
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00240-7
10.1016/j.tranon.2024.102113
102113
Original Research
Collagen extracellular matrix promotes gastric cancer immune evasion by activating IL4I1-AHR signaling
Zhang Xiaowei a
Zhao Yang b
Chen Xu chenx1@jzmu.edu.cn
a⁎
a General Surgery Ward, the First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning, China
b Intensive Medical Ward, the First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning, China
⁎ Corresponding author. chenx1@jzmu.edu.cn
30 8 2024
11 2024
30 8 2024
49 10211323 5 2024
11 8 2024
25 8 2024
© 2024 The Authors. Published by Elsevier Inc.
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/).
Highlights

• Collagen in ECM promotes CAR-T cell exhaustion and immune evasion in gastric cancer.

• 3D collagen environment activates PI3K/AKT signaling, leading to IL4I1 upregulation.

• High IL4I1 expression predicts poor response to CAR-T therapy.

• High ECM stiffness correlates with reduced cytotoxic T cell activity and immune evasion.

Background

Gastric cancer (GC) remains a significant global health challenge with poor prognosis, partly due to its ability to evade the immune system. The extracellular matrix (ECM), particularly collagen, plays a crucial role in tumor immune evasion, but the underlying mechanisms are not fully understood. This study investigates the role of collagen ECM in promoting immune evasion in gastric cancer by activating the IL4I1-AHR signaling pathway.

Methods

We cultured gastric cancer cells in 3D collagen gels and assessed their immune evasion capabilities by co-culturing with HER2-specific CAR-T cells. The expression of IL4I1 and its metabolites was analyzed, and the role of integrin αvβ1 in mediating the effects of collagen was explored. Additionally, the impact of IL4I1-induced AHR activation on CAR-T cell exhaustion was evaluated, both in vitro and in vivo.

Results

We found that gastric cancer cells cultured on collagen exhibited increased resistance to CAR-T cell cytotoxicity, which was associated with upregulated immune checkpoint molecules and downregulated effector cytokines on CAR-T cells. This was linked to increased IL4I1 expression, which was further induced by integrin αvβ1 signaling within the 3D collagen environment. IL4I1 metabolites, particularly KynA, promoted CAR-T cell exhaustion by activating the AHR pathway, leading to decreased cytotoxicity and tumor growth inhibition.

Conclusions

Our study reveals a novel mechanism by which the collagen ECM facilitates immune evasion in gastric cancer through the activation of IL4I1-AHR signaling, contributing to CAR-T cell exhaustion. Targeting this pathway could potentially enhance the efficacy of CAR-T cell therapy in gastric cancer.

Keywords

Gastric cancer
Extracellular matrix
Collagen
Immune evasion
IL4I1
AHR signaling
CAR-T cell therapy
==== Body
pmcIntroduction

Gastric cancer is one of the most prevalent cancers worldwide, with a 5-year relative survival rate of approximately 20% [1,2]. Despite improvements in treatment methods, patient prognosis remains challenging [[3], [4], [5]]. Cancer's hallmark is its ability to evade immune surveillance through mechanisms including immune checkpoint dysregulation and MHC class I downregulation [[6], [7], [8]]. Despite notable progress in treating gastric cancer through recent advances in immunotherapies like PD-1/PD-L1 checkpoint blockade and CAR-T cell therapy, enduring responses remain infrequent among patients [9]. Although the mechanisms underlying CD8+ T cell exhaustion, which leads to the ineffectiveness of immunotherapies such as checkpoint blockade or CAR-T therapy, are well-documented, the role of the extracellular matrix (ECM) in this process remains less clear.

The ECM is known to actively support tumor persistence through interactions with tumor cells and other components of the tumor microenvironment (TME) [10]. Type I collagen, which contributes to a stiff microenvironment around cancer cells, has been discussed in some studies [11]. However, the specific role of Type I collagen in the progression of CD8 T cell exhaustion is still not well understood. The stiffness of the ECM, a critical element of the TME, significantly influences both cancer progression and the efficacy of immunotherapy [12]. This stiffness results from remodeling processes that are regulated by enzymes such as matrix metalloproteinases and lysyl oxidases, leading to the accumulation and cross-linking of ECM proteins [11]. Such increased stiffness creates a physical barrier that impedes the infiltration of immune cells and affects the delivery of immunotherapeutic agents, including those targeting immune checkpoints like PD-1, CTLA-4, and PD-L1 [13]. Additionally, ECM stiffness can modify the expression of immune checkpoint molecules, thereby facilitating immune evasion by cancer cells [14]. For instance, in a stiff ECM, cancer cells may express higher levels of PD-L1, reducing the effectiveness of checkpoint inhibitors that target PD-1 or PD-L1 [15]. The suppression of immune responses by ECM stiffness underscores a significant challenge in developing effective cancer immunotherapies and highlights the need for further research into how the mechanical properties of the ECM affect immune cell function and how altering these properties could enhance the response to immunotherapy.

Here, we demonstrate that tumor cells cultured on collagen facilitate tumor-specific CAR-T cell exhaustion compared to conventional 2D culture, characterized by upregulated immune checkpoint molecules and downregulated effector cytokines on CAR CD8+ T cells. Mechanistically, CD8+ T cell exhaustion occurs due to tumor cells within the 3D collagen environment upregulating IL4I1, an enzyme associated with tryptophan metabolism. Consequently, IL4I1 triggers the activation of the AHR molecule in T lymphocytes through its metabolites I3A and KynA. This sequence of events subsequently intensifies the exhaustion of CAR-T cells. Finally, our organoid model indicates that high IL4I1 expression in tumor predicts poor response to CAR-T therapy. Our study identifies collagen and IL4I1 as potential markers of resistance to CAR-T therapy and validates multiple therapeutic targets in combination with CAR-T therapy.

Result

Collagen ECM-cultured tumor cells promote CAR-T cell exhaustion

To elucidate the role of the ECM in driving gastric cancer immune evasion, we first implanted gastric cancer cells HGC27 and MGC803 into 3D collagen gels at varying concentrations. Subsequently, we co-cultured these tumor cells with HER2-specific CAR-T cells to assess the immune evasion capability of gastric cancer cells (Fig. 1A). Interestingly, with increasing collagen concentration in the 3D gel system, HGC27 and MGC803 gastric cancer cells exhibited enhanced resistance to CAR-T cell cytotoxicity (Figs. 1B and S1A). Furthermore, we did not detect differences in gene expression of MHC class I between these cells (Fig. S1B). When T cells were co-cultured with tumor cells from a 3D collagen gel environment, as opposed to the traditional 2D-cultured tumor co-culture, we observed a more pronounced exhausted phenotype in the T cells. This was characterized by elevated expression of immune checkpoint molecules and decreased levels of effector cytokines (Fig. 1C, D). Interestingly, we found that tumor cells cultured with other types of ECM co-cultured with T cells did not induce exhaustion in the T cells (Fig. S1C–F). Similarly, we found that CAR-T cells with 2D cultured tumor cells showed better second-phase killing ability than those with collagen-cultured tumor cells (Fig. 1E, F), it implies that collagen-cultured tumor cells are more likely to make CAR-T cells exhausted and less effective.Fig. 1 Collagen ECM-cultured tumor cells promote CAR-T cell exhaustion

A, Graphical representation of short-term specific cytotoxicity experiment in vitro. Briefly, 5 × 104 Her2+ MGC803 or HGC27 which was pre-cultured on gels of different dose collagen co-cultured with CAR T cells at an E:T ratio of 1:1 for two days. At the end of the first round of co-culture, the viable CAR-T cells were counted and the CAR-T-mediated specific cytotoxicity was detected by Annexin V-FITC/PI Apoptosis Detection Kit. B, Cytotoxic activity of HER2-specific chimeric antigen receptor-T cells against MGC803 or HGC27. C-D, the expression of multiple inhibitory receptors (IRs) (C) and cytokines (D) of HER2-specific chimeric antigen receptor-T cells against MGC803 or HGC27. E-F, Graphical representation of long-term specific cytotoxicity experiment in vitro. Briefly, 5 × 104 Her2+ MGC803 or HGC27 which pre-cultured on dish or collagen gel co-cultured with 2D-CAR or 3D-CAR T cells at an E:T ratio of 1:1 for two days. At the end of the first round of co-culture, the viable CAR-T cells were counted and the CAR-T-mediated specific cytotoxicity was detected by Annexin V-FITC/PI Apoptosis Detection Kit, and a second round of co-culture using the first round of CAR+ T cells with the same number of tumor cells at the same E:T ratio (1:1). a second round of co-culture assays were performed (F). n = 3 biological independent experiments (A to F). **P < 0.01, ***P < 0.001 or NS (no significant difference, by two-tailed Student's t test (C, D and F) or one-way ANOVA and Bonferroni's test (B). The data represent means ± SD.

Fig 1

IL4I1 is upregulated in collagen ECM-cultured tumor cells

Previous studies have reported that liver cancer cells cultured in 3D collagen gel exhibit an upregulation of IL4I1 compared to cells cultured in conventional 2D monolayers [16]. However, the role of IL4I1 in gastric cancer tumor immunity remains scarcely explored. Therefore, we further examined the expression of IL4I1 in tumor cells cultured in both 2D and 3D collagen gels, and observed an increase in the protein levels of IL4I1 in cells cultured within the 3D collagen environment (Fig. 2A). Subsequently, we employed sgRNA to knock out IL4I1 expression in the gastric cancer cell lines, and we confirmed the effectiveness of the knockout through western blot (Fig. S2A). The IL4I1-knockout cells and sgNC (non-targeting control) cells were then cultured within 3D collagen gels, followed by co-culturing with HER2-specific CAR-T cells. Interestingly, MGC803 and HGC27 gastric cancer cells with IL4I1-knockout cells exhibited decreased resistance to CAR-T cell cytotoxicity, compare to sgNC (non-targeting control) cells (Fig. 2B). We observed a decrease in the expression of checkpoint markers associated with T cell exhaustion on the co-cultured HER2-specific CAR-T cells in IL4I1-knockout cells compared with sgNC cells, accompanied by an augmentation in the production of IFN-γ and TNF-α, indicating an enhanced immune response (Fig. S3A, B). Here, we further validated this process in vivo. Firstly, we established a patient-derived xenograft (PDX) model using tumor tissue from a gastric cancer patient. Subsequently, we applied the previously established protocol to perform gene editing on the PDX, then sgNC PDX and the another PDX with sgIL4I1, into mice [17]. After the adoptive transfer of HER2-specific CD8+ T cells into NSG mice bearing a 5 × 5 mm PDX, we observed that in the absence of CAR-T cell therapy, the sgIL4I1 tumors formed nearly identical sizes compared to the sgNC group (Figs. 2C and S3C). However, under CAR-T cell therapy, sgIL4I1 resulted in a reduced tumor burden (Fig. 2C). Moreover, we isolated CAR-T cells for flow-cytometric analysis. the disruption of IL4I1 in tumor effectively inhibited CAR- T cell exhaustion in mice (Fig. 2D), accompanied by increased cytokine levels (Fig. 2E). Together, these results suggest that IL4I1 lead to CD8+ CAR-T cell exhaustion.Fig. 2 IL4I1 is upregulated in Collagen ECM-cultured tumor cells

A, Immunofluorescence analysis of IL4I1 expression in MGC803 or HGC27 derived from the 2D and 3D groups. Scale bar, 20 μm. B-C, The cytotoxic activity of HER2-specific chimeric antigen receptor-T cells against MGC803 or HGC27 which were transfected with sgNC, sgIL4I1-1 and sgIL4I1-2. C, NSG mice were transplanted with PDX. At 30 days post engraftment, NSG mice were adoptively transferred with 1  ×  106 human HER2-CAR or Mock T cells, then at 30 days post engraftment, mice were taken out in each group and killed for assessment of tumor weight(C). D-E, the expression of multiple IRs(D) and cytokines(E) of HER2-specific chimeric antigen receptor-T cells in PDX tissue. n = 3 biological independent experiments (B) and n = 6 biological independent experiments (C to E). **P < 0.01, ***P < 0.001 or NS (no significant difference, by one-way ANOVA and Bonferroni's test (B to E). The data represent means ± SD.

Fig 2

3D collagen induces IL4I1 expression through integrin αvβ1, thereby promoting tumor immune evasion

A previous report suggests that 3D fibrin gels can induce breast cancer cell stemness by integrin signaling [18,19]. We hypothesize that type I collagen employs transmembrane receptor protein integrin αvβ1 to mediate the biomechanical forces in the tumor microenvironment, which in turn induces IL4I1 expression. This elucidates the potent immune evasion capabilities of collagen-cultured gastric cancer cells. We initially examined the expression of integrin in HGC27 and MGC803 cells, observing a significant upregulation of integrin β1 in 3D-cultured tumor cells (Fig. 3A). Subsequently, we assessed the expression of integrin αvβ1 protein in gastric cancer cells cultured for 3 days in a 3D collagen cell culture system. As anticipated, an elevated expression of integrin αvβ1 was observed in the 3D-cultured tumor cells (Fig. 3B). The integrin αvβ1 inhibitor, αvβ1 integrin-IN-1, blocked integrin αvβ1 and significantly suppressed cellular IL4I1 expression in the 3D collagen system (Fig. 3C). It also inhibited the cytotoxicity of CAR-T cells in the co-culture system (Fig. 3D), indicating that integrin αvβ1 plays a crucial role in collagen-induced immune evasion of tumor cells. Consistently, we found that integrin αvβ1 inhibitor strikingly abolished collagen-induced CD8+T cell exhaustion, as evidenced by downregulation of the expression of PD-1, LAG-3 and TIM-3 (Fig. 3E), but upregulation of IFN-γ and TNF-a (Fig. 3F). Similarly, Integrin αvβ1 signaling inhibition also reversed T cell exhaustion induced by collagen in vivo (Fig. 3G, H), concomitant with enhanced adoptive transfer–produced inhibitory effect on tumor growth (Fig. 3I). In summary, these results suggest that collagen, through integrin αvβ1 signaling, promotes the upregulation of IL4I1 in gastric cancer, thereby inducing tumor immune evasion.Fig. 3 3D collagen induces IL4I1 expression through integrin αvβ1, thereby promoting tumor immune evasion

A, qPCR analysis of Integrins expression in MGC803 or HGC27 derived from the 2D and 3D groups. B, Immunofluorescence analysis of integrin αvβ1 expression in MGC803 or HGC27 derived from the 2D and 3D groups. Scale bar, 20 μm. C, Immunofluorescence analysis of IL4I1 expression in MGC803 or HGC27 treated with or without integrin αvβ1 inhibitor. Scale bar, 20 μm. D, Her2+ MGC803 or HGC27 which was treated with or without integrin αvβ1 inhibitor co-cultured with CAR-T cells at an E:T ratio of 1:1 for two days. CAR-T-mediated specific cytotoxicity was detected by Annexin V-FITC/PI Apoptosis Detection Kit. E-F, the expression of multiple IRs (E) and cytokines (F) of HER2-specific chimeric antigen receptor-T cells against MGC803 or HGC27 which was treated with or without integrin αvβ1 inhibitor. G-I, NSG mice were transplanted with PDX. At 30 days post-engraftment, NSG mice were adoptively transferred with 1 × 106 human HER2-CAR or Mock T cells, combined with either PBS or integrin αvβ1 inhibitor. The expression of IRs (G) and cytokines (H) of HER2-specific chimeric antigen receptor-T cells in PDX tissue. then at 60 days post-engraftment, mice were taken out in each group and killed for assessment of tumor weight (I). n = 3 biological independent experiments (A to F) and n = 6 biological independent experiments (G to H). **P < 0.01, ***P < 0.001 or NS (no significant difference, by Two-tailed Student's t-test. The data represent means ± SD.

Fig 3

3D collagen gel promotes tumor immune evasion through the integrin αvβ1/AKT/SP1/IL4I1 signaling pathway

Reports indicate that ECM, such as collagen, can induce the PI3K/AKT signaling pathway through integrin [20]. In this study, we observed an upregulation of phosphorylated AKT in gastric cancer cells cultured in 3D collagen matrices (Fig. 4A). Furthermore, blocking integrin αvβ1 in the 3D collagen culture system inhibited the expression of p-AKT, suggesting that collagen activates AKT via integrin αvβ1 (Fig. 4A). Additionally, after inhibiting AKT activity with MK-2206, we noticed a decrease level of IL4I1expression in the gastric cancer cells within the 3D collagen culture system (Fig. 4B), along with enhanced CAR-T cytotoxicity (Fig. 4C). Previous study has shown that the AKT signaling pathway can phosphorylate SP1 and promotes its nuclear localization, and various reports have identified SP1 as a direct regulator of IL4I1 [16]. We hypothesize that 3D collagen gels induce IL4I1 expression through the integrin αvβ1/AKT/SP1 pathway. We observed a significant increase level of nuclear SP1 in gastric cancer cells cultured in 3D collagen, which could be inhibited by either blocking integrin αvβ1 or MK-2206 (Fig. 4D). Knocking down SP1 in gastric cancer cells cultured in the collagen gel also resulted in decreased IL4I1 expression (Fig. 4E). Moreover, our ChIP and dual-luciferase assays revealed that SP1 is enriched at the promoter site of IL4I1, enhancing activity of IL4I1 promoter (Fig. 4F, G), suggesting that the nuclear translocation of SP1 activated by AKT may induce IL4I1 activation and negatively regulate the anti-tumor immunity in gastric cancer cells. Furthermore, we adoptively transferred CAR-T cells to mice bearing sgSP1 or sgNC gastric cancer PDX (5 × 5 mm), and isolated CAR-T cells for flow-cytometric analysis at two weeks. Indeed, the SP1 knockout treatment enhanced the efficiency of adoptively transferred CAR-T cells, as evidenced by less exhausted T cells and retarded tumor growth (Fig. 4H–J).Fig. 4 3D collagen gel promotes tumor immune evasion through the integrin αvβ1/AKT/SP1/IL4I1 signaling pathway.

A, Immunofluorescence analysis of p-AKT expression in MGC803 or HGC27 treated with or without integrin αvβ1 inhibitor. Scale bar, 20 μm. B, Immunofluorescence analysis of p-AKT expression in MGC803 or HGC27 treated with or without AKT inhibitor. Scale bar, 20 μm. C, Her2+ MGC803 or HGC27 which was treated with or without AKT inhibitor co-cultured with CAR-T cells at an E:T ratio of 1:1 for two days. CAR-T-mediated specific cytotoxicity was detected by Annexin V-FITC/PI Apoptosis Detection Kit. D, Immunofluorescence analysis of SP1 expression in MGC803 or HGC27 treated with or without AKT inhibitor and integrin αvβ1 inhibitor. Scale bar, 20 μm. E, Immunofluorescence analysis of IL4I1 expression in MGC803 or HGC27 transfected with or without sgSP1. Scale bar, 20 μm. F, MGC803 or HGC27 derived from the 2D and 3D groups. ChIP–qPCR analysis was performed with an antibody to SP1 and IL4I1-promotor-specific primers. G, MGC803 cells were cotransfected with a IL4I1 promoter-luciferase reporter PGL3 and Flag–SP1 plasmid for 24 h. Cells were then cultured with 2D or 3D gel for another 48 h, followed by analysis of luciferase activity. H-J, NSG mice were transplanted with PDX transfected with or without sgSP1. At 30 days post engraftment, NSG mice were adoptively transferred with 1  ×  106 human HER2-CAR or Mock T cells, then at 30 days post engraftment, mice were taken out in each group and killed for the expression of IRs (H), cytokines (I) and assessment of tumor weight (J).n = 3 biological independent experiments (A to G) and n = 6 biological independent experiments (H to J). **P < 0.01, ***P < 0.001 or NS (no significant difference, by one-way ANOVA and Bonferroni's test (F to J). Two-tailed Student's t-test (C). The data represent means ± SD.

Fig 4

Collagen ECM-cultured tumor cells derived KynA promotes CAR-T exhaustion

To date, as IL4I1 inhibitors have not been applicable for cancer therapy, our focus has shifted to potential downstream targets of IL4I1 to enhance the efficacy of CAR-T against solid tumors like gastric cancer. IL4I1 converts tryptophan (Trp) into indole-3-pyruvic acid (I3P), subsequently generating metabolites such as kynurenic acid (KynA) [21]. Consistently, we observed elevated levels of KynA in gastric cancer cell cultured within 3D collagen gel compare with 2D cell (Fig. 5A). Under 3D collagen conditions, the upregulation of IL4I1 expression was significantly higher compared to the other two tryptophan-metabolism enzymes, indoleamine 2,3-dioxygenase 1,2 (IDO1, IDO2), and tryptophan 2,3-dioxygenase (TDO2) (Fig. 5B). Furthermore, the levels of KynA produced by 3D gastric cancer cell with IL4I1 knockout were also lower than those produced by sgNC cells (Fig. 5C). To determine if metabolites from 3D tumor cells drive CD8+ CAR-T cell exhaustion, we cultured freshly isolated CD8+ T cells from healthy peripheral blood with added KynA. Remarkably, KynA notably increased PD-1, TIM3 and LAG3 expression on CD8+ T cells, accompanied by reduced cytokine levels (Fig. 5D, E). These results imply that tumor derived KynA promotes CD8+ T cell exhaustion, contributing to diminished anti-tumor immunity in 3D collagen gel.Fig. 5 Collagen ECM-cultured tumor cells derived KynA promotes CAR-T exhaustion

A, KynA levels measured by LC-MS in lysates of MGC803 or HGC27 derived from the 2D and 3D groups. B, qPCR analysis of tryptophan metabolism related enzymes expression in MGC803 or HGC27 derived from the 2D and 3D groups. C, KynA levels measured by LC-MS in lysates of MGC803 or HGC27 which were transfected with sgNC, sgIL4I1-1 and sgIL4I1-2. D-E, the expression of IRs (D) and cytokines (E) of HER2-specific chimeric antigen receptor-T cells which was treated with KynA. n = 3 biological independent experiments (A to E). **P < 0.01, ***P < 0.001 or NS (no significant difference, by one-way ANOVA and Bonferroni's test (C). Two-tailed Student's t-test (A,B,D,E). The data represent means ± SD.

Fig 5

IL4I1-induced AHR activation within T cells promotes CAR-T exhaustion

Various Trp metabolites can activate the aryl hydrocarbon receptor (AHR) to inhibit T cell function [22,23]. Interestingly, we found that KynA significantly activate CAR-T cell nuclear translocation of AHR (Fig. 6A). Moreover, CAR-T cells co-cultured with 3D tumor cells exhibited higher AHR nuclear translocation compared to those co-cultured with 2D cells (Fig. 6B). To verify the role of intracellular AHR activation in CAR-T exhaustion, we knocked down IL4I1 in 3D cultured cells and observed an inability to induce AHR nuclear translocation in T cells (Fig. 6C). Furthermore, we employed the AHR inhibitor SR-1 in the 3D co-culture to block AHR activity. We demonstrated that SR-1 treatment significantly enhanced T cell to kill tumor cells in 3D environment (Fig. 6D). To assess IL4I1-AHR-induced CAR-T resistance in vivo, we established a human gastric cancer PDX and adoptively transferred CAR-T cells. We found that the blockade of AHR in PDX led to decreased growth of the tumor size as compared to the PBS control group (Fig. 6E); Moreover, AHR inhibitor strikingly abolished tumor-induced CD8+T cell exhaustion, as evidenced by downregulation of the expression of PD-1, LAG-3 and TIM-3, but upregulation of IFN-γ and TNF-α (Fig. 6F, G). Finally, utilizing GEO database, we discovered that gastric cancer patients with high IL4I1/AHR expression exhibited worse overall survival compared to those with low IL4I1 expression (Fig. 6H). Moreover, we found that patients with high expression of COL1A1 and integrin αvβ1 had poor prognoses (Fig. S5A–C). In conclusion, these findings suggest that IL4I1/AHR may modulate the progression of gastric cancer with high collagen content by regulating CAR-T cell exhaustion.Fig. 6 IL4I1-induced AHR activation within T cells promotes CAR-T exhaustion

A, Immunofluorescence analysis of AHR expression in CAR-T treated with or without KynA. Scale bar, 10 μm. B, Immunofluorescence analysis of AHR expression in in HER2-specific chimeric antigen receptor-T cells against 2D or 3D tumor. Scale bar, 10 μm. C, Immunofluorescence analysis of AHR expression in in HER2-specific chimeric antigen receptor-T cells, which was treated with or without SR1 against 3D tumor. Scale bar, 10 μm. D, Cytotoxic activity of HER2-specific chimeric antigen receptor-T cells, which was treated with or without SR1 against 3D tumor. E, Size of the PDX in mice, which was transferred or not with HER2-specific chimeric antigen receptor-T cells and treated with or not with SR1. F, IRs of HER2-specific chimeric antigen receptor-T cells, which was treated with or without SR1 against PDX. G, cytokines of HER2-specific chimeric antigen receptor-T cells, which was treated with or without SR1 against PDX. H, Survival analysis of IL4I1 based on the GSE15459 (n = 200) or GSE28541 (n = 40) databases. n = 3 biological independent experiments (A to D) and n = 6 biological independent experiments (E to G). **P < 0.01, ***P < 0.001 or NS (no significant difference, by one-way ANOVA and Bonferroni's test (E to G). Two-tailed Student's t-test (D). The data represent means ± SD.

Fig 6

Discussion

Indeed, immunotherapy has proven to be highly effective in clinical practice and has rapidly become part of the standard treatment for various cancers, often used in conjunction with chemotherapy and radiation therapy [24,25]. Immunotherapy can be broadly categorized into two major classes. The first class involves therapies that activate or enhance the body's immune system through small molecules or antibodies [26,27]. These therapies include immune checkpoint inhibitors (such as CTLA-4 antibodies, PD-1 antibodies, and PD-L1 antibodies), immune agonists (including CD40 antibodies and CD40L antibodies), tumor mRNA vaccines, oncolytic viruses, and more [[28], [29], [30], [31]]. The second class focuses on adaptive transfer of immune cells that specifically recognize and target tumor cells, such as CAR-T cells, CAR-NK cells, CAR-M cells and TCR-T [32,33].

However, despite the widespread application of immunotherapy in clinical settings, it is effective for only a subset of patients [24]. A significant proportion of patients do not respond to immunotherapy or develop resistance shortly after treatment [33,34]. Furthermore, while CAR-T cell therapy has shown remarkable efficacy in hematologic malignancies, its effectiveness in solid tumors has been limited [35]. The challenges of CAR-T cell therapy, particularly in solid tumors like gastric cancer, highlight the need for a deeper understanding of the regulatory mechanisms of tumor immunity within the tumor microenvironment (TME) [36].

Our findings highlight the significant role of collagen proteins in the extracellular matrix (ECM) in the process of immune evasion in gastric cancer. We observed that tumor cells cultured on collagen, as opposed to traditional flat culture surfaces, promote the depletion of tumor-specific CAR-T cells and inhibit their ability to kill gastric cancer cells. Mechanistically, within the 3D collagen environment, tumor cells are stimulated by extracellular cues, activating the mechanosensitive integrin signaling pathway on the tumor cell membrane. This activation leads to downstream PI3K/AKT activation. Activated AKT phosphorylates SP1, a transcription factor, causing its nuclear translocation and subsequent activation of IL4I1, an enzyme involved in tryptophan metabolism. This results in a highly active tryptophan metabolism within 3D cells.

Evading immune surveillance is crucial for tumor growth. Dorota Kuczek et al. found that high levels of collagen may enhance the expression of TGF in cancer cells, thereby inhibiting the activity of infiltrating CD8 cells in tumors [37]. Additionally, DC functionality is regulated by extracellular matrix stiffness. DCs cultured on a stiff matrix similar to pathological tissues (12 kPa) may fail to elicit immune responses compared to DCs cultured on a soft matrix resembling healthy tissue (2 kPa). This can severely impact the clearance of pathogens or abnormal cells [38]. High extracellular matrix stiffness-induced Piezo1 activation may promote macrophage polarization and enhance their immune-suppressive phenotype, resulting in reduced cytotoxic T cell abundance and proliferation [39]. Alba Nicolas-Boluda et al. used preclinical mouse models of pancreatic cancer, breast cancer, and cholangiocarcinoma and found a negative correlation between ECM stiffness and T cell infiltration in tumors and the efficacy of PD-1 blockade therapy [14]. Therefore, these studies are consistent with our findings, suggesting that high collagen density may directly or indirectly promote immune evasion and resistance to CAR-T immunotherapy in cancer.

Tryptophan metabolism is a well-known mechanism of acquired immune resistance in immunotherapy. Enzymes like IL4I1, IDO, and TDO inhibit immune responses in various cancer types, including breast cancer, melanoma, lung cancer, colorectal cancer, and gastric cancer [40,41]. Elevated expression of tryptophan metabolism enzymes can directly or indirectly suppress the effector function of CD8+ T cells [42]. Strategies targeting this metabolic pathway can reverse tumor immune resistance mechanisms.

Previous studies have shown that tryptophan metabolism enzymes can activate downstream AHR, thereby directly or indirectly inhibiting T cell function [43,44]. Apart from tryptophan metabolism products, tobacco smoke can also stimulate AHR activation, inducing PD-L1 expression in lung cancer models, leading to immune evasion. AHR activity can effectively predict the response to PD-L1 blockade [45]. The impact of ECM stiffening on immune surveillance may depend on the balance between different pathways in different cell types. Moreover, the potential mechanisms linking ECM stiffness to mechanical signaling through integrins and the regulation of tryptophan metabolism are still poorly understood. Further research is needed to address and clarify the impact of the synergistic effects of collagen fibers and tryptophan metabolism on cancer immunotherapy in clinical patient cohorts.

Materials and methods

Cell lines and cell culture

Human gastric cancer cell line MGC803, HGC27 and HEK293T (X100478) were purchased from the China Center for Type Culture Collection (Beijing, China). All the above cell lines were cultured in DMEM (Gibco, USA) containing 10 % fetal bovine serum (FBS) (Gibco, USA) and 1% Penicillin-Streptomycin solution (Gibco, USA). All cells were grown at 37°C in a 5% CO2 incubator. Cells were tested for mycoplasma detection, inter-species cross contamination and authenticated by isoenzyme and short-tandem repeat (STR) analyses in the Cell Resource Centre of Peking Union Medical College before the study.

Animal studies

NOD scid gamma (NSG) mice (Charles River) aged 6–8 weeks old were purchased from Charles River Co. These animals were maintained in the Animal Facilities of the Jinzhou Medical University under pathogen-free conditions. All studies involving mice were approved by the Animal Care and Use Committee of the Jinzhou Medical University. In summary, the newly harvested CRC tissues (F0 generation) underwent three rounds of washing in PBS supplemented with 100 units/mL of penicillin and streptomycin. Subsequently, the specimen was cut into pieces approximately 5 × 5 mm in size. To create the F1 generation, a single tumor fragment was transplanted into the right flank of a mouse that had been given anesthesia. The tumors of the F1 generation were palpable two to four weeks following implantation. Tumors that grew to a volume of 1 cm3 were removed, chopped into many pieces measuring 3 by 3 mm each, and then placed into fresh NSG mice to generate the following generation. NSG mice were engrafted on the right flank of stomach cancer patients as xenografts for the PDX mouse model. Once the tumors had grown to 5 mm by 5 mm, mice were injected with 1 × 106 CAR T cells via the tail vein. In order to determine the number and level of exhaustion of CAR-T cells within the tumor microenvironment, we measured the tumor weight and conducted a flow cytometry study on day 40.

CAR-expressing lentivirus production and plasmids

The study used the CAR constructs as previously described (31999649). In short, Human Epithelial Growth Receptor 2 (36855335), eGFP, and the CD28 and CD3-zeta signaling domains were integrated into the HER2 structure. A marker of CAR+ T cells was eGFP expression. 293T cells were maintained in a 10 cm dish until 80% confluence in order to produce lentiviruses. Next, transfection was performed in accordance with the instructions provided by the manufacturer using the lipo2000 kit (Thermo, USA). In summary, 500 µl of transfection buffer was mixed with 20 µg of plasmid (10 µg of CAR plasmid, 3 µg of pMD2.G, and 7 µg of psPAX2), and then 40 µl of lipo2000 transfection reagent was added. 293T cells were gently infected with the transfection mixture after they had been incubated for 10 min at room temperature. Without agitating the cells, the old medium was taken out and replaced with 7 ml of previously warmed medium after 6 h. After 48 h, the virus-containing supernatant was gathered and filtered through a 0.45 um filter to get rid of cell debris. The viral supernatant was then aliquoted, concentrated, and kept in storage at -80°C.

Chromatin immunoprecipitation (ChIP) assay

Chromatin immunoprecipitation assays were performed by abcam Co.,Ltd. Briefly, ten million cells were washed twice in cold PBS buffer and cross-linked with 1% formaldehyde for 10 min at room temperature and then quenched by addition of glycine (125 mmol/L final concentration). Afterwards, samples were lysed and chromatins were obtained on ice. Chromatins were sonicated to get soluble sheared chromatin (average DNA length of 200–500 bp). 20 μL chromatin was saved at −20°C. For input DNA, 100 μL chromatin was used for immunoprecipitation by anti-SP1 antibodies (Abcam, EPR22648-50) and IgG antibodies (ab17870, Abcam) respectively. 10 μg of antibody was used in the immunoprecipitation reactions at 4°C overnight. The next day, 30 μL of protein beads was added and the samples were further incubated for 3 h. The beads were next washed once with 20 mM Tris/HCL (pH 8.1), 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS; twice with 10 mM Tris/HCL (pH 8.1), 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid; and twice with TE buffer 1 × (10 mM Tris-Cl at pH 7.5, 1 mM EDTA). Bound material was then eluted from the beads in 300 μL of elution buffer (100 mM NaHCO3, 1% SDS), treated first with RNase A (final concentration 8 μg/mL) during 6 h at 65°C and then with proteinase K (final concentration 345 μg/mL) overnight at 45°C. The immunoprecipitated DNA was used for PCR identification.

Cytotoxicity assay in vitro

To measure the short-term specific cytotoxicity experiment in vitro, 2D cultured or 3D cultured 5 × 104 Her2+ MGC803 or HGC27 was planked 12 h ahead of time in 24-well plates. Then the Her specific-CAR T cells were added to the plates at an E:T ratio of 0.1:1 for 72 h, respectively. After 72 h, plates were centrifuged at 500g for 5 mins and cytotoxicity experiment in vitro was determined by flow cytometry.

Real-time PCR

Total RNA was extracted from cells using Trizol (Invitrogen) and transcribed to cDNA by using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). The primer sequences are shown as the Table S1. Real-time PCR was performed using ABI 7500 (Applied Biosystems). Values are means ± SD from three independent experiments which were performed in duplicate.

Western blotting

Cells were collected, sonicated, then lysed in RIPA lysis buffer. An assay kit for BCA (bicinchoninic acid) was used to measure the protein concentrations (Beyotime, China). Subsequently, the protein was separated onto nitrocellulose using an SDS-PAGE (SDS–polyacrylamide gel electrophoresis) gel. Nitrocellulose membranes were blocked in 5% bovine serum albumin and probed with antibodies overnight: anti-actin (Cell Signaling, 1:1,000); anti-SP1 (Cell Signaling, 1:1,000); anti-IL4I1 (GeneTex; 1:1,000). Enhanced chemiluminescence was seen subsequent to secondary antibodies linked to horseradish peroxidase (Thermo Fisher, MA).

Immunofluorescence

Cells were fixed in 4% paraformaldehyde, then permeabilized with 0.2% Triton X-100. Tumor sections or fixed cells were blocked in 5% BSA and then incubated for an overnight period at 4°C with the primary antibodies. Following a wash, the cells were left to be treated at room temperature for two hours with secondary antibodies. Ultimately, the slides were mounted for confocal examination after being counterstained with 4′,6-Diamidino-2′-phenylindole (DAPI). Using Image J 9.0 software, the intensity of immunofluorescence was examined.

Flow cytometry

Collagenase type Ⅳ (Merck, USA; 1 mg/ml) and DNase Ⅰ (Merck, USA; 5 μg/ml) were used to break down the tumor tissues into individual cells. Following digestion, red blood cells (RBCs) were eliminated by exposing the single-cell suspensions to RBC lysis buffer. This was achieved by passing the suspensions through a 70 µm cell strainer and centrifuging the pellets. Before being stained with the appropriate antibodies, all samples (tumor single cells or in vivo cultured cells) were dyed for ten minutes at room temperature using Live/Dead dye. Cells were treated with Cell Activation Cocktail (Biolegend, USA) for 4 h at 37°C in order to stain intracellular cytokines. After that, the cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher, USA), and they were stained with antibodies against TNF-α, IL-2, or IFN-γ. Software called FlowJo was used to analyze data. Table S2 displays the antibodies that were employed.

Luciferase assays

The Renilla luciferase plasmid (pRL-SV40) containing 100 μg, the firefly luciferase plasmid (PGL4.10-IL4I1) promoter-luciferase reporter vector, and 1 μg of pCMVh-SP1 were transfected into HEK293T cells for a duration of 12 h. Cell lysates were examined using the Dual-Luciferase Reporter Assay (Beyotime, USA). The activity of firefly luciferase was compared to that of Renilla luciferase.

Liquid chromatography with Q-exactive mass spectrometry

Cells or cell culture media were lysed in an extraction solvent (80% methanol/water) for 30 min at -80°C in order to determine the amount of Kyn. The supernatant extracts were subjected to liquid chromatography using Q-exactive mass spectrometry following centrifugation at 12,000g for 10 min at 4°C. Using Thermo Fisher's Xcalibur Qual browser, the area beneath each compound's curve was integrated to quantify the data. Using a 50-ppm window, the precise mass ion of each metabolite and its following isotopic ions were extracted (EIC).

Transduction and enrichment of PDXs

The PDX tumors that had already been established were surgically removed and then broken down into individual cells using a gentleMACS tissue dissociator along with a human tumor dissociation kit from Miltenyi in Germany. The red blood cells were ruptured using ACK lysing buffer (STEMCELL Technologies, Canada), Subsequently, the cells were implanted into a 50% Matrigel (BD) solution in the flank of female NSG mice aged 6-8 weeks.

Statistics and reproducibility

All experiments were performed at least three biological repeats except as indicated in the figure legends and no statistical method was used to predetermine sample size. No data were excluded from analyses. The mice were randomly assigned to different groups. The analysis was conducted using the Graphpad Prism 8.0 software. Results are expressed as mean ± SD as indicated, and analyzed by Student's t-test followed by two-tailed Paired t-test or one-way ANOVA followed by Bonferroni as indicated. The p-value < 0.05 was considered statistically significant. The Spearman's correlation test was used to examine the relationship between the expression of MAFG and number of tumor infiltrating T cells.

Funding information

This study was supported by grants from the Science and Technology Plan Project of Liaoning Province(2023JH2/101300069).

Informed consent

N/A.

Registry and the registration no. of the study/trial

N/A.

Ethics statements

The experiments were performed in accordance with the ethical guidelines of the Declaration of Helsinki. Approval of the Research Protocol by the Institutional Review Board: This study was approved by the First Affiliated Hospital of Jinzhou Medical University (2023109). All the patients provided written informed consent to participate in this study.

All animal procedures were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals of Jinzhou Medical University and all efforts were made to minimize animal suffering. The study protocol was approved by the experimental animal welfare and ethics committee of the First Affiliated Hospital of Jinzhou Medical University (241091).

CRediT authorship contribution statement

Xiaowei Zhang: Writing – original draft, Methodology, Investigation, Formal analysis. Yang Zhao: Writing – review & editing, Methodology. Xu Chen: Investigation, Funding acquisition, Formal analysis, Conceptualization, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare no conflict of interest.

Appendix Supplementary materials

Image, image 1

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102113.
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