix. The cells in the TME can be divided into tumor-promoting cells and tumor-suppressing cells.They exert their effects through different mechanisms in the microenvironment, but their functions are dynamically changing. Compared to the primary lesion, the expression of immune checkpoint molecules in the lung metastatic foci is increased, and there is an increase in immunosuppressive cells

Molecular characteristics of osteosarcoma microenvironment

The bone microenvironment is closely related to the progression and malignancy of osteosarcoma. The analysis of TME landscapes in osteosarcoma suggested that patients with non-metastasis showed higher immune scores and better prognosis compared with those with metastatic osteosarcoma. In addition, high levels of tumor-infiltrating immune cells may suppress metastasis [38]. Wu et al. conducted a comprehensive genomic and immune characterization of post-treatment primary, local recurrence, and metastasis osteosarcoma specimens from 48 pediatric and adult patients. The results suggested that the high levels of genomic rearrangements and moderate point mutation burdens in osteosarcoma, which was not associated with immune infifiltrate levels and neoantigen expression. Besides, there are likely multiple immune-suppressive mechanisms in play in osteosarcoma [75]. The immunosuppressive mechanisms in osteosarcoma, including (1) Highly mutated and rearranged osteosarcoma genome may not generate sufficient neoantigens to elicit an immune response, (2) Aneuploidy and copy number changes alterations and (3) Deregulation of tumor-intrinsic immunosuppressive pathways such as IFNG, MAPK/PI3K/mTOR, and JAK/STAT signal pathways [76]. Yang et al. summarized the factors, including mesenchymal stem cells, hypoxia and acidic condition, chemokines and extracellular vesicles, that affect osteosarcoma metastasis in bone microenvironment through several signaling pathways including the PI3K/Akt, Wnt/β-Catenin, MAPK/ERK, Hedgehog, and Notch signaling pathways[PMID: 32977425]. TAM-derived cathepsins may mediate the activation of the nuclear factor-kappa B (NF-kB) signaling pathway and the signal transducer and activator of transcription 3 (STAT3) to facilitate therapeutic resistance [77]. Sundara et al. studied 85 formalin-fixed, paraffin-embedded blocks from 25 osteosarcoma patients and found that the high density of tumor-infiltrating T cells in metastatic osteosarcoma lesions compared to primary tumors and local relapses. Besides, positive PD-L1 expression was found in 13% of primary tumors, 25% of relapses and 48% of metastases and correlated with a high T-cell infiltration [56]. Wan et al. identified five immune subtypes in 87 osteosarcoma samples, and each of them was associated with distinct molecular characteristics and clinical outcomes [78]. The study of RNA sequencing of 100,987 individual cells from seven primary, two recurrent, and two lung metastatic osteosarcoma lesions provides a deeper insight into the cellular and molecular characteristics of osteosarcoma and its TME properties. The results showed that proinflammatory FABP4+ macrophages infiltration was found in lung metastatic osteosarcoma. And a large number of inactivated and exhausted T cells were observed, especially CD8+ T cells that highly express the inhibitory receptor TIGIT and lymphocyteactivation gene 3 (LAG-3). Compared to primary lesions, pulmonary metastatic lesions expressed more immune checkpoint and immunoregulatory molecules, including PD-1, PD-L1, LAG-3, TIM-3, indoleamine 2,3-dioxygenase (IDO1) and IFN-γ [33].

Given the suppressive immune microenvironment and high heterogeneity of osteosarcoma, achieving satisfactory therapeutic outcomes with immunotherapy may require approaches from the following aspects: (1) enhancing the immunogenicity of tumors to allow more immune cells to infiltrate the tumor tissue; (2) revitalizing the activity of anti-tumor immune cells within the microenvironment, turning “cold” tumors into “hot” tumors; (3) identifying biomarkers that can predict the responsiveness to immunotherapy, to select a population of osteosarcoma patients who are likely to benefit from such treatments; (4) considering the diverse immunosuppressive pathways in osteosarcoma, a single immunotherapy might have limited effects, while combined therapies could potentially break through this bottleneck.

The relationship between TME and prognosis in osteosarcoma

In order to detect the correlation between microenvironment and clinical parameters, Hong et al. calculated immune and stromal scores based on the ESTIMATE algorithm in 83 osteosarcoma samples and found that 137 genes were dysregulated, including 134 upregulated genes and three downregulated genes [79]. Hu et al. analyzed the data of 89 osteosarcoma samples and identified 769 TME-related genes by comparing the high-immune score and low-immune score osteosarcoma patients, which confirmed that patients with higher immune score had a favorable overall survival (OS) and disease free survival (DFS) [80]. Sheng et al. reviewed the potential mechanisms underlying osteosarcoma metastasis including microenvironment, osteoclast, angiogenesis, metabolism, immunity, and noncoding RNAs. For the immunity and metastasis, TAMs, TILs interactions between of PD-1 and PD-L1 putatively participate in immune response during osteosarcoma metastatic progression [81]. In addition, analysis of 85 osteosarcoma samples from TCGA dataset indicated that osteosarcoma cases with elevated immune cell infiltration in the microenvironment showed better prognosis [61].

In general, the TME of osteosarcoma is immunosuppressive, as increased expression of immunosuppressive molecules such as PD-1 and PD-L1 has been detected, especially in metastatic lesions. In theory, treatment with ICIs can be effective for osteosarcoma patients. Besides, osteosarcoma is a low immunogenic tumor that cannot attract a sufficient number of immune cells and tumor-specific T cells to infiltrate the tumor microenvironment. Therefore, combination strategies to improve the immunogenicity of osteosarcoma and then administering immunotherapy may have stronger anti-tumor effects. And discovering more potential tumor antigen that is specifically or at least relatively exclusively expressed in osteosarcoma will be beneficial for the development of immunotherapy strategies based on these targets.

Studies of ICIs for osteosarcoma

PD-1/PD-L1

Osteosarcoma has been shown to have variable PD-L1 expression and frequent deletions of MHC class I, which may promote immune evasion [56]. Metastatic osteosarcoma express higher PD-L1 than primary tumors. Blockade of PD-1/PD-L1 interactions significantly promotes the anti-tumor activity of tumor-infiltrating CTLs in vitro and in vivo [82]. Nivolumab (anti-PD-1 antibody) inhibits osteosarcoma metastasis in human PBMC-engrafted mouse models. The further study of mechanisms indicated that nivolumab promotes tumor lymphocyte infiltration (CD4+ and CD8+ lymphocytes) and enhances the cytolytic activity of CD8 lymphocytes in lung metastases [83]. Davis et al. initiated a phase I/II clinical study to test the safety, pharmacokinetics, and anti-tumor activity of nivolumab in children and young adults with recurrent or refractory non-CNS solid tumors or lymphoma [84]. The preliminary research results showed that nivolumab only has 10 -30% response rate in lymphoma but no response in other tumor types (including osteosarcoma), although the treatment related side effects were acceptable [84]. SARC028 trial (NCT02301039) is the first study to assess the activity and safety of anti-PD-1-antibodies in the treatment of soft-tissue sarcoma and bone sarcoma. This trail recruited 86 patients, and 80 of whom were treated with 200 mg intravenous pembrolizumab every three weeks. The results showed that two (5%) of 40 patients with bone sarcoma had an objective response, including one (5%) of 22 patients with osteosarcoma and one (20%) of five patients with chondrosarcoma. And the adverse events were acceptable [85]. Another single-arm, open-label, phase II trial (NCT03013127) was performed to evaluate the anti-tumor activity and safety of the PD-1 antibody pembrolizumab in patients with unresectable, relapsed osteosarcoma. Total 12 patients were enrolled in this study and accepted treatment of pembrolizumab 200 mg every 21 days. The results demonstrated that pembrolizumab was well tolerated but did not show clinical benefit [86]. Besides, a phase II study (NCT02406781) was conducted to test the safety and anti-tumor effects of pembrolizumab in combination with metronomic cyclophosphamide in patients with advanced osteosarcoma. It is a pity that only one patient experienced partial response (PR). Expression level of PD-L1 is not directly related to anti-tumor efficacy [87]. PD-1/PD-L1 antibodies not only block the interaction of PD-1/PD-L1, but also show anti-tumor effect through other ways. Liu et al. reported that atezolizumab (anti PD-L1 antibody) inhibits proliferation and induces immune-independent apoptosis of osteosarcoma cells through increasing the release of ROS and cytochrome-c. The excessive release of ROS could induce autophagy. They performed further studies in vitro and in vivo and demonstrated that blocking the protective autophagy induced by atezolizumab could significantly amplify its anti-tumor effect on osteosarcoma cells [88]. The safe dose of atezolizumab was confirmed in pediatric and young adult patients, supportive of weight-based dosing in pediatric patients [89]. However, the therapeutic effect of atezolizumab monotherapy was limited [90].

CTLA-4

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) participates in the negative regulation of T cell activation and proliferation, which suppresses anti-tumor response [91]. The study suggested that CTLA-4 genetic polymorphisms also potentially associated with osteosarcoma risk [92, 93]. A phase I clinical trial was conducted by Merchant et al. to test the safety, pharmacokinetics, and immunogenicity of ipilimumab (anti-CTLA-4 antibody) in pediatric patients with advanced solid tumors, including osteosarcoma. The results suggested that ipilimumab showed incredible safety and increased numbers of activated and cycling T cells, but not regulatory T cells [94].

CD47

There is a large amount of macrophage infiltration in osteosarcoma, and the phagocytic effect of macrophages on tumor cells is inhibited by CD47 molecule. CD47 as a transmembrane protein inhibits macrophage phagocytosis when it binds to macrophage SIRPα [95]. Under physiological conditions, this is protection for normal tissues. But for tumors, CD47 is a checkpoint molecule that is overexpressed on tumor cells and inhibits macrophage anti-tumor activity. CD47 is also highly expressed in osteosarcoma and associated with the progression of tumor [96]. In vitro and in vivo osteosarcoma models demonstrated that anti-CD47 monoclonal antibody can block the CD47-SIRPα signaling pathway, thus enhancing the anti-tumor ability of macrophages [96].

TIM-3

TIM-3 was originally found to be expressed on the surface of f type 1 T helper (Th1) cells [97]. TIM-3, as a negative regulator, binds to its ligand galectin-9 (Gal-9) to induce the depletion of Th1 cells [98]. Subsequently, TIM-3 was found to be expressed on the surface of various immune cells, including CD8+T, CD4+T, Treg, macrophage, NK cells and DCs, playing different roles [98]. Blocking the TIM-3/Gal-9 signaling pathway can significantly increase the function of T cells. Therefore, the negative regulation of TIM-3 has attracted much attention for the anti-tumor therapy. TIM-3 and Gal-9 are expressed in osteosarcoma tissues, and the interaction of TIM-3 and Gal-9 promotes the apoptosis of CD4+ and CD8+ T cells in the TME of osteosarcoma, which is related with poor prognosis in osteosarcoma patients [99]. TIM-3 blockade impaired tumor growth in osteosarcoma models and decreased the number of tumor-infiltrating CD4+ T cells while increasing the numbers and functional activation of tumor-infiltrating CD8+ T cells [57]. To date, clinical research outcomes regarding TIM-3 inhibitors have not been published for osteosarcoma. But TIM-3 is a significant potential target for future advancements.

In addition to TIM-3, the expression of LAG-3 and IDO1 are significantly expressed in pulmonary metastatic foci of osteosarcoma and is closely related to immune suppression [33]. Another inhibitory molecule HHLA2, a newly defined B7 family member, widely expressed in osteosarcoma, which is associated with metastases and worse survival [100]. And the immunoreceptor inhibitory checkpoint marker TIGIT was highly expressed by the CD8+ T, CD4+ T, Treg and NKT cells in osteosarcoma lesions. Blocking TIGIT signaling can significantly enhance the cytotoxicity effects of CD3+ T cells with high expression of TIGIT against osteosarcoma cells, indicating potential therapeutic value of targeted TIGIT therapy for osteosarcoma [4]. Currently, there is more research on traditional immune checkpoints, such as PD-1, PD-L2 and CTLA-4, but emerging immune checkpoint molecules have tremendous potential and are worth further investigation in osteosarcoma. We summarized the clinical trials of ICIs in patients with osteosarcoma (Table 1). But the current research results show that immune checkpoint inhibitors have unsatisfactory anti-tumor effects in osteosarcoma. Perhaps exploring the underlying mechanisms of immunotherapy at a deeper level can provide new directions and assistance for the application of immunotherapy in osteosarcoma.

Table 1 Summary of clinical trials of ICIs in osteosarcoma

Registration NO	Clinical Trial	Target	Participant Group	State	Stage	
NCT03013127	Pembrolizumab in Patients With Relapsed Or Metastatic Osteosarcoma Not Eligible for Curative Surgery	PD−1	Pembrolizumab	Terminated	Phase 2	
NCT03006848	Avelumab in Patients With Recurrent or Progressive Osteosarcoma	PD-L1	Avelumab	Has results	Phase 2	
NCT04751383	Magrolimab and Dinutuximab in Patients With Relapsed or Refractory Neuroblastoma or Relapsed Osteosarcoma	CD47

GD2

	Arm A: Dinutuximab + Magrolimab

Arm B: Dinutuximab + Magrolimab + Surgery

	Suspended	Phase 1	
NCT05019703	Atezolizumab and Cabozantinib in Adolescents and Young Adults Patients With Recurrent or Metastatic Osteosarcoma	PD-L1

VEGFR2/MET

	Biological: Atezolizumab

Drug: Cabozantinib

	Recruiting	Phase 2	
NCT04044378	Famitinib Plus Camrelizumab & Famitinib Alone & Famitinib Plus Ifosfamide in Patients With Advanced Osteosarcoma	PD−1

VEGFR2/

PDGFR/c-kit

	Arm A: Famitinib + Camrelizumab

Arm B: Famitinib

Arm C: Famitinib + Ifosfamide

	Withdrawn	Phase 1/2	
NCT04668300	Oleclumab and Durvalumab in Patients With Recurrent, Refractory, or Metastatic Sarcoma	PD-L1

CD73

	Oleclumab + Durvalumab	Recruiting	Phase 2	
NCT02500797	Nivolumab With or Without Ipilimumab in Patients With Metastatic or Unresectable Sarcoma	PD−1

CTLA−4

	Arm A: Nivolumab

Arm B: Nivolumab + Ipilimumab

	Has results	Phase 2	
NCT02304458	Nivolumab With or Without Ipilimumab in Younger Patients With Recurrent or Refractory Solid Tumors or Sarcomas	PD−1

CTLA−4

	Arm A: Nivolumab

Arm B: Nivolumab + Ipilimumab

	Has results	Phase 1/2	
NCT02301039	Pembrolizumab in Patients With Advanced Sarcomas	PD−1	Pembrolizumab	Has results	Phase 2	
NCT02982486	Nivolumab Plus Ipilimumab in Patients With Non-resectable Sarcoma and Endometrial Carcinoma	PD−1

CTLA−4

	Nivolumab + Ipilimumab	Unknown	Phase 2	
NCT02406781	MK3475 and Metronomic Cyclophosphamide in Patients With Advanced Sarcomas	PD−1	Pembrolizumab + Metronomic Cyclophosphamide	Has results	Phase 2	
NCT03359018	Apatinib Plus Anti-PD1 Therapy in Patients With Advanced Osteosarcoma	PD-L1

VEGFR

	Apatinib + SHR−1210	Completed	Phase 2	
NCT02982941	Enoblituzumab in Children With B7-H3-expressing Solid Tumors	B7-H3	Enoblituzumab	Completed	Phase 1	
NCT03006848	Avelumab in Patients With Recurrent or Progressive Osteosarcoma	PD-L1	Avelumab	Has results	Phase 2	
NCT03628209	Nivolumab or Nivolumab and Azacitidine in Patients With Recurrent, Resectable Osteosarcoma	PD−1	Arm A: Nivolumab

Arm B: Nivolumab + Azacitidine

	Recruiting	Phase 1/2	
NCT02541604	Atezolizumab in Pediatric and Young Adult Participants With Solid Tumors	PD-L1	Atezolizumab	Has results	Phase 1/2	
NCT02815995	Immunotherapeutic Agents in Multiple Sarcoma Subtypes	PD-L1

CTLA−4

	Durvalumab + Tremelimumab	Completed	Phase 2	
NCT03190174	Nivolumab Plus ABI−009 in Patients With Advanced Sarcoma and Certain Cancers	PD−1

mTOR

	Nivolumab + ABI−009	Completed	Phase 1/2	
NCT03277924	Sunitinib and/or Nivolumab Plus Chemotherapy in Patients With Advanced Soft Tissue and Bone Sarcomas	PD−1

VEGFR/PDGFRβ/FLT3/RET/CSF−1R

chemotherapy

	Arm A: Sunitinib + Chemotherapy

Arm B: Sunitinib + Nivolumab + Chemotherapy

	Recruiting	Phase 1/2	
NCT03449108	LN−145 or LN−145-S1 in Treating Patients With Relapsed or Refractory Ovarian Cancer, Triple Negative Breast Cancer (TNBC), Anaplastic Thyroid Cancer, Osteosarcoma, or Other Bone and Soft Tissue Sarcomas	PD−1

CTLA−4

	Autologous Tumor Infiltrating Lymphocytes + Nivolumab + pilimumab	Active, not recruiting	Phase 2	
NCT03676985	PD-L1 Antibody in Limited Stage of High-grade Osteosarcoma	PD-L1	ZKAB001	Unknown	Phase 1/2	
NCT04074564	MASCT-I Combined With Apatinib and/or Camrelizumab in Bone and Soft Tissue Sarcoma	PD−1

VEGFR2

	MASCT-I + Camrelizumab + Apatinib	Recruiting	Early Phase 1	
NCT04294511	Camrelizumab in Combination With Neoadjuvant Chemotherapy in Osteosarcoma	PD−1

chemotherapy

	Camrelizumab + chemotherapy	Unknown	Phase 2	
NCT04351308	MAPI + Camrelizumab Versus API + Apatinib Versus MAPI in Patients With a Poor Response to Preoperative Chemotherapy for Newly Diagnosed High-grade Osteosarcoma	PD−1

VEGFR2

	Arm A: chemotherapy

Arm B: chemotherapy + Apatinib

Arm C: chemotherapy + Camrelizumab

	Unknown	Phase 2	
NCT04359550	ZKAB001 for Maintenance Therapy in Patients With High-grade Osteosarcoma After Adjuvant Chemotherapy	PD-L1	ZKAB001	Unknown	Phase 3	
NCT04544995	Niraparib and Dostarlimab in Pediatric Participants With Solid Tumors	PD−1

PARP

	Niraparib + Dostarlimab	Suspended	Phase 1	
NCT04730349	Bempegaldesleukin in Combination With Nivolumab in Children, Adolescents and Young Adults With Recurrent or Treatment-resistant Cancer	PD−1

CD122

	Nivolumab + NKTR−214	Has results	Phase 1/2	
NCT04803877	Regorafenib and Nivolumab in Osteosarcoma	PD−1

VEGFR1/VEGFR2/VEGFR3/PDGFR-β/Kit/RET/Raf−1

	Nivolumab + Regorafenib	Active, not recruiting	Phase 2	
NCT05182164	Pembrolizumab and Cabozantinib in Patients With Advanced Sarcomas	PD−1

VEGFR2/MET

	Pembrolizumab + Cabozantinib	Recruiting	Phase 2	
NCT05302921	Neoadjuvant Dual Checkpoint Inhibition and Cryoablation in Relapsed/Refractory Pediatric Solid Tumors	PD−1

CTLA−4

	Nivolumab + Ipilimumab + Cryoablation	Active, not recruiting	Phase 2	
NCT03307616	Nivolumab With and Without Ipilimumab and Radiation Therapy in Treating Patients With Recurrent or Resectable Undifferentiated Pleomorphic Sarcoma or Dedifferentiated Liposarcoma Before Surgery	PD−1

CTLA−4

	Arm A: Nivolumab

Arm B: Nivolumab + Ipilimumab

Arm C: Nivolumab + Radiation Therapy

Arm D: Nivolumab + Ipilimumab + Radiation Therapy

	Active, not recruiting	Phase 2	
NCT03282344	NKTR−214 in Combination With Nivolumab in Patients With Metastatic and/or Locally Advanced Sarcoma	PD−1

CD122

	NKTR−214 + Nivolumab	Active, not recruiting	Phase 2	

Studies of CAR-modified cellular therapies for osteosarcoma

CAR-T therapy

Adoptive T cell transfer, particularly CAR-engineered T cells, is one of the current research hotspots. At present, research on adoptive cell therapy represented by CAR-T is flourishing in various tumors, especially in hematological tumors, which have achieved good therapeutic effects. CAR-T also has shown great potential in the treatment of osteosarcoma. Targeting suitable molecules is the prerequisite for the anti-tumor effects of CAR-T. Human epidermal growth factor receptor-2 (HER2) CAR-T cells, disialoganglioside (GD2) CAR-T cells, and B7H3 CAR-T cells have been discussed the most in osteosarcoma [101].

HER2

HER2 expression has been reported in 60-70% of primary osteosarcoma [102] and is associated with adverse clinical outcomes [103]. Due to the low expression level of HER2, anti-HER2 monoclonal antibodies were ineffective for osteosarcoma patients with HER2-positive [104]. Therefore, Ahmed et al. developed HER2-specific CAR-T cells and tested the their ability to proliferate, produce immunostimulatory cytokines, and kill HER2-positive osteosarcoma cell lines in vitro. The HER2-positive CAR-T cells showed markedly regression of osteosarcoma both in locoregional and lung metastatic models [105]. In addition, HER2-specific CAR-T cells also decreased sarcosphere formation of drug resistant tumor-initialing cells [106]. In 2015, Ahmed et al. reported the results of a phase I/II clinical trail (NCT00902044) which to evaluate the safety and efficacy of HER2-CAR-T cells in patients with refractory or metastatic HER2-positive sarcoma. This trail included 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). Patients received escalating doses of HER2-CAR-T cells (1 × 104 to 1 × 108 /m2) without the administration of IL-2 or lymphodepleting chemotherapy. The results demonstrated that infusion of up to 1 × 108 /m2 HER2-CAR-T cells was well tolerated, and 4 of 17 evaluable patients had stable disease for 12 weeks to 14 months. After removal of residual metastasis, three patients remain in remission at 6, 12, and 16 months, and one showing > 90% necrosis.The median OS time of all 19 patients is 10.3 months (range 5.1 to 29.1 months). Further analysis found that the CAR-T cells persisted for up to 18 weeks in peripheral blood, and they were detected in tumor sites [107].

GD2

Osteosarcoma cells express high level GD2 antigen that can be a potential target [108]. GD2-specific CAR-T cells exhibited therapeutic potency in Ewing sarcoma priclincal models [109]. Chulanetra et al. constructed CAR modified T cells targeting GD2 and demonstrated that the osteosarcoma cells expressing high levels of GD2 were be effectively targeted and killed by GD2-CAR-T cells in vitro. Besides, the combination of GD2-CAR-T cells with suboptimal chemotherapeutic treatment with docorubicin showed stronger anti-tumor efficacy. Unfortunately, further analysis suggested that the osteosarcoma cells and GD2-CAR-T cells were induced to up-regulate expression of PD-L1 and PD-1, respectively. And the interaction of PD-1 and PD-L1 promoted apoptosis of CAR-T cells [110]. This phenomenon suggests that the combination of PD-1/PD-l1 antibody and CAR-T cells may exhibit better anti-osteosarcoma effects.

ALCAM

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a 105 kDa trans-membrane glycoprotein that belongs to the immunoglobulin superfamily. The study demonstrated that ALCAM was associated with tumorigenesis in osteosarcoma [111]. ALCAM acts as a potential therapeutic target for osteosarcoma patients with ALCAM positivity. Wang et al. developed CAR-T cells targeting ALCAM and evaluated the anti-tumor efficacy. ALCAM-CAR-T cells exhibited specific and potent cytotoxicity against human osteosarcoma cells in vitro; the cytotoxicity was positively correlated with the level of ALCAM expression on the osteosarcoma cells. In murine models, ALCAM-CAR-T cells inhibited the tumor growth with no obvious toxicity [112].

B7-H3

B7-H3 (CD276) is a checkpoint molecule that contributes to tumor immune evasion, metastasis and poor prognosis [113, 114]. B7-H3 highly expressed on pediatric solid tumors, including osteosarcoma. Majzner et al. constructed a novel second-generation CAR-T cells directing B7-H3 and tested the anti-tumor efficacy in a MG63.3 xenograft model of osteosarcoma with strong metastatic potential. The results demonstrated that B7-H3-CAR-T cells mediated complete regression, eradication of osteosarcoma and lead to a significant survival advantage compared with control group. In addition, they found that CAR-T cell activity is dependent on B7-H3 molecule density, which reduced the off-target effects of CAR-T cells [115]. Zhang et al. conducted the third-generation CAR-T cells targeting B7-H3 and proved the killing effects for tumor cells in vitro and in vivo using the patientderived xenografts (PDXs) model of osteosarcoma [116–125].

EphA2

EphA2 is a tyrosine kinase receptor for Ephrin signalling during embryonic development. In osteosarcoma, overexpression of EphA2 has association with oncogenic signalling, the promotion of angiogenesis and tumor progression [117]. EphA2 is a promising target because of the high expression in tumors and low expression in normal tissue [118]. Hsu et al. generated EphA2-CAR-T cells that effectively killed EphA2-positive osteosarcoma cell lines in vitro. And in subcutaneous osteosarcoma mouse models, EphA2-CAR-T cells induced significant reduction or elimination of osteosarcoma and extended survival in a dose and delivery route-dependent manner. Besides, EphA2-CAR-T cells showed superior efficacy for liver and lung metastatic osteosarcoma cells when delivered systemically [119].

IL-11Rα

Interleukin-11 (IL-11), a member of a family of pleiotropic cytokines [120], specifically binds to IL-11 receptor α-chain (IL-11Rα) and active the signaling pathways involved in adipogenesis, osteoclastogenesis, neurogenesis, and megakaryocyte maturation and platelet production [121]. The overexpression of IL-11Rα was found in several cancer types, including osteosarcoma [122]. Based on that, Huang et al. developed IL-11Rα-CAR-T cells and tested the tumor suppression activity on osteosarcoma in vitro and in vivo. IL-11Rα-CAR-T cells not only killed the osteosarcoma cell lines expressing IL-11Rα, but also results in the regression of osteosarcoma lung metastases. Besides, the cytotoxicity of IL-11Rα-CAR-T cells correlated with level of IL-11Ra expression on osteosarcoma cells [123].

IGF1R and ROR1

Insulin-like growth factor 1 receptor (IGF1R) is a tetrameric transmembrane receptor tyrosine kinase and widely expressed by several solid tumors and hematologic malignancies. The binding of ligand and IGF1R contributes to the proliferation, survival, transformation, metastasis, and angiogenesis [124]. Overexpression of tyrosine kinase-like orphan receptor 1 (ROR1) involved in tumor cell migration and invasiveness [125]. And the limited expression of ROR1 in normal tissues is another advantage for it to be an attractive therapeutic target in sarcomas [126]. The preclinical study conducted by Huang et al. demonstrated that IGF1R and ROR1 CAR-T cells exhibited significantly tumor growth inhibition and survival extension on sarcoma [127].

NKG2D

CAR-T cells therapies are increasingly attractive for various malignant tumor. However, the severe cytokine release syndrome (CRS) caused by T-cell activation and expansion limited the clinical application. To solve the problem, Fernandez et al. used memory T cells to express an CAR. They chose natural killer cell group 2D (NKG2D) as the target of CAR-T cells [128]. NKG2D receptor is heterogeneously expressed in primary and metastatic osteosarcoma cells, but is rarely expressed by healthy tissues [129]. CD45RA+memory T cells expressing an NKG2D CAR effectively killed the osteosarcoma cells in vitro and inhibited the tumor growth in mouse models of metastatic osteosarcoma with no toxicity to healthy tissues [130]. Similarly, to diminish the risk of CRS, Lu et al. developed a CAR-T adaptor molecule (CAM)-based therapy using a bispecific low-molecular weight ligand EC17 (FITC- folic acid). EC17 penetrates solid tumors within minutes and strongly bind with folate receptor (FR), whereas unbound EC17 will be rapidly cleared [131]. Administration of intermitte dosing and/or dose-titration of EC17 CAM could drive CAR-T cell activation, proliferation, and persistence and control the amount of cytokine releasing, which is a safety mechanism for the application of CAR-T cells. For FR+ osteosarcoma cells, EC17 controlled CAR-T cells showed potent anti-tumor activity in mice models [132].

CAR-M therapy

Macrophages, as the most abundant immune cell component infiltrating the osteosarcoma microenvironment, hold great potential as a tool for adoptive immunotherapy. Compared with other immune cells, one major advantage of using macrophages for ACT is the propensity in migration and infiltration into tumors [133]. CAR macrophage (CAR-M) studies are mainly at the nascent stage with one phase I trial ongoing which uses autologous CAR-M targeting HER2 overexpressing solid tumors [134]. Pre-clinical study showed that anti-CD19 and anti-HER2 CAR-Ms phagocytose antigen-bearing tumor cells in an antigen-specific manner in vitro. In lung metastases mouse models, CAR-M treatment significantly inhibited tumor growth and prolonged the overall survival of the tumor-bearing mice [134]. In addition, CAR-Ms promoted an inflammatory state within the TME and were capable of antigen cross-presentation to tumor-specific CD8+ T cells [135]. In osteosarcoma, CAR-M is a promising tool, which specifically recognize and eliminate tumor cells [136, 137]. Moreover, engineered CAR-Ms have leaded to increase of anti-tumor cytokines, such as IL-6, and chemokines, such as CXCL18, in the TME of osteosarcoma [138, 139]. The production of these beneficial cytokines fosters help to convert cold tumors into hot tumors [140].

CAR-NK therapy

As previously mentioned, NK cells possess potent anti-osteosarcoma effects, but NK cells lack the appropriate machinery to recognize tumor antigens without binding Fc-γ receptors through CD16 to IgG-coated targets. When the NK cells are loaded a CAR targeting specific osteosarcoma antigen, this limitation can be overcome [141]. Currently, there are a number of clinical trials involving CAR-NK that have been planned or are ongoing. All these clinical trials are at the phase I/II trial stage [142]. At present, the early clinical trial results have not yet demonstrated consistent efficacy and safety. The trial in adults with chronic lymphocytic leukemia demonstrated that CAR-NK targeting CD19 are safe and effective [143]. However, the results from studies on targets other than CD19 in CAR-NK have been less than satisfactory, including both in vivo studies and clinical research. GD2-specific CAR-NK cells for Ewing sarcoma failed to eliminate GD2-positive sarcoma in xenografts models [71]. NK cells have a relatively short half-life, typically less than 10 days [144], which is a double-edge sword in CAR-NK therapy. This confers an advantage in case severe toxicity occurs, but also creates a challenge that repeated administrations may be needed to achieve durable response. Compared with CAR-T cells, CAR-NK therapy show several advantages, including (1) the ability to be derived from established cell lines or allogeneic NK cells without the major histocompatibility complex (MHC) restriction; (2) CAR-NK cells eliminate cancer cells through both CAR-dependent and CAR-independent manners; (3) reduced toxicity, particularly in terms of cytokine-release syndrome and neurotoxicity.

So far, CAR-NK and CAR-M therapies have not made promising progress in the research of osteosarcoma. It need to point out, these two therapies could theoretically compensate for the shortcomings of CAR-T treatment, but there is still a long way to go before their clinical application. At present, CAR-T has shown clear anti-tumor effects in preclinical studies for osteosarcoma, and a large number of clinical trials are still ongoing, with GD2, HER2, and B7-H3 as the main targets (Table 2). Compared to other cellular adoptive therapies, CAR-T research in osteosarcoma may achieve breakthrough progress first. We are looking forward to having inspiring results reported in the future.

Table 2 Summary of clinical trials of adoptive cell therapies in osteosarcoma

Registration NO	Clinical Trial	Target	Participant Group	State	Stage	
NCT04539366	GD2-Targeted Modified T-cells in Children, Adolescents, and Young Adults With Relapsed/​Refractory Osteosarcoma and Neuroblastoma	GD2	GD2-CART cells	Suspended	Phase 1	
NCT02107963	T Cells Expressing an Anti-GD2 Chimeric Antigen Receptor in Children and Young Adults With GD2 + Solid Tumors	GD2	GD2-CART cells	Completed	Phase 1	
NCT03209869	Ex-Vivo Expanded and Activated Haploidentical NK Cells and Hu14.18-IL2 for Patients With Relapsed or Refractory Neuroblastoma and Osteosarcoma	GD2	Ex vivo Expanded and Activated Haploidentical Donor NK Cells + hu14.18-IL2	Withdrawn	Phase 1	
NCT03635632	C7R-GD2.CART Cells for Patients With Relapsed or Refractory Neuroblastoma and Other GD2 Positive Cancers	GD2	C7R-GD2.CART cells	Recruiting	Phase 1	
NCT01953900	iC9-GD2-CAR-VZV-CTLs for Refractory or Metastatic GD2-positive Sarcoma and Neuroblastoma	GD2	GD2-CART cells + VZV vaccine	Active, not recruiting	Phase 1	
NCT02107963	T Cells Expressing an Anti-GD2 Chimeric Antigen Receptor in Children and Young Adults With GD2 + Solid Tumors	GD2	GD2-CART cells	Completed	Phase 1	
NCT03373097	Anti-GD2 CAR T Cells in Pediatric Patients Affected by High Risk and/or Relapsed/Refractory Neuroblastoma or Other GD2-positive Solid Tumors	GD2	GD2-CART cells	Recruiting	Phase 1/2	
NCT03721068	CAR T-Cells Targeting the GD2 With IL−15 + iCaspase9 for Relapsed/Refractory Neuroblastoma or Relapsed/Refractory Osteosarcoma	GD2	iC9.GD2.CAR.IL−15 T cells	Recruiting	Phase 1	
NCT00902044	HER2 Chimeric Antigen Receptor Expressing T Cells in Advanced Sarcoma	HER2	HER2-CART cells	Active, not recruiting	Phase 1	
NCT04995003	HER2 Chimeric Antigen Receptor T Cells in Combination With Checkpoint Blockade in Patients With Advanced Sarcoma	HER2	Arm A: HER2-CART cells + Pembrolizumab

Arm B: HER2-CART cells + Nivolumab

	Recruiting	Phase 1	
NCT04483778	B7H3 CAR T Cell Immunotherapy for Recurrent/Refractory Solid Tumors in Children and Young Adults	B7-H3	Arm A: SCRI-CARB7H3(s)

Arm B: SCRI-CARB7H3(s)x19

Arm C: SCRI-CARB7H3(s)x19 + Pembrolizumab

	Active, not recruiting	Phase 1	
NCT04897321	B7-H3-Specific Chimeric Antigen Receptor Autologous T-Cell Therapy for Pediatric Patients With Solid Tumors	B7-H3	B7-H3 CART cells	Recruiting	Phase 1	
NCT04864821	CD276 Targeted Autologous Chimeric Antigen Receptor T Cell Infusion in Patients With CD276 Positive Advanced Solid Tumor	B7-H3	CD276-CART cells	Unknown	Early Phase 1	
NCT03618381	EGFR806 CAR T Cell Immunotherapy for Recurrent/Refractory Solid Tumors in Children and Young Adults	EGFR	Arm A: EGFR 806CAR-EGFRt

Arm B: EGFR 806CAR-EGFRt and CD19CAR-T2A-HER2tG

	Recruiting	Phase 1	
NCT03462316	NY-ESO−1-specific T Cell Receptor T Cell in Sarcoma	NY-ESO−1	NY-ESO−1 TCR Specific T cell	Active, not recruiting	Phase 1	
NCT03356782	4th Generation Safety-engineered CAR T Cells Targeting Sarcomas	CD133, GD2, Muc1, CD117 or other marker	Sarcoma-specific CART cells	Recruiting	Phase 1/2	
NCT04433221	Combination Immunotherapy Targeting Sarcomas	GD2, PSMA, HER2, CD276 or other markers	Multiple sarcoma-specific CART cells and sarcoma vaccines	Recruiting	Phase 1/2	
NCT05312411	FITC-E2 CAR T Cells in Combination With Parenterally Administered Folate-Fluorescein (UB-TT170) For Osteogenic Sarcoma		UB-TT170 following SCRI-E2CAR EGFrtv1	Recruiting	Phase 1	
NCT05621668	T-Cell Membrane-Anchored Tumor Targeted Il12 (Attil12)- T-Cell Therapy in Subjects With Advanced/​Metastatic Soft Tissue and Bone Sarcoma		AttIL2-T cell therapy	Recruiting	Phase 1	
NCT05703854	CAR.70-engineered IL15-transduced Cord Blood-derived NK Cells in Conjunction With Lymphodepleting Chemotherapy for the Management of Advanced Renal Cell Carcinoma, Mesothelioma and Osteosarcoma		CAR.70/IL15-transduced CB-derived NK cells	Recruiting	Phase 1/2	
NCT02508038	α/β CD19 + Depleted Haploidentical Transplantation + Zometa for Pediatric Hematologic Malignancies and Solid Tumors		TCRαβ+/CD19 + depleted Haploidentical HSCT + Zoledronate	Recruiting	Phase 1	
NCT03449108	LN−145 or LN−145-S1 in Treating Patients With Relapsed or Refractory Ovarian Cancer, Triple Negative Breast Cancer, Anaplastic Thyroid Cancer, Osteosarcoma, or Other Bone and Soft Tissue Sarcomas		Autologous Tumor Infiltrating Lymphocytes LN−145 or LN−145-S1	Active, not recruiting	Phase 2	
NCT02100891	Haploidentical Transplant and Donor Natural Killer Cells for Solid Tumors		Allogeneic HCT + Donor NK Cell Infusion	Active, not recruiting	Phase 2	

Other immunotherapies for osteosarcoma

GD2 and HER2 as candidate target antigens due to their high expression level on osteosarcoma cells. However, clinical trials of trastuzumab (anti-HER2) or dinutuximab (anti-GD2) for metastatic or refractory osteosarcoma were not successful [145, 146]. Therefore, Park et al. constructed the bispecific antibodies (BsAbs) targeting CD3 and GD2 or HER2. GD2-BsAb and HER2-BsAb successfully directed T cells into tumor tissues and exerted potent anti-tumor activity against osteosarcoma. Besides, PD-1/PD-L1 blockade significantly enhanced anti-tumor activity of GD2-BsAb and HER2-BsAb [147]. Currently, only a small number of clinical studies of BsAbs have been conducted in osteosarcoma, summarized in Table 3. Besides, Mason et al. performed a phase I trial in canine osteosarcoma to evaluate the anti-tumor activity of recombinant Listeria vaccines expressing a chimeric human HER2/neu fusion protein (ADXS31-164). They found that ADXS31-164 induced HER2/neu-specifific immunity and reduced the incidence of metastatic disease and prolonged OS [148]. DCs are the most potent professional antigen-presenting cells and play a crucial role in the osteosarcoma microenvironment. Enhancing the anti-tumor capabilities of DCs holds promise as a novel therapeutic approach for osteosarcoma. Kawano et al. suggested that combined DCs and anti-TGF-β antibodies to treat osteosarcoma and detected enhanced systematic immune responses in mice models [149]. The synergistic action of DCs and agonistic antibodies against the glucocorticoid-induced tumor necrosis factor receptor (anti-GITR) can significantly amplify the systemic immune response. This strategy not only facilitates the elimination of regulatory T cells but also effectively inhibites osteosarcoma growth in vivo [150]. A variety of vaccines have shown encouraging results, including the CD1c+ DC vaccine, which harnesses the power of antigen-presenting DCs, and the innovative approach of vaccinating with polyinosinic: polycytidylic acid (poly I: C). This treatment activates and loads tumor antigens onto CD103+ myeloid/conventional DC1s, enhancing the immune system’s capacity to target cancer cells [151].

Table 3 Summary of clinical trials of BsAbs in osteosarcoma

Registration NO	Clinical Trial	Target	Participant Group	State	Stage	
NCT03860207	Humanized 3F8 Bispecific Antibody (Hu3F8-BsAb) in Patients With Relapsed/Refractory Neuroblastoma, Osteosarcoma and Other Solid Tumor Cancers	GD2

CD3

	Humanized 3F8-BsAb	Terminated	Phase 1/2	
NCT02173093	Activated T Cells Armed With GD2 Bispecific Antibody in Children and Young Adults With Neuroblastoma and Osteosarcoma	GD2

CD3

	IL−2 + GM-CSF + GD2Bi-aATC	Unknown	Phase 1/2	
NCT03406949	MGD009/MGA012 Combination in Relapsed/Refractory Cancer	B7-H3

CD3

PD−1

	Obrindatamab + Retifanlimab	Completed	Phase 1	
NCT02628535	MGD009 in B7-H3-expressing Tumors	B7-H3

CD3

	Obrindatamab	Terminated	Phase 1	

Research on immunotherapy for osteosarcoma is mostly in the early stages, and current results show that these treatment strategies have some therapeutic potential, but further validation and improvement are still needed. We present the current immunotherapy in osteosarcoma in Fig. 2.

Fig. 2 The main immunotherapy strategies in osteosarcoma. In osteosarcoma, the relatively well-studied immunotherapies include: immune checkpoint inhibitors (with targets such as PD-1/PD-L1, CTLA-4, TIM-3, CD47 and TIGIT), adoptive cell therapies (such as CAR-T, CAR-M and CAR-NK), dendritic cell vaccines, and bispecific antibodies (targeting HER2 or GD2 and CD3). TAA: tumor-associated antigen

Combination therapy strategies

Combination of anti-PD-1/PD-L1 and anti-CTLA-4 treatment

Due to the presence of various immunosuppressive pathways in the microenvironment of osteosarcoma, treatment with a single-target ICI has not achieved satisfactory results. Like other solid tumors, trying a combination therapy of two ICIs in osteosarcoma may have better anti-tumor effects. At present, the combined application of ICIs is still mainly focused on classic anti-PD-1/PD-L1 and anti-CTLA-4 antibodies. Although combination therapy may bring breakthroughs in efficacy, its side effects also need to be paid more attention.

A study demonstrated that anti-PD-L1 treatment for metastatic osteosarcoma upregulate additional inhibitory receptors including CTLA-4, which contributed to the resistance of anti-PD-L1 therapy. Therefore, a combination therapy of anti-PD-L1 with anti-CTLA-4 antibody completely controlled metastatic osteosarcoma in an osteosarcoma mouse model [152]. A case report demonstrated that the combination of an anti-PD-1 antibody (nivolumab) and an anti-CTLA-4 antibody (ipilimumab) inhibited the progression of metastatic tumor for a young man with a metastatic osteosarcoma not responsive to several lines of standard chemotherapy. Before the application of dual checkpoint inhibition treatment, they detected the amplification of PD-L1 and PD-L2 in resected tumor tissue through next-generation sequencing. Thus it can be seen, it is important that identifying biomarkers to select patients with high response rates to immunotherapy [153]. The trial compared the efficacy of anti-PD-1 antibody only and the combination therapy of anti-PD-1 antibody plus anti-CTLA-4 antibody. Enrolled patients were allocated to receive either nivolumab monotherapy (43 patients) or nivolumab plus ipilimumab (42 patients). Nivolumab monotherapy showed limited efficacy for patients with advanced sarcoma. Nivolumab combined with ipilimumab demonstrated promising response rate with a manageable safety profile, which need further study [154]. 62 patients diagnosed as advanced or metastatic sarcoma were enrolled into the clinical trial (NCT02815995) to evaluate the safety and efficacy of the combination of durvalumab (anti-PD-L1 antibody) and tremelimumab anti-CTLA-4 antibody) [155]. Patients received 1500 mg intravenous durvalumab and 75 mg intravenous tremelimumab for four cycles, followed by durvalumab alone every 4 weeks for up to 12 months. The PFS at 12 weeks was 49%. Grade 3–4 treatment-related adverse events were reported in 21 patients [155]. It can be supposed that the combination therapy of different immune checkpoint inhibitors is a promising option for patients with osteosarcoma who have progressed after multi line therapy.

Combination of anti-PD-1/PD-L1 and anti-angiogenic treatment

Angiogenesis is significantly correlated with tumor progression and is an important part of the formation of the immunosuppressive microenvironment in osteosarcoma. Combining anti-angiogenic therapy with immune checkpoint inhibitor treatment is beneficial for lifting immunosuppression. At the same time, anti-angiogenic therapy promotes the normalization of tumor blood vessels, helping more anti-tumor drugs and immune cells to enter the tumor microenvironment and exert their effects.

Immunosuppression promoted by tumor angiogenesis, and more immune cells are related with angiogenesis [156]. Therefore, simultaneously targeting tumor blood vessels and antagonizing immune suppression may propose new therapeutic strategies for sarcoma. At 2020, Martin-Broto et al. reported the results of a phase Ib/II trial evaluating nivolumab (anti-PD-1 antibody) and sunitinib (inhibitor of anti-angiogenesis) combination in advanced soft tissue sarcomas (NCT03277924). 16 sarcoma patients (including osteosarcoma) entered into phase Ib study, and the results determined the recommended dose of sunitinib for phase II was 37.5 mg as induction and then 25 mg in combination with nivolumab. The 6-month PFS rate for soft-tissue sarcoma patients was 48% (95% CI: 41–55%). The most frequent treatment-related toxicities were fatigue (63.5%) and increased aspartate aminotransferase (48.0%). The most common grade 3 or 4 side effects were transaminitis (17.3%) and neutropenia (11.5%) [157]. We look forward to the publication of overall survival data of soft-tissue sarcoma patients and research results on osteosarcoma cohort. Yao et al. conducted a phase I trial (NCT04074564) to assess the safety and efficacy of multi-antigen stimulated cell therapy-I in combination with camrelizumab (anti-PD-1 antibody) and apatinib (a highly selective inhibitor targeting VEGFR2) in patients with unresectable recurrent or metastatic bone and soft-tissue sarcoma who had previously undergone at least one line of systemic therapy. Totally, 19 patients, including 6 osteosarcoma patients, were enrolled into the study. For the 6 patients with osteosarcoma, the ORR was 33.3%, the disease control response (DCR) was 50.0%, and median PFS was 5.7 months. The most common treatment-related adverse event was decreased neutrophil count. 11 (57.9%) experienced grade ≥ 3 treatmentrelated adverse events. No treatment-related deaths occurred [158]. Besides, Xie et al. reported the results of a single-arm phase II trial evaluating the safety and efficacy of apatinib (anti-VEGFR2 inhibitor) plus camrelizumab (anti-PD-1 antibody) in patients with chemotherapy-refractory osteosarcoma (NCT03359018). 43 patients enrolled this trial, and the ORR was 20.9% and two patients with durable disease control were observed. 13 of 43 patients were progression free at 6 months and the 6-month PFS rate was 50.9% [159]. In addition to ICIs, Park et al. conducted the preclinical study demonstrating that VEGF blockade enhanced the infiltration of tumor-antigens (GD2/HER2/GPC3) armed T cells into tumor microenvironment and increased CD8+ T cells survival and dispersion [160].

Combination of target-specific and non target-specific treatment

Target-specific therapies are notable for their strong targeting capabilities and significant effects, while non target-specific therapies enhance the overall immune response to fight cancer, potentially offering a broader range of applications. Non target-specific immune response, such as vaccine, DCs, IL-2 and TGF-β, combined with target-specific immunotherapy may exhibit stronger anti-tumor efficacy.

A study examined the anti-tumor efficacy of the combination of tumor lysate-pulsed DCs and CTLA-4 antibody in a C3H mouse osteosarcoma model. The combination therapy potently inhibited the accumulation of Tregs and promoted the infiltration of CD8+ T cells inside the metastatic settings. The synergistic effect of tumor lysate-pulsed DCs and CTLA-4 blockade is manifested in inhibiting the growth of metastatic tumors and prolonging survival time [161]. Guo et al. developed a new bifunctional fusion protein composed of a monoclonal antibody against PD-L1 fused with the extracellular domain of TGF-β receptor. However, the results of the phase Ib study showed no significantly improvement of the ORR in patients with recurrent osteosarcoma, while the side effects are acceptable [162]. Bempegaldesleukin, a CD122-preferential IL-2 pathway agonist, is associated with increased proliferation and activation of TILs among patients with solid tumors [163]. Bempegaldesleukin may be beneficial for optimizing the responsiveness of metastatic sarcoma to checkpoint blockade. Based on that, a study was conducted to evaulate the safety and efficacy of bempegaldesleukin combination with nivolumab for 84 patients with selected locally advanced or metastatic high-grade sarcoma, including 10 osteosarcoma patients. The combination strategy showed limited activity for most sarcoma. In other sarcoma subtypes, CD8+ T cell infiltration, PD-1 expression and upregulation of immunerelated pathways correlated with improved ORR. However, in osteosarcoma, although it has a higher tumor mutational burden, there is no significant response to the combination therapy [164].

As mentioned earlier, T cells are edited to express specific tumor antigens, allowing them to recognize and kill tumor cells. GD2-CAR-T therapy is being explored in extensive research in osteosarcoma, but the survival time of CAR-T cells in body is indeed limited. When T cells recognize viruses, they can exist in the body for many years. And when the same virus or virus vaccine enters the body again, these T cells can be quickly activated and cleared of the virus. Tanaka et al. generated the GD2-CAR-modified varicella zoster virus (VZV)-specific T cells and conducted a phase I trail (NCT01953900) to evaluate the activity of combined application of CAR-T cells and vaccine in advanced osteosarcoma and neuroblastoma [165]. At present, the results of this study have not been reported. It is hoped that through the multifaceted exploration of researchers, strategy can be found to control tumors for osteosarcoma patients, especially those with recurrent or metastatic disease.

Immunotherapy-combination stratagies have certain therapeutic potential, and discovering and verifying effective treatment combinations that achieve a “1 + 1 > 2” synergistic effect remains challenging. The exploration of advantageous combinations for immunotherapy-combination stratagy is a long and arduous task.

Conclusions and future directions

Osteosarcoma is the most common primary bone malignancy for children and young adults. The current standard care has improved the prognosis of patients with localized osteosarcoma, but the overall survival has not substantially improved over the past decades. For metastatic or relapsed osteosarcoma, even active treatment results in a 5-year survival rate of 20%. Therefore, it is urgent to find new and effective strategies to further improve the survival rate of osteosarcoma patients. The immunosuppressive TME of osteosarcoma, especially the lung metastatic foci, is infiltrated with a variety of cells that promote immunosuppression and express numerous immunosuppressive molecules, providing opportunities for the application of immunotherapy in osteosarcoma. However, it is precisely due to the unique inhibitory immune microenvironment of osteosarcoma, the responsiveness to immunotherapy is unsatisfactory. The immune checkpoint blockers (such as anti-PD-1/PD-L1 antibody and anti-CTLA-4 antibody) treatment only showed a limited therapeutic effect on osteosarcoma. CAR-T therapy in osteosarcoma is still in early stages of research. CAR-T therapy has not yet achieved the same success in clinical trials for osteosarcoma as it has in hematological malignancies, and other CAR-engineered adoptive cell therapies, such as CAR-NK and CAR-M, are still in the preclinical research phase. Based on current preclinical and clincial data on immunotherapy, there is still a long way to go for the application of immunotherapy in osteosarcoma. We need to explore more other biomarkers to predict the efficacy of immunotherapy in patients and choose the subpopulations that will be beneficial from immunotherapy. In addition, a single immunotherapy has low response rate in osteosarcoma. Combination therapy based on immunotherapy that converting a immune cold into an inflamed microenvironment may be a future direction.

Abbreviations

PFS Progression-free survival

ORR Objective response rate

ICIs Immune checkpoint inhibitors

CAR-T Chimeric antigen receptor T

ACT Adoptive cell therapy

TME Tumor microenvironment

TAMs Tumor-associated macrophages

TANs Tumor-associated neutrophils

MDSCs Myeloid-derived suppressor cells

NK cells Natural killer cells

DCs Dendritic cells

CTCs Circulating tumor cells

TGF-β Transforming growth factor-beta

Arg-1 Arginase-1

iNOS Inducible nitric oxide synthase

ROS Reactive oxygen species

VEGF Vascular endothelial growth factor

HGF Hepatocyte growth factor

EMT Epithelial-mesenchymal transition

TIM-3 T-cell immunoglobulin and mucin domain-containing protein 3

TIGIT T cell immunoreceptor with Ig and ITIM domains

NETs Neutrophil extracellular traps

CSCs Cancer stem cells

MSCs Mesenchymal stem cells

EVs Extracellular vesicles

IFN-γ Interferon-γ

TILs Tumor-infiltrating lymphocytes

APCs Antigen-presenting cells

TNF-α Tumor necrosis factor-α

CTLs Cytotoxic T lymphocytes

C1Q Complete component 1q

NF-kB Nuclear factor-kappa B

STAT3 Signal transducer and activator of transcription 3

LAG-3 Lymphocyteactivation gene 3

IDO1 Indoleamine 2,3-dioxygenase

OS Overall survival

DFS Disease free survival

PR Partial response

CTLA-4 Cytotoxic T lymphocyte-associated antigen-4

Th1 Type 1 T helper

Gal-9 Galectin-9

HER2 Human epidermal growth factor receptor-2

GD2 Disialogangliosides

ALCAM Activated leukocyte cell adhesion molecule

PDXs Patientderived xenografts

IL-11 Interleukin-11

IL-11Rα IL-11 receptor α-chain

IGF1R Insulin-like growth factor 1 receptor

ROR1 Tyrosine kinase-like orphan receptor 1

CRS Cytokine release syndrome

NKG2D Natural killer cell group 2D

CAM CART adaptor molecule

FR Folate receptor

CAR-M CAR macrophage

MHC Major histocompatibility complex

BsAbs Bispecific antibodies

GITR Glucocorticoid-induced tumor necrosis factor receptor

DCR Disease control response

VZV Varicella zoster virus

Acknowledgements

Not applicable.

Author contributions

SY searched the literatures and wrote the manuscript. XY designed the study and participated in the discussion. All authors reviewed the manuscript.

Funding

This review was supported by National Natural Science Foundation of China (Grant No. 82203007) and the China Postdoctoral Science Foundation (No. 2023MD744162).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have read and approved the final manuscript for publication.

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Beird HC Osteosarcoma Nat Rev Dis Primers 2022 8 1 77 10.1038/s41572-022-00409-y 36481668
Beird HC, et al. Osteosarcoma Nat Rev Dis Primers. 2022;8(1):77.36481668 10.1038/s41572-022-00409-y
2. Czarnecka AM et al. Molecular Biology of Osteosarcoma. Cancers (Basel), 2020. 12(8).
3. Isakoff MS Osteosarcoma: current treatment and a collaborative pathway to Success J Clin Oncol 2015 33 27 3029 35 10.1200/JCO.2014.59.4895 26304877
Isakoff MS, et al. Osteosarcoma: current treatment and a collaborative pathway to Success. J Clin Oncol. 2015;33(27):3029–35.26304877 10.1200/JCO.2014.59.4895
4. Zhou Y Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma Nat Commun 2020 11 1 6322 10.1038/s41467-020-20059-6 33303760
Zhou Y, et al. Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma. Nat Commun. 2020;11(1):6322.33303760 10.1038/s41467-020-20059-6
5. Anderson ME Update on Survival in Osteosarcoma Orthop Clin North Am 2016 47 1 283 92 10.1016/j.ocl.2015.08.022 26614941
Anderson ME. Update on Survival in Osteosarcoma. Orthop Clin North Am. 2016;47(1):283–92.26614941 10.1016/j.ocl.2015.08.022
6. Arndt CA Common musculoskeletal tumors of childhood and adolescence Mayo Clin Proc 2012 87 5 475 87 10.1016/j.mayocp.2012.01.015 22560526
Arndt CA, et al. Common musculoskeletal tumors of childhood and adolescence. Mayo Clin Proc. 2012;87(5):475–87.22560526 10.1016/j.mayocp.2012.01.015
7. Bacci G High grade osteosarcoma of the extremities with lung metastases at presentation: treatment with neoadjuvant chemotherapy and simultaneous resection of primary and metastatic lesions J Surg Oncol 2008 98 6 415 20 10.1002/jso.21140 18792969
Bacci G, et al. High grade osteosarcoma of the extremities with lung metastases at presentation: treatment with neoadjuvant chemotherapy and simultaneous resection of primary and metastatic lesions. J Surg Oncol. 2008;98(6):415–20.18792969 10.1002/jso.21140
8. Sayles LC Genome-informed targeted therapy for Osteosarcoma Cancer Discov 2019 9 1 46 63 10.1158/2159-8290.CD-17-1152 30266815
Sayles LC, et al. Genome-informed targeted therapy for Osteosarcoma. Cancer Discov. 2019;9(1):46–63.30266815 10.1158/2159-8290.CD-17-1152
9. Duffaud F Efficacy and safety of regorafenib in adult patients with metastatic osteosarcoma: a non-comparative, randomised, double-blind, placebo-controlled, phase 2 study Lancet Oncol 2019 20 1 120 33 10.1016/S1470-2045(18)30742-3 30477937
Duffaud F, et al. Efficacy and safety of regorafenib in adult patients with metastatic osteosarcoma: a non-comparative, randomised, double-blind, placebo-controlled, phase 2 study. Lancet Oncol. 2019;20(1):120–33.30477937 10.1016/S1470-2045(18)30742-3
10. Long GV Overall survival and response with Nivolumab and Relatlimab in Advanced Melanoma NEJM Evid 2023 2 4 EVIDoa2200239 10.1056/EVIDoa2200239 38320023
Long GV, et al. Overall survival and response with Nivolumab and Relatlimab in Advanced Melanoma. NEJM Evid. 2023;2(4):EVIDoa2200239.38320023 10.1056/EVIDoa2200239
11. Reck M Pembrolizumab versus Chemotherapy for PD-L1-Positive non-small-cell Lung Cancer N Engl J Med 2016 375 19 1823 33 10.1056/NEJMoa1606774 27718847
Reck M, et al. Pembrolizumab versus Chemotherapy for PD-L1-Positive non-small-cell Lung Cancer. N Engl J Med. 2016;375(19):1823–33.27718847 10.1056/NEJMoa1606774
12. Ferris RL Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck N Engl J Med 2016 375 19 1856 67 10.1056/NEJMoa1602252 27718784
Ferris RL, et al. Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. N Engl J Med. 2016;375(19):1856–67.27718784 10.1056/NEJMoa1602252
13. Grupp SA Chimeric antigen receptor-modified T cells for acute lymphoid leukemia N Engl J Med 2013 368 16 1509 18 10.1056/NEJMoa1215134 23527958
Grupp SA, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509–18.23527958 10.1056/NEJMoa1215134
14. Porter DL Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia Sci Transl Med 2015 7 303 303ra139 10.1126/scitranslmed.aac5415 26333935
Porter DL, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7(303):303ra139.26333935 10.1126/scitranslmed.aac5415
15. Kochenderfer JN Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor J Clin Oncol 2015 33 6 540 9 10.1200/JCO.2014.56.2025 25154820
Kochenderfer JN, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol. 2015;33(6):540–9.25154820 10.1200/JCO.2014.56.2025
16. Liang H Advancements in osteosarcoma management: integrating immune microenvironment insights with immunotherapeutic strategies Front Cell Dev Biol 2024 12 1394339 10.3389/fcell.2024.1394339 38915446
Liang H, et al. Advancements in osteosarcoma management: integrating immune microenvironment insights with immunotherapeutic strategies. Front Cell Dev Biol. 2024;12:1394339.38915446 10.3389/fcell.2024.1394339
17. Zheng Y Mesenchymal stem cells in the osteosarcoma microenvironment: their biological properties, influence on tumor growth, and therapeutic implications Stem Cell Res Ther 2018 9 1 22 10.1186/s13287-018-0780-x 29386041
Zheng Y, et al. Mesenchymal stem cells in the osteosarcoma microenvironment: their biological properties, influence on tumor growth, and therapeutic implications. Stem Cell Res Ther. 2018;9(1):22.29386041 10.1186/s13287-018-0780-x
18. Liu T Self-seeding circulating tumor cells promote the proliferation and metastasis of human osteosarcoma by upregulating interleukin-8 Cell Death Dis 2019 10 8 575 10.1038/s41419-019-1795-7 31366916
Liu T, et al. Self-seeding circulating tumor cells promote the proliferation and metastasis of human osteosarcoma by upregulating interleukin-8. Cell Death Dis. 2019;10(8):575.31366916 10.1038/s41419-019-1795-7
19. Huang Q The role of tumor-associated macrophages in osteosarcoma progression - therapeutic implications Cell Oncol (Dordr) 2021 44 3 525 39 10.1007/s13402-021-00598-w 33788151
Huang Q, et al. The role of tumor-associated macrophages in osteosarcoma progression - therapeutic implications. Cell Oncol (Dordr). 2021;44(3):525–39.33788151 10.1007/s13402-021-00598-w
20. Duluc D Interferon-gamma reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages Int J Cancer 2009 125 2 367 73 10.1002/ijc.24401 19378341
Duluc D, et al. Interferon-gamma reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages. Int J Cancer. 2009;125(2):367–73.19378341 10.1002/ijc.24401
21. Luo ZW Macrophages in Osteosarcoma Immune Microenvironment: implications for Immunotherapy Front Oncol 2020 10 586580 10.3389/fonc.2020.586580 33363016
Luo ZW, et al. Macrophages in Osteosarcoma Immune Microenvironment: implications for Immunotherapy. Front Oncol. 2020;10:586580.33363016 10.3389/fonc.2020.586580
22. He Z Zhang S Tumor-Associated macrophages and their Functional Transformation in the hypoxic Tumor Microenvironment Front Immunol 2021 12 741305 10.3389/fimmu.2021.741305 34603327
He Z, Zhang S. Tumor-Associated macrophages and their Functional Transformation in the hypoxic Tumor Microenvironment. Front Immunol. 2021;12:741305.34603327 10.3389/fimmu.2021.741305
23. Shao XJ Inhibition of M2-like macrophages by all-trans retinoic acid prevents cancer initiation and stemness in osteosarcoma cells Acta Pharmacol Sin 2019 40 10 1343 50 10.1038/s41401-019-0262-4 31296953
Shao XJ, et al. Inhibition of M2-like macrophages by all-trans retinoic acid prevents cancer initiation and stemness in osteosarcoma cells. Acta Pharmacol Sin. 2019;40(10):1343–50.31296953 10.1038/s41401-019-0262-4
24. Aran D Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage Nat Immunol 2019 20 2 163 72 10.1038/s41590-018-0276-y 30643263
Aran D, et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol. 2019;20(2):163–72.30643263 10.1038/s41590-018-0276-y
25. Herroon MK Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms Oncotarget 2013 4 11 2108 23 10.18632/oncotarget.1482 24240026
Herroon MK, et al. Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms. Oncotarget. 2013;4(11):2108–23.24240026 10.18632/oncotarget.1482
26. Tang F Surgical Treatment of Osteosarcoma Induced Distant Pre-metastatic Niche in Lung to facilitate the colonization of circulating Tumor cells Adv Sci (Weinh) 2023 10 28 e2207518 10.1002/advs.202207518 37585564
Tang F, et al. Surgical Treatment of Osteosarcoma Induced Distant Pre-metastatic Niche in Lung to facilitate the colonization of circulating Tumor cells. Adv Sci (Weinh). 2023;10(28):e2207518.37585564 10.1002/advs.202207518
27. Haist M et al. The functional crosstalk between Myeloid-Derived Suppressor Cells and Regulatory T Cells within the immunosuppressive Tumor Microenvironment. Cancers (Basel), 2021. 13(2).
28. Ran S Wilber A Novel role of immature myeloid cells in formation of new lymphatic vessels associated with inflammation and tumors J Leukoc Biol 2017 102 2 253 63 10.1189/jlb.1MR1016-434RR 28408396
Ran S, Wilber A. Novel role of immature myeloid cells in formation of new lymphatic vessels associated with inflammation and tumors. J Leukoc Biol. 2017;102(2):253–63.28408396 10.1189/jlb.1MR1016-434RR
29. Gabrilovich DI Myeloid-derived suppressor cells Cancer Immunol Res 2017 5 1 3 8 10.1158/2326-6066.CIR-16-0297 28052991
Gabrilovich DI. Myeloid-derived suppressor cells. Cancer Immunol Res. 2017;5(1):3–8.28052991 10.1158/2326-6066.CIR-16-0297
30. Umansky V et al. The role of myeloid-derived suppressor cells (MDSC) in Cancer Progression. Vaccines (Basel), 2016. 4(4).
31. Tsubakihara Y, Moustakas A. Epithelial-mesenchymal transition and metastasis under the control of transforming growth factor beta. Int J Mol Sci, 2018. 19(11).
32. Wang C CD300ld on neutrophils is required for tumour-driven immune suppression Nature 2023 621 7980 830 9 10.1038/s41586-023-06511-9 37674079
Wang C, et al. CD300ld on neutrophils is required for tumour-driven immune suppression. Nature. 2023;621(7980):830–9.37674079 10.1038/s41586-023-06511-9
33. Ligon JA et al. Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes. J Immunother Cancer, 2021. 9(5).
34. Li F Interaction gene set between osteoclasts and regulatory CD4(+) T cells can accurately predict the prognosis of patients with osteosarcoma Cancer Sci 2023 114 7 3014 26 10.1111/cas.15821 37150900
Li F, et al. Interaction gene set between osteoclasts and regulatory CD4(+) T cells can accurately predict the prognosis of patients with osteosarcoma. Cancer Sci. 2023;114(7):3014–26.37150900 10.1111/cas.15821
35. Li X Tim3/Gal9 interactions between T cells and monocytes result in an immunosuppressive feedback loop that inhibits Th1 responses in osteosarcoma patients Int Immunopharmacol 2017 44 153 9 10.1016/j.intimp.2017.01.006 28103502
Li X, et al. Tim3/Gal9 interactions between T cells and monocytes result in an immunosuppressive feedback loop that inhibits Th1 responses in osteosarcoma patients. Int Immunopharmacol. 2017;44:153–9.28103502 10.1016/j.intimp.2017.01.006
36. Lucca LE Dominguez-Villar M Modulation of regulatory T cell function and stability by co-inhibitory receptors Nat Rev Immunol 2020 20 11 680 93 10.1038/s41577-020-0296-3 32269380
Lucca LE, Dominguez-Villar M. Modulation of regulatory T cell function and stability by co-inhibitory receptors. Nat Rev Immunol. 2020;20(11):680–93.32269380 10.1038/s41577-020-0296-3
37. Wu L et al. Tumor-Associated neutrophils in Cancer: going pro. Cancers (Basel), 2019. 11(4).
38. Yang B Identification of prognostic biomarkers associated with metastasis and immune infiltration in osteosarcoma Oncol Lett 2021 21 3 180 10.3892/ol.2021.12441 33574919
Yang B, et al. Identification of prognostic biomarkers associated with metastasis and immune infiltration in osteosarcoma. Oncol Lett. 2021;21(3):180.33574919 10.3892/ol.2021.12441
39. Fu Y Development and validation of a Hypoxia-Associated Prognostic signature related to Osteosarcoma Metastasis and Immune Infiltration Front Cell Dev Biol 2021 9 633607 10.3389/fcell.2021.633607 33816483
Fu Y, et al. Development and validation of a Hypoxia-Associated Prognostic signature related to Osteosarcoma Metastasis and Immune Infiltration. Front Cell Dev Biol. 2021;9:633607.33816483 10.3389/fcell.2021.633607
40. Papayannopoulos V Neutrophil extracellular traps in immunity and disease Nat Rev Immunol 2018 18 2 134 47 10.1038/nri.2017.105 28990587
Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol. 2018;18(2):134–47.28990587 10.1038/nri.2017.105
41. Lin Y Development and validation of neutrophil extracellular traps-derived signature to predict the prognosis for osteosarcoma patients Int Immunopharmacol 2024 127 111364 10.1016/j.intimp.2023.111364 38101221
Lin Y, et al. Development and validation of neutrophil extracellular traps-derived signature to predict the prognosis for osteosarcoma patients. Int Immunopharmacol. 2024;127:111364.38101221 10.1016/j.intimp.2023.111364
42. Tang H Osteosarcoma neutrophil extracellular trap network-associated gene recurrence and metastasis model J Cancer Res Clin Oncol 2024 150 2 48 10.1007/s00432-023-05577-2 38285218
Tang H, et al. Osteosarcoma neutrophil extracellular trap network-associated gene recurrence and metastasis model. J Cancer Res Clin Oncol. 2024;150(2):48.38285218 10.1007/s00432-023-05577-2
43. Brown HK Tellez-Gabriel M Heymann D Cancer stem cells in osteosarcoma Cancer Lett 2017 386 189 95 10.1016/j.canlet.2016.11.019 27894960
Brown HK, Tellez-Gabriel M, Heymann D. Cancer stem cells in osteosarcoma. Cancer Lett. 2017;386:189–95.27894960 10.1016/j.canlet.2016.11.019
44. Chang AI Involvement of mesenchymal stem cells in cancer progression and metastases Curr Cancer Drug Targets 2015 15 2 88 98 10.2174/1568009615666150126154151 25619387
Chang AI, et al. Involvement of mesenchymal stem cells in cancer progression and metastases. Curr Cancer Drug Targets. 2015;15(2):88–98.25619387 10.2174/1568009615666150126154151
45. Chang X New perspective into mesenchymal stem cells: molecular mechanisms regulating osteosarcoma J Bone Oncol 2021 29 100372 10.1016/j.jbo.2021.100372 34258182
Chang X, et al. New perspective into mesenchymal stem cells: molecular mechanisms regulating osteosarcoma. J Bone Oncol. 2021;29:100372.34258182 10.1016/j.jbo.2021.100372
46. Lagerweij T, Perez-Lanzon M, Baglio SR. A preclinical mouse model of Osteosarcoma to define the Extracellular vesicle-mediated communication between Tumor and mesenchymal stem cells. J Vis Exp, 2018(135).
47. Khare D Mesenchymal stromal cell-derived exosomes affect mRNA expression and function of B-Lymphocytes Front Immunol 2018 9 3053 10.3389/fimmu.2018.03053 30622539
Khare D, et al. Mesenchymal stromal cell-derived exosomes affect mRNA expression and function of B-Lymphocytes. Front Immunol. 2018;9:3053.30622539 10.3389/fimmu.2018.03053
48. Zhang Q Exosomes originating from MSCs stimulated with TGF-beta and IFN-gamma promote Treg differentiation J Cell Physiol 2018 233 9 6832 40 10.1002/jcp.26436 29336475
Zhang Q, et al. Exosomes originating from MSCs stimulated with TGF-beta and IFN-gamma promote Treg differentiation. J Cell Physiol. 2018;233(9):6832–40.29336475 10.1002/jcp.26436
49. Jia XH Activation of mesenchymal stem cells by macrophages promotes tumor progression through immune suppressive effects Oncotarget 2016 7 15 20934 44 10.18632/oncotarget.8064 26988913
Jia XH, et al. Activation of mesenchymal stem cells by macrophages promotes tumor progression through immune suppressive effects. Oncotarget. 2016;7(15):20934–44.26988913 10.18632/oncotarget.8064
50. Wang JW Exosomal miR-1228 from Cancer-Associated fibroblasts promotes Cell Migration and Invasion of Osteosarcoma by directly targeting SCAI Oncol Res 2019 27 9 979 86 10.3727/096504018X15336368805108 30180920
Wang JW, et al. Exosomal miR-1228 from Cancer-Associated fibroblasts promotes Cell Migration and Invasion of Osteosarcoma by directly targeting SCAI. Oncol Res. 2019;27(9):979–86.30180920 10.3727/096504018X15336368805108
51. Mazumdar A et al. Osteosarcoma-Derived Extracellular vesicles induce lung fibroblast reprogramming. Int J Mol Sci, 2020. 21(15).
52. Xu Y Osteosarcoma cells secrete CXCL14 that activates integrin alpha11beta1 on fibroblasts to form a lung metastatic niche Cancer Res 2024 84 7 994 1012 10.1158/0008-5472.CAN-23-1307 38295227
Xu Y, et al. Osteosarcoma cells secrete CXCL14 that activates integrin alpha11beta1 on fibroblasts to form a lung metastatic niche. Cancer Res. 2024;84(7):994–1012.38295227 10.1158/0008-5472.CAN-23-1307
53. Wolf-Dennen K Gordon N Kleinerman ES Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions Oncoimmunology 2020 9 1 1747677 10.1080/2162402X.2020.1747677 32313728
Wolf-Dennen K, Gordon N, Kleinerman ES. Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions. Oncoimmunology. 2020;9(1):1747677.32313728 10.1080/2162402X.2020.1747677
54. Corre I et al. The Osteosarcoma Microenvironment: a Complex but Targetable Ecosystem. Cells, 2020. 9(4).
55. Han Q Shi H Liu F CD163(+) M2-type tumor-associated macrophage support the suppression of tumor-infiltrating T cells in osteosarcoma Int Immunopharmacol 2016 34 101 6 10.1016/j.intimp.2016.01.023 26938675
Han Q, Shi H, Liu F. CD163(+) M2-type tumor-associated macrophage support the suppression of tumor-infiltrating T cells in osteosarcoma. Int Immunopharmacol. 2016;34:101–6.26938675 10.1016/j.intimp.2016.01.023
56. Sundara YT Increased PD-L1 and T-cell infiltration in the presence of HLA class I expression in metastatic high-grade osteosarcoma: a rationale for T-cell-based immunotherapy Cancer Immunol Immunother 2017 66 1 119 28 10.1007/s00262-016-1925-3 27853827
Sundara YT, et al. Increased PD-L1 and T-cell infiltration in the presence of HLA class I expression in metastatic high-grade osteosarcoma: a rationale for T-cell-based immunotherapy. Cancer Immunol Immunother. 2017;66(1):119–28.27853827 10.1007/s00262-016-1925-3
57. Sun CY T cell exhaustion drives osteosarcoma pathogenesis Ann Transl Med 2021 9 18 1447 10.21037/atm-21-3928 34733999
Sun CY, et al. T cell exhaustion drives osteosarcoma pathogenesis. Ann Transl Med. 2021;9(18):1447.34733999 10.21037/atm-21-3928
58. Le T Su S Shahriyari L Immune classification of osteosarcoma Math Biosci Eng 2021 18 2 1879 97 10.3934/mbe.2021098 33757216
Le T, Su S, Shahriyari L. Immune classification of osteosarcoma. Math Biosci Eng. 2021;18(2):1879–97.33757216 10.3934/mbe.2021098
59. Collin M Bigley V Human dendritic cell subsets: an update Immunology 2018 154 1 3 20 10.1111/imm.12888 29313948
Collin M, Bigley V. Human dendritic cell subsets: an update. Immunology. 2018;154(1):3–20.29313948 10.1111/imm.12888
60. Le T et al. Data-Driven Mathematical Model of Osteosarcoma. Cancers (Basel), 2021. 13(10).
61. Zhang C Profiles of immune cell infiltration and immune-related genes in the tumor microenvironment of osteosarcoma Aging 2020 12 4 3486 501 10.18632/aging.102824 32039832
Zhang C, et al. Profiles of immune cell infiltration and immune-related genes in the tumor microenvironment of osteosarcoma. Aging. 2020;12(4):3486–501.32039832 10.18632/aging.102824
62. Jones KB Dendritic cells drive osteosarcomagenesis through newly identified Oncogene and Tumor suppressor Cancer Discov 2019 9 11 1484 6 10.1158/2159-8290.CD-19-0994 31676561
Jones KB. Dendritic cells drive osteosarcomagenesis through newly identified Oncogene and Tumor suppressor. Cancer Discov. 2019;9(11):1484–6.31676561 10.1158/2159-8290.CD-19-0994
63. Kansara M Infiltrating myeloid cells drive Osteosarcoma Progression via GRM4 regulation of IL23 Cancer Discov 2019 9 11 1511 9 10.1158/2159-8290.CD-19-0154 31527131
Kansara M, et al. Infiltrating myeloid cells drive Osteosarcoma Progression via GRM4 regulation of IL23. Cancer Discov. 2019;9(11):1511–9.31527131 10.1158/2159-8290.CD-19-0154
64. He YT In vitro generation of cytotoxic T lymphocyte response using dendritic cell immunotherapy in osteosarcoma Oncol Lett 2016 12 2 1101 6 10.3892/ol.2016.4714 27446401
He YT, et al. In vitro generation of cytotoxic T lymphocyte response using dendritic cell immunotherapy in osteosarcoma. Oncol Lett. 2016;12(2):1101–6.27446401 10.3892/ol.2016.4714
65. Himoudi N Lack of T-cell responses following autologous tumour lysate pulsed dendritic cell vaccination, in patients with relapsed osteosarcoma Clin Transl Oncol 2012 14 4 271 9 10.1007/s12094-012-0795-1 22484634
Himoudi N, et al. Lack of T-cell responses following autologous tumour lysate pulsed dendritic cell vaccination, in patients with relapsed osteosarcoma. Clin Transl Oncol. 2012;14(4):271–9.22484634 10.1007/s12094-012-0795-1
66. Miwa S Phase 1/2 study of immunotherapy with dendritic cells pulsed with autologous tumor lysate in patients with refractory bone and soft tissue sarcoma Cancer 2017 123 9 1576 84 10.1002/cncr.30606 28241093
Miwa S, et al. Phase 1/2 study of immunotherapy with dendritic cells pulsed with autologous tumor lysate in patients with refractory bone and soft tissue sarcoma. Cancer. 2017;123(9):1576–84.28241093 10.1002/cncr.30606
67. Prager I Watzl C Mechanisms of natural killer cell-mediated cellular cytotoxicity J Leukoc Biol 2019 105 6 1319 29 10.1002/JLB.MR0718-269R 31107565
Prager I, Watzl C. Mechanisms of natural killer cell-mediated cellular cytotoxicity. J Leukoc Biol. 2019;105(6):1319–29.31107565 10.1002/JLB.MR0718-269R
68. Zhang ML PD–L1/PD–1 axis serves an important role in natural killer cell–induced cytotoxicity in osteosarcoma Oncol Rep 2019 42 5 2049 56 31485666
Zhang ML, et al. PD–L1/PD–1 axis serves an important role in natural killer cell–induced cytotoxicity in osteosarcoma. Oncol Rep. 2019;42(5):2049–56.31485666
69. Lazarova M Steinle A Impairment of NKG2D-Mediated tumor immunity by TGF-beta Front Immunol 2019 10 2689 10.3389/fimmu.2019.02689 31803194
Lazarova M, Steinle A. Impairment of NKG2D-Mediated tumor immunity by TGF-beta. Front Immunol. 2019;10:2689.31803194 10.3389/fimmu.2019.02689
70. Chauvin JM, Zarour HM. TIGIT in cancer immunotherapy. J Immunother Cancer, 2020. 8(2).
71. Kailayangiri S Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G Oncoimmunology 2017 6 1 e1250050 10.1080/2162402X.2016.1250050 28197367
Kailayangiri S, et al. Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G. Oncoimmunology. 2017;6(1):e1250050.28197367 10.1080/2162402X.2016.1250050
72. Sarvaria A Madrigal JA Saudemont A B cell regulation in cancer and anti-tumor immunity Cell Mol Immunol 2017 14 8 662 74 10.1038/cmi.2017.35 28626234
Sarvaria A, Madrigal JA, Saudemont A. B cell regulation in cancer and anti-tumor immunity. Cell Mol Immunol. 2017;14(8):662–74.28626234 10.1038/cmi.2017.35
73. Li GQ Application of Immune Infiltration Signature and machine learning model in the Differential diagnosis and prognosis of Bone-Related Malignancies Front Cell Dev Biol 2021 9 630355 10.3389/fcell.2021.630355 33937231
Li GQ, et al. Application of Immune Infiltration Signature and machine learning model in the Differential diagnosis and prognosis of Bone-Related Malignancies. Front Cell Dev Biol. 2021;9:630355.33937231 10.3389/fcell.2021.630355
74. Tu J Single-cell RNA datasets and bulk RNA datasets analysis demonstrated C1Q + tumor-associated macrophage as a major and antitumor immune cell population in osteosarcoma Front Immunol 2023 14 911368 10.3389/fimmu.2023.911368 36814925
Tu J, et al. Single-cell RNA datasets and bulk RNA datasets analysis demonstrated C1Q + tumor-associated macrophage as a major and antitumor immune cell population in osteosarcoma. Front Immunol. 2023;14:911368.36814925 10.3389/fimmu.2023.911368
75. Wu CC Immuno-genomic landscape of osteosarcoma Nat Commun 2020 11 1 1008 10.1038/s41467-020-14646-w 32081846
Wu CC, et al. Immuno-genomic landscape of osteosarcoma. Nat Commun. 2020;11(1):1008.32081846 10.1038/s41467-020-14646-w
76. Wu CC Livingston JA Genomics and the Immune Landscape of Osteosarcoma Adv Exp Med Biol 2020 1258 21 36 10.1007/978-3-030-43085-6_2 32767232
Wu CC, Livingston JA. Genomics and the Immune Landscape of Osteosarcoma. Adv Exp Med Biol. 2020;1258:21–36.32767232 10.1007/978-3-030-43085-6_2
77. Larionova I Interaction of tumor-associated macrophages and cancer chemotherapy Oncoimmunology 2019 8 7 1596004 10.1080/2162402X.2019.1596004 31143517
Larionova I, et al. Interaction of tumor-associated macrophages and cancer chemotherapy. Oncoimmunology. 2019;8(7):1596004.31143517 10.1080/2162402X.2019.1596004
78. Wan B Analysis of Immune Gene expression subtypes reveals Osteosarcoma Immune Heterogeneity J Oncol 2021 2021 p6649412 10.1155/2021/6649412
Wan B, et al. Analysis of Immune Gene expression subtypes reveals Osteosarcoma Immune Heterogeneity. J Oncol. 2021;2021:p6649412.10.1155/2021/6649412
79. Hong W Immune-related prognosis biomarkers associated with osteosarcoma microenvironment Cancer Cell Int 2020 20 83 10.1186/s12935-020-1165-7 32190007
Hong W, et al. Immune-related prognosis biomarkers associated with osteosarcoma microenvironment. Cancer Cell Int. 2020;20:83.32190007 10.1186/s12935-020-1165-7
80. Hu C Comprehensive analysis of prognostic tumor microenvironment-related genes in osteosarcoma patients BMC Cancer 2020 20 1 814 10.1186/s12885-020-07216-2 32854645
Hu C, et al. Comprehensive analysis of prognostic tumor microenvironment-related genes in osteosarcoma patients. BMC Cancer. 2020;20(1):814.32854645 10.1186/s12885-020-07216-2
81. Sheng G Osteosarcoma Metastasis Front Oncol 2021 11 780264 10.3389/fonc.2021.780264 34956899
Sheng G, et al. Osteosarcoma Metastasis Front Oncol. 2021;11:780264.34956899 10.3389/fonc.2021.780264
82. Lussier DM Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions J Immunother 2015 38 3 96 106 10.1097/CJI.0000000000000065 25751499
Lussier DM, et al. Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions. J Immunother. 2015;38(3):96–106.25751499 10.1097/CJI.0000000000000065
83. Zheng B PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse J Hematol Oncol 2018 11 1 16 10.1186/s13045-018-0560-1 29409495
Zheng B, et al. PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse. J Hematol Oncol. 2018;11(1):16.29409495 10.1186/s13045-018-0560-1
84. Davis KL Nivolumab in children and young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): a multicentre, open-label, single-arm, phase 1–2 trial Lancet Oncol 2020 21 4 541 50 10.1016/S1470-2045(20)30023-1 32192573
Davis KL, et al. Nivolumab in children and young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): a multicentre, open-label, single-arm, phase 1–2 trial. Lancet Oncol. 2020;21(4):541–50.32192573 10.1016/S1470-2045(20)30023-1
85. Tawbi HA Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial Lancet Oncol 2017 18 11 1493 501 10.1016/S1470-2045(17)30624-1 28988646
Tawbi HA, et al. Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol. 2017;18(11):1493–501.28988646 10.1016/S1470-2045(17)30624-1
86. Boye K Pembrolizumab in advanced osteosarcoma: results of a single-arm, open-label, phase 2 trial Cancer Immunol Immunother 2021 70 9 2617 24 10.1007/s00262-021-02876-w 33580363
Boye K, et al. Pembrolizumab in advanced osteosarcoma: results of a single-arm, open-label, phase 2 trial. Cancer Immunol Immunother. 2021;70(9):2617–24.33580363 10.1007/s00262-021-02876-w
87. Le Cesne A Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study Eur J Cancer 2019 119 151 7 10.1016/j.ejca.2019.07.018 31442817
Le Cesne A, et al. Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study. Eur J Cancer. 2019;119:151–7.31442817 10.1016/j.ejca.2019.07.018
88. Liu Z Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma Cell Death Dis 2021 12 2 164 10.1038/s41419-021-03449-6 33558476
Liu Z, et al. Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma. Cell Death Dis. 2021;12(2):164.33558476 10.1038/s41419-021-03449-6
89. Shemesh CS Population pharmacokinetics, exposure-safety, and immunogenicity of atezolizumab in pediatric and young adult patients with cancer J Immunother Cancer 2019 7 1 314 10.1186/s40425-019-0791-x 31753029
Shemesh CS, et al. Population pharmacokinetics, exposure-safety, and immunogenicity of atezolizumab in pediatric and young adult patients with cancer. J Immunother Cancer. 2019;7(1):314.31753029 10.1186/s40425-019-0791-x
90. Geoerger B Atezolizumab for children and young adults with previously treated solid tumours, non-hodgkin lymphoma, and Hodgkin lymphoma (iMATRIX): a multicentre phase 1–2 study Lancet Oncol 2020 21 1 134 44 10.1016/S1470-2045(19)30693-X 31780255
Geoerger B, et al. Atezolizumab for children and young adults with previously treated solid tumours, non-hodgkin lymphoma, and Hodgkin lymphoma (iMATRIX): a multicentre phase 1–2 study. Lancet Oncol. 2020;21(1):134–44.31780255 10.1016/S1470-2045(19)30693-X
91. Egen JG Kuhns MS Allison JP CTLA-4: new insights into its biological function and use in tumor immunotherapy Nat Immunol 2002 3 7 611 8 10.1038/ni0702-611 12087419
Egen JG, Kuhns MS, Allison JP. CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat Immunol. 2002;3(7):611–8.12087419 10.1038/ni0702-611
92. He J Association between CTLA-4 genetic polymorphisms and susceptibility to osteosarcoma in Chinese Han population Endocrine 2014 45 2 325 30 10.1007/s12020-013-0050-8 24078408
He J, et al. Association between CTLA-4 genetic polymorphisms and susceptibility to osteosarcoma in Chinese Han population. Endocrine. 2014;45(2):325–30.24078408 10.1007/s12020-013-0050-8
93. Liu J Effect of cytotoxic T-lymphocyte antigen-4, TNF-alpha polymorphisms on osteosarcoma: evidences from a meta-analysis Chin J Cancer Res 2013 25 6 671 8 24385694
Liu J, et al. Effect of cytotoxic T-lymphocyte antigen-4, TNF-alpha polymorphisms on osteosarcoma: evidences from a meta-analysis. Chin J Cancer Res. 2013;25(6):671–8.24385694
94. Merchant MS Phase I clinical trial of Ipilimumab in Pediatric patients with Advanced Solid tumors Clin Cancer Res 2016 22 6 1364 70 10.1158/1078-0432.CCR-15-0491 26534966
Merchant MS, et al. Phase I clinical trial of Ipilimumab in Pediatric patients with Advanced Solid tumors. Clin Cancer Res. 2016;22(6):1364–70.26534966 10.1158/1078-0432.CCR-15-0491
95. Tsai RK Discher DE Inhibition of self engulfment through deactivation of myosin-II at the phagocytic synapse between human cells J Cell Biol 2008 180 5 989 1003 10.1083/jcb.200708043 18332220
Tsai RK, Discher DE. Inhibition of self engulfment through deactivation of myosin-II at the phagocytic synapse between human cells. J Cell Biol. 2008;180(5):989–1003.18332220 10.1083/jcb.200708043
96. Xu JF CD47 blockade inhibits tumor progression human osteosarcoma in xenograft models Oncotarget 2015 6 27 23662 70 10.18632/oncotarget.4282 26093091
Xu JF, et al. CD47 blockade inhibits tumor progression human osteosarcoma in xenograft models. Oncotarget. 2015;6(27):23662–70.26093091 10.18632/oncotarget.4282
97. Wen Y Immune checkpoints in osteosarcoma: recent advances and therapeutic potential Cancer Lett 2022 547 215887 10.1016/j.canlet.2022.215887 35995141
Wen Y, et al. Immune checkpoints in osteosarcoma: recent advances and therapeutic potential. Cancer Lett. 2022;547:215887.35995141 10.1016/j.canlet.2022.215887
98. Zhao L TIM-3: an update on immunotherapy Int Immunopharmacol 2021 99 107933 10.1016/j.intimp.2021.107933 34224993
Zhao L, et al. TIM-3: an update on immunotherapy. Int Immunopharmacol. 2021;99:107933.34224993 10.1016/j.intimp.2021.107933
99. Ge W Tim-3 as a diagnostic and prognostic biomarker of osteosarcoma Tumour Biol 2017 39 7 1010428317715643 10.1177/1010428317715643 28671022
Ge W, et al. Tim-3 as a diagnostic and prognostic biomarker of osteosarcoma. Tumour Biol. 2017;39(7):1010428317715643.28671022 10.1177/1010428317715643
100. Koirala P HHLA2, a member of the B7 family, is expressed in human osteosarcoma and is associated with metastases and worse survival Sci Rep 2016 6 31154 10.1038/srep31154 27531281
Koirala P, et al. HHLA2, a member of the B7 family, is expressed in human osteosarcoma and is associated with metastases and worse survival. Sci Rep. 2016;6:31154.27531281 10.1038/srep31154
101. DeRenzo C Gottschalk S Genetically modified T-Cell therapy for Osteosarcoma: into the Roaring 2020s Adv Exp Med Biol 2020 1257 109 31 10.1007/978-3-030-43032-0_10 32483735
DeRenzo C, Gottschalk S. Genetically modified T-Cell therapy for Osteosarcoma: into the Roaring 2020s. Adv Exp Med Biol. 2020;1257:109–31.32483735 10.1007/978-3-030-43032-0_10
102. Tabak SA Khalifa SE Fathy Y HER-2 immunohistochemical expression in bone sarcomas: a New Hope for Osteosarcoma patients Open Access Maced J Med Sci 2018 6 9 1555 60 10.3889/oamjms.2018.318 30337965
Tabak SA, Khalifa SE, Fathy Y. HER-2 immunohistochemical expression in bone sarcomas: a New Hope for Osteosarcoma patients. Open Access Maced J Med Sci. 2018;6(9):1555–60.30337965 10.3889/oamjms.2018.318
103. Wang SL Prognostic significance of the expression of HER family members in primary osteosarcoma Oncol Lett 2018 16 2 2185 94 30008917
Wang SL, et al. Prognostic significance of the expression of HER family members in primary osteosarcoma. Oncol Lett. 2018;16(2):2185–94.30008917
104. Colomer R Herceptin: from the bench to the clinic Cancer Invest 2001 19 1 49 56 10.1081/CNV-100000074 11291556
Colomer R, et al. Herceptin: from the bench to the clinic. Cancer Invest. 2001;19(1):49–56.11291556 10.1081/CNV-100000074
105. Ahmed N Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression Mol Ther 2009 17 10 1779 87 10.1038/mt.2009.133 19532139
Ahmed N, et al. Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression. Mol Ther. 2009;17(10):1779–87.19532139 10.1038/mt.2009.133
106. Rainusso N Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma Cancer Gene Ther 2012 19 3 212 7 10.1038/cgt.2011.83 22173710
Rainusso N, et al. Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma. Cancer Gene Ther. 2012;19(3):212–7.22173710 10.1038/cgt.2011.83
107. Ahmed N Human epidermal growth factor receptor 2 (HER2) -Specific chimeric Antigen receptor-modified T cells for the Immunotherapy of HER2-Positive sarcoma J Clin Oncol 2015 33 15 1688 96 10.1200/JCO.2014.58.0225 25800760
Ahmed N, et al. Human epidermal growth factor receptor 2 (HER2) -Specific chimeric Antigen receptor-modified T cells for the Immunotherapy of HER2-Positive sarcoma. J Clin Oncol. 2015;33(15):1688–96.25800760 10.1200/JCO.2014.58.0225
108. Picci P Relationship of chemotherapy-induced necrosis and surgical margins to local recurrence in osteosarcoma J Clin Oncol 1994 12 12 2699 705 10.1200/JCO.1994.12.12.2699 7989947
Picci P, et al. Relationship of chemotherapy-induced necrosis and surgical margins to local recurrence in osteosarcoma. J Clin Oncol. 1994;12(12):2699–705.7989947 10.1200/JCO.1994.12.12.2699
109. Charan M GD2-directed CAR-T cells in combination with HGF-targeted neutralizing antibody (AMG102) prevent primary tumor growth and metastasis in ewing sarcoma Int J Cancer 2020 146 11 3184 95 10.1002/ijc.32743 31621900
Charan M, et al. GD2-directed CAR-T cells in combination with HGF-targeted neutralizing antibody (AMG102) prevent primary tumor growth and metastasis in ewing sarcoma. Int J Cancer. 2020;146(11):3184–95.31621900 10.1002/ijc.32743
110. Chulanetra M GD2 chimeric antigen receptor modified T cells in synergy with sub-toxic level of doxorubicin targeting osteosarcomas Am J Cancer Res 2020 10 2 674 87 32195035
Chulanetra M, et al. GD2 chimeric antigen receptor modified T cells in synergy with sub-toxic level of doxorubicin targeting osteosarcomas. Am J Cancer Res. 2020;10(2):674–87.32195035
111. Federman N Enhanced growth inhibition of osteosarcoma by cytotoxic polymerized liposomal nanoparticles targeting the alcam cell surface receptor Sarcoma 2012 2012 p126906 10.1155/2012/126906
Federman N, et al. Enhanced growth inhibition of osteosarcoma by cytotoxic polymerized liposomal nanoparticles targeting the alcam cell surface receptor. Sarcoma. 2012;2012:p126906.10.1155/2012/126906
112. Wang Y Anti-CD166/4-1BB chimeric antigen receptor T cell therapy for the treatment of osteosarcoma J Exp Clin Cancer Res 2019 38 1 168 10.1186/s13046-019-1147-6 30995926
Wang Y, et al. Anti-CD166/4-1BB chimeric antigen receptor T cell therapy for the treatment of osteosarcoma. J Exp Clin Cancer Res. 2019;38(1):168.30995926 10.1186/s13046-019-1147-6
113. Picarda E Ohaegbulam KC Zang X Molecular pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy Clin Cancer Res 2016 22 14 3425 31 10.1158/1078-0432.CCR-15-2428 27208063
Picarda E, Ohaegbulam KC, Zang X. Molecular pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy. Clin Cancer Res. 2016;22(14):3425–31.27208063 10.1158/1078-0432.CCR-15-2428
114. Tekle C B7-H3 contributes to the metastatic capacity of melanoma cells by modulation of known metastasis-associated genes Int J Cancer 2012 130 10 2282 90 10.1002/ijc.26238 21671471
Tekle C, et al. B7-H3 contributes to the metastatic capacity of melanoma cells by modulation of known metastasis-associated genes. Int J Cancer. 2012;130(10):2282–90.21671471 10.1002/ijc.26238
115. Majzner RG Cells Targeting CART B7-H3, a Pan-cancer Antigen, Demonstrate Potent Preclinical Activity against Pediatric Solid Tumors and Brain tumors Clin Cancer Res 2019 25 8 2560 74 10.1158/1078-0432.CCR-18-0432 30655315
Majzner RG, Cells Targeting CART, et al. B7-H3, a Pan-cancer Antigen, Demonstrate Potent Preclinical Activity against Pediatric Solid Tumors and Brain tumors. Clin Cancer Res. 2019;25(8):2560–74.30655315 10.1158/1078-0432.CCR-18-0432
116. Zhang Q B7-H3 targeted CAR-T cells show highly efficient anti-tumor function against osteosarcoma both in vitro and in vivo BMC Cancer 2022 22 1 1124 10.1186/s12885-022-10229-8 36320072
Zhang Q, et al. B7-H3 targeted CAR-T cells show highly efficient anti-tumor function against osteosarcoma both in vitro and in vivo. BMC Cancer. 2022;22(1):1124.36320072 10.1186/s12885-022-10229-8
117. Fritsche-Guenther R De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signalling pathway Histopathology 2010 57 6 836 50 10.1111/j.1365-2559.2010.03713.x 21166698
Fritsche-Guenther R, et al. De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signalling pathway. Histopathology. 2010;57(6):836–50.21166698 10.1111/j.1365-2559.2010.03713.x
118. Posthumadeboer J Surface proteomic analysis of osteosarcoma identifies EPHA2 as receptor for targeted drug delivery Br J Cancer 2013 109 8 2142 54 10.1038/bjc.2013.578 24064975
Posthumadeboer J, et al. Surface proteomic analysis of osteosarcoma identifies EPHA2 as receptor for targeted drug delivery. Br J Cancer. 2013;109(8):2142–54.24064975 10.1038/bjc.2013.578
119. Hsu K Chimeric Antigen receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone tumours Cancer Gene Ther 2021 28 3–4 321 34 10.1038/s41417-020-00221-4 32873870
Hsu K, et al. Chimeric Antigen receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone tumours. Cancer Gene Ther. 2021;28(3–4):321–34.32873870 10.1038/s41417-020-00221-4
120. Campbell CL Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma Am J Pathol 2001 158 1 25 32 10.1016/S0002-9440(10)63940-5 11141475
Campbell CL, et al. Increased expression of the interleukin-11 receptor and evidence of STAT3 activation in prostate carcinoma. Am J Pathol. 2001;158(1):25–32.11141475 10.1016/S0002-9440(10)63940-5
121. Schwertschlag US Hematopoietic, immunomodulatory and epithelial effects of interleukin-11 Leukemia 1999 13 9 1307 15 10.1038/sj.leu.2401514 10482979
Schwertschlag US, et al. Hematopoietic, immunomodulatory and epithelial effects of interleukin-11. Leukemia. 1999;13(9):1307–15.10482979 10.1038/sj.leu.2401514
122. Lewis VO The interleukin-11 receptor alpha as a candidate ligand-directed target in osteosarcoma: consistent data from cell lines, orthotopic models, and human tumor samples Cancer Res 2009 69 5 1995 9 10.1158/0008-5472.CAN-08-4845 19244100
Lewis VO, et al. The interleukin-11 receptor alpha as a candidate ligand-directed target in osteosarcoma: consistent data from cell lines, orthotopic models, and human tumor samples. Cancer Res. 2009;69(5):1995–9.19244100 10.1158/0008-5472.CAN-08-4845
123. Huang G Genetically modified T cells targeting interleukin-11 receptor alpha-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases Cancer Res 2012 72 1 271 81 10.1158/0008-5472.CAN-11-2778 22075555
Huang G, et al. Genetically modified T cells targeting interleukin-11 receptor alpha-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases. Cancer Res. 2012;72(1):271–81.22075555 10.1158/0008-5472.CAN-11-2778
124. Pollak M The insulin and insulin-like growth factor receptor family in neoplasia: an update Nat Rev Cancer 2012 12 3 159 69 10.1038/nrc3215 22337149
Pollak M. The insulin and insulin-like growth factor receptor family in neoplasia: an update. Nat Rev Cancer. 2012;12(3):159–69.22337149 10.1038/nrc3215
125. Cui B Targeting ROR1 inhibits epithelial-mesenchymal transition and metastasis Cancer Res 2013 73 12 3649 60 10.1158/0008-5472.CAN-12-3832 23771907
Cui B, et al. Targeting ROR1 inhibits epithelial-mesenchymal transition and metastasis. Cancer Res. 2013;73(12):3649–60.23771907 10.1158/0008-5472.CAN-12-3832
126. Hudecek M The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor Blood 2010 116 22 4532 41 10.1182/blood-2010-05-283309 20702778
Hudecek M, et al. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood. 2010;116(22):4532–41.20702778 10.1182/blood-2010-05-283309
127. Huang X IGF1R- and ROR1-Specific CAR T cells as a potential therapy for high risk sarcomas PLoS ONE 2015 10 7 e0133152 10.1371/journal.pone.0133152 26173023
Huang X, et al. IGF1R- and ROR1-Specific CAR T cells as a potential therapy for high risk sarcomas. PLoS ONE. 2015;10(7):e0133152.26173023 10.1371/journal.pone.0133152
128. Fernandez L Activated and expanded natural killer cells target osteosarcoma tumor initiating cells in an NKG2D-NKG2DL dependent manner Cancer Lett 2015 368 1 54 63 10.1016/j.canlet.2015.07.042 26276724
Fernandez L, et al. Activated and expanded natural killer cells target osteosarcoma tumor initiating cells in an NKG2D-NKG2DL dependent manner. Cancer Lett. 2015;368(1):54–63.26276724 10.1016/j.canlet.2015.07.042
129. Spear P NKG2D ligands as therapeutic targets Cancer Immun 2013 13 8 23833565
Spear P, et al. NKG2D ligands as therapeutic targets. Cancer Immun. 2013;13:8.23833565
130. Fernandez L Memory T cells expressing an NKG2D-CAR efficiently Target Osteosarcoma cells Clin Cancer Res 2017 23 19 5824 35 10.1158/1078-0432.CCR-17-0075 28659311
Fernandez L, et al. Memory T cells expressing an NKG2D-CAR efficiently Target Osteosarcoma cells. Clin Cancer Res. 2017;23(19):5824–35.28659311 10.1158/1078-0432.CCR-17-0075
131. Amato RJ A phase I study of folate immune therapy (EC90 vaccine administered with GPI-0100 adjuvant followed by EC17) in patients with renal cell carcinoma J Immunother 2013 36 4 268 75 10.1097/CJI.0b013e3182917f59 23603861
Amato RJ, et al. A phase I study of folate immune therapy (EC90 vaccine administered with GPI-0100 adjuvant followed by EC17) in patients with renal cell carcinoma. J Immunother. 2013;36(4):268–75.23603861 10.1097/CJI.0b013e3182917f59
132. Lu YJ Preclinical evaluation of bispecific adaptor molecule controlled folate receptor CAR-T cell therapy with Special Focus on Pediatric malignancies Front Oncol 2019 9 151 10.3389/fonc.2019.00151 30941303
Lu YJ, et al. Preclinical evaluation of bispecific adaptor molecule controlled folate receptor CAR-T cell therapy with Special Focus on Pediatric malignancies. Front Oncol. 2019;9:151.30941303 10.3389/fonc.2019.00151
133. Sanchez-Paulete AR Targeting macrophages with CAR T cells delays solid tumor progression and enhances Antitumor Immunity Cancer Immunol Res 2022 10 11 1354 69 10.1158/2326-6066.CIR-21-1075 36095236
Sanchez-Paulete AR, et al. Targeting macrophages with CAR T cells delays solid tumor progression and enhances Antitumor Immunity. Cancer Immunol Res. 2022;10(11):1354–69.36095236 10.1158/2326-6066.CIR-21-1075
134. Klichinsky M Human chimeric antigen receptor macrophages for cancer immunotherapy Nat Biotechnol 2020 38 8 947 53 10.1038/s41587-020-0462-y 32361713
Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol. 2020;38(8):947–53.32361713 10.1038/s41587-020-0462-y
135. Gajewski TF Schreiber H Fu YX Innate and adaptive immune cells in the tumor microenvironment Nat Immunol 2013 14 10 1014 22 10.1038/ni.2703 24048123
Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 2013;14(10):1014–22.24048123 10.1038/ni.2703
136. Koksal H Treating osteosarcoma with CAR T cells Scand J Immunol 2019 89 3 e12741 10.1111/sji.12741 30549299
Koksal H, et al. Treating osteosarcoma with CAR T cells. Scand J Immunol. 2019;89(3):e12741.30549299 10.1111/sji.12741
137. Moreno C, Alternative CAR, Therapies et al. Recent approaches in Engineering chimeric Antigen receptor Immune cells to Combat Cancer. Biomedicines, 2022. 10(7).
138. Brady RV Thamm DH Tumor-associated macrophages: prognostic and therapeutic targets for cancer in humans and dogs Front Immunol 2023 14 1176807 10.3389/fimmu.2023.1176807 37090720
Brady RV, Thamm DH. Tumor-associated macrophages: prognostic and therapeutic targets for cancer in humans and dogs. Front Immunol. 2023;14:1176807.37090720 10.3389/fimmu.2023.1176807
139. Chen S Harnessing and enhancing macrophage phagocytosis for Cancer Therapy Front Immunol 2021 12 635173 10.3389/fimmu.2021.635173 33790906
Chen S, et al. Harnessing and enhancing macrophage phagocytosis for Cancer Therapy. Front Immunol. 2021;12:635173.33790906 10.3389/fimmu.2021.635173
140. de Visser KE Joyce JA The evolving tumor microenvironment: from cancer initiation to metastatic outgrowth Cancer Cell 2023 41 3 374 403 10.1016/j.ccell.2023.02.016 36917948
de Visser KE, Joyce JA. The evolving tumor microenvironment: from cancer initiation to metastatic outgrowth. Cancer Cell. 2023;41(3):374–403.36917948 10.1016/j.ccell.2023.02.016
141. Quamine AE et al. Approaches to Enhance Natural Killer Cell-based immunotherapy for Pediatric Solid tumors. Cancers (Basel), 2021. 13(11).
142. Wu Y Tian Z Wei H Developmental and Functional Control of Natural Killer cells by cytokines Front Immunol 2017 8 930 10.3389/fimmu.2017.00930 28824650
Wu Y, Tian Z, Wei H. Developmental and Functional Control of Natural Killer cells by cytokines. Front Immunol. 2017;8:930.28824650 10.3389/fimmu.2017.00930
143. Liu E Use of CAR-Transduced Natural Killer cells in CD19-Positive lymphoid tumors N Engl J Med 2020 382 6 545 53 10.1056/NEJMoa1910607 32023374
Liu E, et al. Use of CAR-Transduced Natural Killer cells in CD19-Positive lymphoid tumors. N Engl J Med. 2020;382(6):545–53.32023374 10.1056/NEJMoa1910607
144. Zhang Y In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection Immunology 2007 121 2 258 65 10.1111/j.1365-2567.2007.02573.x 17346281
Zhang Y, et al. In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection. Immunology. 2007;121(2):258–65.17346281 10.1111/j.1365-2567.2007.02573.x
145. Ebb D Phase II trial of trastuzumab in combination with cytotoxic chemotherapy for treatment of metastatic osteosarcoma with human epidermal growth factor receptor 2 overexpression: a report from the children’s oncology group J Clin Oncol 2012 30 20 2545 51 10.1200/JCO.2011.37.4546 22665540
Ebb D, et al. Phase II trial of trastuzumab in combination with cytotoxic chemotherapy for treatment of metastatic osteosarcoma with human epidermal growth factor receptor 2 overexpression: a report from the children’s oncology group. J Clin Oncol. 2012;30(20):2545–51.22665540 10.1200/JCO.2011.37.4546
146. Yu AL Phase I trial of a human-mouse chimeric anti-disialoganglioside monoclonal antibody ch14.18 in patients with refractory neuroblastoma and osteosarcoma J Clin Oncol 1998 16 6 2169 80 10.1200/JCO.1998.16.6.2169 9626218
Yu AL, et al. Phase I trial of a human-mouse chimeric anti-disialoganglioside monoclonal antibody ch14.18 in patients with refractory neuroblastoma and osteosarcoma. J Clin Oncol. 1998;16(6):2169–80.9626218 10.1200/JCO.1998.16.6.2169
147. Park JA Cheung NV GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma J Hematol Oncol 2020 13 1 172 10.1186/s13045-020-01012-y 33303017
Park JA, Cheung NV. GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma. J Hematol Oncol. 2020;13(1):172.33303017 10.1186/s13045-020-01012-y
148. Mason NJ Immunotherapy with a HER2-Targeting Listeria induces HER2-Specific immunity and demonstrates potential therapeutic effects in a phase I Trial in Canine Osteosarcoma Clin Cancer Res 2016 22 17 4380 90 10.1158/1078-0432.CCR-16-0088 26994144
Mason NJ, et al. Immunotherapy with a HER2-Targeting Listeria induces HER2-Specific immunity and demonstrates potential therapeutic effects in a phase I Trial in Canine Osteosarcoma. Clin Cancer Res. 2016;22(17):4380–90.26994144 10.1158/1078-0432.CCR-16-0088
149. Kawano M Anti-TGF-beta antibody combined with dendritic cells produce antitumor effects in osteosarcoma Clin Orthop Relat Res 2012 470 8 2288 94 10.1007/s11999-012-2299-2 22415727
Kawano M, et al. Anti-TGF-beta antibody combined with dendritic cells produce antitumor effects in osteosarcoma. Clin Orthop Relat Res. 2012;470(8):2288–94.22415727 10.1007/s11999-012-2299-2
150. Kawano M Dendritic cells combined with anti-GITR antibody produce antitumor effects in osteosarcoma Oncol Rep 2015 34 4 1995 2001 10.3892/or.2015.4161 26239052
Kawano M, et al. Dendritic cells combined with anti-GITR antibody produce antitumor effects in osteosarcoma. Oncol Rep. 2015;34(4):1995–2001.26239052 10.3892/or.2015.4161
151. Zhou Y et al. Vaccine efficacy against primary and metastatic cancer with in vitro-generated CD103(+) conventional dendritic cells. J Immunother Cancer, 2020. 8(1).
152. Lussier DM Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma J Immunother Cancer 2015 3 21 10.1186/s40425-015-0067-z 25992292
Lussier DM, et al. Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma. J Immunother Cancer. 2015;3:21.25992292 10.1186/s40425-015-0067-z
153. Nuytemans L NGS-analysis to the rescue: dual checkpoint inhibition in metastatic osteosarcoma - a case report and review of the literature Acta Clin Belg 2021 76 2 162 7 10.1080/17843286.2019.1683129 31635553
Nuytemans L, et al. NGS-analysis to the rescue: dual checkpoint inhibition in metastatic osteosarcoma - a case report and review of the literature. Acta Clin Belg. 2021;76(2):162–7.31635553 10.1080/17843286.2019.1683129
154. D’Angelo SP Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials Lancet Oncol 2018 19 3 416 26 10.1016/S1470-2045(18)30006-8 29370992
D’Angelo SP, et al. Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials. Lancet Oncol. 2018;19(3):416–26.29370992 10.1016/S1470-2045(18)30006-8
155. Somaiah N Durvalumab plus Tremelimumab in advanced or metastatic soft tissue and bone sarcomas: a single-centre phase 2 trial Lancet Oncol 2022 23 9 1156 66 10.1016/S1470-2045(22)00392-8 35934010
Somaiah N, et al. Durvalumab plus Tremelimumab in advanced or metastatic soft tissue and bone sarcomas: a single-centre phase 2 trial. Lancet Oncol. 2022;23(9):1156–66.35934010 10.1016/S1470-2045(22)00392-8
156. Motz GT Coukos G The parallel lives of angiogenesis and immunosuppression: cancer and other tales Nat Rev Immunol 2011 11 10 702 11 10.1038/nri3064 21941296
Motz GT, Coukos G. The parallel lives of angiogenesis and immunosuppression: cancer and other tales. Nat Rev Immunol. 2011;11(10):702–11.21941296 10.1038/nri3064
157. Martin-Broto J et al. Nivolumab and sunitinib combination in advanced soft tissue sarcomas: a multicenter, single-arm, phase Ib/II trial. J Immunother Cancer, 2020. 8(2).
158. Zhou Y A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas BMC Med 2023 21 1 470 10.1186/s12916-023-03132-x 38031088
Zhou Y, et al. A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas. BMC Med. 2023;21(1):470.38031088 10.1186/s12916-023-03132-x
159. Xie L et al. Apatinib plus camrelizumab (anti-PD1 therapy, SHR-1210) for advanced osteosarcoma (APFAO) progressing after chemotherapy: a single-arm, open-label, phase 2 trial. J Immunother Cancer, 2020. 8(1).
160. Park JA et al. Targeting tumor vasculature to improve antitumor activity of T cells armed ex vivo with T cell engaging bispecific antibody. J Immunother Cancer, 2023. 11(3).
161. Kawano M Enhancement of antitumor immunity by combining anti-cytotoxic T lymphocyte antigen-4 antibodies and cryotreated tumor lysate-pulsed dendritic cells in murine osteosarcoma Oncol Rep 2013 29 3 1001 6 10.3892/or.2013.2224 23291864
Kawano M, et al. Enhancement of antitumor immunity by combining anti-cytotoxic T lymphocyte antigen-4 antibodies and cryotreated tumor lysate-pulsed dendritic cells in murine osteosarcoma. Oncol Rep. 2013;29(3):1001–6.23291864 10.3892/or.2013.2224
162. Xie L Exploratory study of an anti-PD-L1/TGF-beta antibody, TQB2858, in patients with refractory or recurrent osteosarcoma and alveolar soft part sarcoma: a report from Chinese sarcoma study group (TQB2858-Ib-02) BMC Cancer 2023 23 1 868 10.1186/s12885-023-11390-4 37715133
Xie L, et al. Exploratory study of an anti-PD-L1/TGF-beta antibody, TQB2858, in patients with refractory or recurrent osteosarcoma and alveolar soft part sarcoma: a report from Chinese sarcoma study group (TQB2858-Ib-02). BMC Cancer. 2023;23(1):868.37715133 10.1186/s12885-023-11390-4
163. Diab A Bempegaldesleukin (NKTR-214) plus Nivolumab in patients with Advanced Solid tumors: phase I dose-escalation study of Safety, Efficacy, and Immune activation (PIVOT-02) Cancer Discov 2020 10 8 1158 73 10.1158/2159-8290.CD-19-1510 32439653
Diab A, et al. Bempegaldesleukin (NKTR-214) plus Nivolumab in patients with Advanced Solid tumors: phase I dose-escalation study of Safety, Efficacy, and Immune activation (PIVOT-02). Cancer Discov. 2020;10(8):1158–73.32439653 10.1158/2159-8290.CD-19-1510
164. D’Angelo SP Pilot study of bempegaldesleukin in combination with nivolumab in patients with metastatic sarcoma Nat Commun 2022 13 1 3477 10.1038/s41467-022-30874-8 35710741
D’Angelo SP, et al. Pilot study of bempegaldesleukin in combination with nivolumab in patients with metastatic sarcoma. Nat Commun. 2022;13(1):3477.35710741 10.1038/s41467-022-30874-8
165. Tanaka M Vaccination targeting native receptors to enhance the function and proliferation of Chimeric Antigen Receptor (CAR)-Modified T cells Clin Cancer Res 2017 23 14 3499 509 10.1158/1078-0432.CCR-16-2138 28183713
Tanaka M, et al. Vaccination targeting native receptors to enhance the function and proliferation of Chimeric Antigen Receptor (CAR)-Modified T cells. Clin Cancer Res. 2017;23(14):3499–509.28183713 10.1158/1078-0432.CCR-16-2138
