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J Pharm Anal
J Pharm Anal
Journal of Pharmaceutical Analysis
2095-1779
2214-0883
Xi'an Jiaotong University

S2095-1779(24)00029-7
10.1016/j.jpha.2024.01.009
100941
Short Communication
In situ repolarization of tumor-associated macrophages with synergic nanoformulation to reverse immunosuppressive TME in mouse breast cancer for cancer therapy
Luo Ruhua abc
Yue Zhongyu ab
Yang Qian ab
Zhang Honghua ab
Xie Tian tianxie@hznu.edu.cn
ab∗∗
Wang Shuling wsling222@163.com
ab∗∗∗
Tian Qingchang tianqc@hznu.edu.cn
ab∗
a School of Pharmacy, Hangzhou Normal University, Hangzhou, 311121, China
b Key Laboratory of Elemene Class Anti-Cancer Chinese Medicines, Engineering Laboratory of Development and Application of Traditional Chinese Medicines, Collaborative Innovation Center of Traditional Chinese Medicines of Zhejiang Province, Hangzhou Normal University, Hangzhou, 311121, China
c Department of Pharmacy, Hankou Hospital, Wuhan, 430012, China
∗ Corresponding author. School of Pharmacy, Hangzhou Normal University, Hangzhou, 311121, China. tianqc@hznu.edu.cn
∗∗ Corresponding author. School of Pharmacy, Hangzhou Normal University, Hangzhou, 311121, China. tianxie@hznu.edu.cn
∗∗∗ Corresponding author. School of Pharmacy, Hangzhou Normal University, Hangzhou, 311121, China. wsling222@163.com
02 2 2024
8 2024
02 2 2024
14 8 10094122 7 2023
26 1 2024
29 1 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

Image 1

Highlights

• M2 phenotype macrophage-targeted Lipo@CpG-FA was used to repolarize TAMs and reverse immunosuppressive TME.

• The synergic nanoformulation reduced M2 macrophages and caused regression and inhibition of 4T1 breast cancers.

• Elemene could inhibit the effect M2 macrophage proliferation by enhancing the therapeutic effects.
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pmcCancer immunotherapy is a novel cancer treatment strategy. Studies increasingly demonstrate that the immunosuppressive tumor microenvironment (TME) can inhibit effector T cells from attacking tumor cells [1], a pivotal issue in solid tumors immunotherapy.

Macrophages are abundant and have an impact on the immune-microenvironment in tumors [2]. Most leukocytes in TME are M2 phenotype macrophages, which can promote tumor growth and cause immunosuppression [3]. M1 phenotype macrophages not only kill tumor cells, but they also play a dominant role in orchestrating cancer-related inflammation (CRI). The conversion of tumor-associated macrophages (TAMs) from M2 phenotype to M1 phenotype appears to be the main route to reversing the immunosuppressive TME.

This work describes a combined therapeutic strategy for transforming “cold” tumors into “hot” tumors by repolarizing M2-TAMs to tumoricidal M1 phenotypes using CpG liposomes and elemene (Ele) liposomes (Fig. S1A). CpG oligodeoxynucleotide (ODN) can stimulate a cascade of innate immune responses. Lipo@CpG-folic acid (FA), an M2 phenotype macrophage-targeted CpG ODNs delivery system, was developed to repolarize tumor-associated macrophages. Ele, an effective sesquiterpene derived from Curcuma longa, has numerous antitumor and curative effects in addition to low toxicity levels and side effects [4]. The combination of Lipo@CpG-FA with FA-Lipo@Ele-AS1411 inhibited 4T1 breast cancers and reversed M2-TAMs-mediated immunosuppression in TME.

In this work, liposomes were prepared using an ultrasonic-dispersion method in which FA-1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG) and Chol-AS1411 were linked to liposomes through incubation (methodology and some other results shown in Supplementary Materialdata). As a result, we produced two types of targeting liposomes: Lipo@CpG-FA and FA-Lipo@Ele-AS1411 (Table S1, and Figs. S1B and C). A laser scanning confocal microscope (LSCM) was used to complete a cellular uptake study. The findings confirmed the role of FA and AS1411 (Table S2) in increasing uptake via active targeting, significantly affecting the cellular uptake of the targeting liposomes (Figs. S1D, S1E, and S2).

The immunostimulatory property of Lipo@CpG-FA was studied on untreated Raw 264.7 cells and M2 phenotype macrophages. Fig. S3 displays that interleukin-4 (IL-4)-induced M2 phenotype macrophages co-cultured with Lipo@CpG-FA demonstrated a tendency toward M1 phenotype transition and had a high pro-inflammatory immune response with up-regulated markers of M1 macrophages (Table S3).

The ability of a panel of toll-like receptor (TLR) pathway agonists to stimulate macrophages with antitumor functions, such as phagocytosis, was then tested. Compared with IL-4 group, Lipo@CpG-FA could reverse the ability of M2 macrophages to M1 and macrophages phagocytized tumor cells (Fig. S3E).

The antitumor effect of Lipo@CpG-FA was demonstrated in a 4T1 tumor-bearing mice model. The study followed all guidelines established by the Animal Ethics and Welfare Committee (AEWC) of Hangzhou Normal University (Approval number: 2021-1260). Lipo@CpG-FA was an effective anti-tumor treatment with no significant effect on mouse body weight and no significant toxic effects (Fig. 1A). Encapsulating CpG into Lipo and Lipo-FA significantly increased the anti-tumor efficacy, as evidenced by the tumor inhibition rate increase to 33.77% ± 3.784% and 73.45% ± 3.784%, respectively.Fig. 1 Lipo@CpG-folic acid (FA) reprogramming immunosuppressive tumor microenvironment (TME) by eliminating M2-TAMs. (A) In vivo antitumor efficacy of Lipo@CpG-FA. Tumor growth curves of 4T1-bearing mice intratumorally injected with Saline, Lipo (blank liposomes), Lipo@CpG, and Lipo@CpG-FA every other day (CpG dose, 4 μg per mouse). Tumor inhibition rate curves of different formulation-treated breast tumors. Body weights of 4T1-bearing mice from different groups. Weights of excised tumor at the end of the experiment. Data represent mean ± standard deviation (SD) (n = 4). ∗∗P < 0.01, ∗∗∗∗P < 0.0001. (B, C) Confocal images showing M1 and M2 macrophage staining of 4T1 tumor tissues from different groups of mice in tumor growth inhibition study (n = 3). (D) Confocal images displaying CD8+ T cells and CD4+ T cells staining of 4T1 tumor tissues. (E) Enzyme-linked immunosorbent assay (ELISA) results of  interleukin (IL)-6, IL-10, IL-12p70, and tumor necrosis factor-α (TNF-α) secretion in 4T1 tumor tissues from mice receiving the indicated treatment. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. The data represent mean ± SD (n = 3). iNOS: inducible nitric oxide synthase; Arg-1: arginase-1.

Fig. 1

LSCM revealed that Lipo@CpG-FA and Lipo@CpG up-regulated M1 macrophage marker proteins, inducible nitric oxide synthase (iNOS) and cluster of differentiation (CD) 86, and down-regulated M2 macrophage marker proteins, arginase-1 (Arg-1) and CD206, in tumor tissue; however, Lipo@CpG-FA group was more pronounced (Figs. 1B and C). These results could be explained by the ability of Lipo@CpG-FA to convert M2 macrophages into M1 macrophages, which are associated with remodeling the immunosuppressive TME and thus inhibiting tumor growth.

The polarization of macrophage cells from M2 phenotype to M1 phenotype was anticipated to reprogram the immunosuppressive TME. To confirm this, we investigated the abundance of effector T cells and the secretion of immune cytokines in tumor tissues. LSCM results demonstrated that compared with Lipo@CpG, Lipo@CpG-FA treatment led to the highest levels of CD4+ T cells and CD8+ T cells in the tumor (Fig. 1D). Furthermore, the tumor increased the secretion levels of cytokines, including tumor necrosis factor-α (TNF-α), interleukin-12p70 (IL-12p70), and interleukin-6 (IL-6) (Fig. 1E). Notably, Lipo@CpG-FA revealed the highest cytokine levels among all treated groups. Reduced secretion of cytokine interleukin-10 (IL-10) alleviated the immunosuppressive TME. The findings demonstrated that Lipo@CpG-FA induced effective immunostimulatory and anti-tumor immunity.

The anti-tumor efficacy of combining Lipo@CpG-FA with AS1411-Lipo@Ele-FA in vitro (Fig. S4) and in mice breast cancer was confirmed (Fig. S5). Compared to saline group, the combination regimens (A and B) demonstrated better antitumor efficacy than the single-agent regimen (Fig. S5B). For regimens A and B, average tumor inhibition rates were 52.55 % and 75.24 %, respectively. The findings revealed that alternating the two drugs enhanced the tumor treatment effect and confirmed the conjecture that Lipo@CpG-FA (4 μg/per) could reverse the immunosuppressive TME. However, a significant difference was observed in the tumor inhibition rate between regimens A and B, suggesting further studies into the underlying mechanisms. The combination regimens had no significant toxic effects in mice and prolonged survival time during treatment (Figs. S5C and E). Remarkably, compared with regimen A, the tumor tissue section samples from regimen B exhibited considerable apoptosis or necrosis (Fig. S5H).

We investigated whether combining Lipo@CpG-FA and AS1411-Lipo@Ele-FA could reduce tumor immunosuppression by eliminating M2-TAMs. Flow cytometry was performed on tumor cells from each group. According to the flow cytometry results (Figs. 2A and B), the saline and Lipo groups had minimal effect on M2-TAMs. In contrast, regimens A and B significantly reduced the number of M2-TAMs in the tumor. M2-TAMs were 12.4% lower after regimen B treatment compared to saline. Meanwhile, the proportion of M1-TAMs and total macrophages in this group had increased to 37.78 % and 39.58 %, respectively, as manifested in Figs. 2C and D. These data suggest that CpG liposomes can convert M2-TAMs to M1 phenotype.Fig. 2 Lipo@CpG-folic acid (FA) together with AS1411-Lipo@elemene (Ele)-FA reprogramming immunosuppression in tumor microenvironment (TME) by eliminating M2-tumor-associated macrophages (TAMs). (A) Representative flow cytometry profiles of M2-TAMs (F4/80+ CD206+ cells) and M1-TAMs (F4/80+ CD86+ cells) in 4T1 tumors after the indicated treatment. (B, C) Representative flow cytometry profiles of M2-TAMs (F4/80+ CD206+ cells) (B) and M1-TAMs (F4/80+ CD86+ cells) (C). ∗P < 0.05, ∗∗∗∗P < 0.0001. (D) Representative flow cytometry profiles of total TAMs in 4T1 tumors after the indicated treatment. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (E–G) Representative flow cytometry profiles of CD4+ T and CD8+ T cells in 4T1 tumors after the indicated treatment. ∗∗P < 0.01, ∗∗∗P < 0.001. (H) Representative flow cytometry profiles of total CD4+ T and CD8+ T cells in 4T1 tumors after the indicated treatment. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. The data represent mean ± standard deviation (SD) (n = 3).

Fig. 2

Interestingly, M2-TAMs were also lower in regimen A than in saline, but the number of M1-TAMs and total macrophages in this group remained unchanged. According to the findings, Ele may have an inhibitory effect on M2 macrophage proliferation. There were insufficient M2-TAMs to be polarized due to the continuous injection of AS1411-Lipo@Ele-FA for seven days (regimen A) and the more prolonged inhibitory effects of Ele. The cumulative amount of CpG administered in this regimen was insufficient to reverse the suppressive TME, indicating that regimen B can effectively eliminate M2 TAMs from the tumor site. This could explain why there was a significant difference in the tumor inhibition rate between regimens A and B.

The activated effector T cells migrate into the tumor, resulting in anti-tumor immunity. Furthermore, the flow cytometry results in Figs. 2E–H confirmed that combining Lipo@CpG-FA and AS1411-Lipo@Ele-FA resulted in the highest levels of CD4+ and CD8+ T cells at 2.42% and 1.67%, respectively. The findings demonstrated that combining CpG with Ele induced potent antitumor immunity levels.

A mutiny of macrophages in solid tumors can reprogram the immunosuppressive TME. We designed and prepared two types of nanoformulation: Lipo@CpG-FA and AS1411-Lipo@Ele-FA. Lipo@CpG-FA demonstrated its ability to effectively target M2-TAMs and convert M2 macrophages into M1 phenotype, reversing the suppressive TME in vitro. It not only activated anti-tumor immune competence but also converted “cold” tumors into “hot” tumors, resulting in CD4+ and CD8+ T cells infiltration. Furthermore, AS1411-Lipo@Ele-FA was able to specifically kill 4T1 cells while also inhibiting M2-TAM proliferation in TME. We inferred that Ele could inhibit M2 macrophages or convert M2 into other unknown macrophages, however, these mechanisms merit further investigation.

CRediT author statement

Ruhua Luo: Methodology, Software, Investigation, Formal analysis, Writing - Original draft preparation; Zhongyu Yue: Data curation, Software, Writing - Original draft preparation; Qian Yang: Data curation, Writing - Original draft preparation; Honghua Zhang: Software, Validation; Tian Xie, Shuling Wang: Conceptualization, Funding acquisition, Resources, Supervision; Qingchang Tian: Methodology, Software, Investigation, Formal analysis, Validation, Funding acquisition.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Acknowledgments

This work was supported by 10.13039/501100004731 Zhejiang Provincial Natural Science Foundation of China (Grant No.: LY20H160008 ), the Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China (Grant No.: LHDMZ22H300001) , and 10.13039/501100001809 National Natural Science Foundation of China (Grant No.: 82074052 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2024.01.009.
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