Spatially resolved endothelial signaling via nampt-itga5 drives immune evasion in stem-like gastric cancer

BackgroundPromising development in immune checkpoint blockade (ICB) therapy has shown remarkable results in the treatment of gastric cancer (GC). However, the objective response rate in GC remains unsatisfactory. Noninvasive imaging to predict responses to ICB therapy via tumor microenvironment (TME) assessment is needed. Accordingly, this study aimed to evaluate the role of 68Ga-FAPI-04 PET/CT in the assessment of the immunosuppressive TME in GC and to cross-correlate imaging findings with responses to ICB therapy.MethodsThe correlation between fibroblast-activation-protein (FAP) expression and immunosuppressive cell infiltration was analyzed using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) database, and GC tissue microarrays. To characterize the TME, TMEscores were calculated based on RNA-seq data from four GC patients. A total of 21 patients with GC underwent 68Ga-FAPI-04 PET/CT before ICB treatment, and two of them were imaged after ICB therapy.ResultsFAP expression was found to be closely correlated with poor prognosis and infiltration of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs), exhausted T cells, and regulatory T cells (Tregs) in GC. We also found a strong relationship (R 2 = 0.9678, p = 0.0162) between 68Ga-FAPI-04 uptake and TMEscore. Further analyses indicated that high 68Ga-FAPI-04 uptake was correlated with reduced therapeutic benefits from ICB therapy.Conclusions 68Ga-FAPI-04 PET/CT may be used to noninvasively image the cancer-associated fibroblasts immunosuppressive TME in vivo and also potentially serve as a predictive biomarker of survival and antitumor immune response among patients who received ICB therapies.

Purpose: Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer mortality worldwide. The range of therapeutic strategies available for the treatment of GC has improved in recent decades. However, the prognosis of patients with advanced GC remains poor even after curative resection, mainly because of recurrence, such as peritoneal dissemination and liver metastases. Previous studies have shown that the interaction between cancer cells and their surrounding stroma plays an important role in tumor development. Cancer-associated fibroblasts (CAFs) have been reported to be involved in invasion and metastasis in cancers, including gastric cancer (GC), via their stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying the tumor-promoting effects and the potential of developing therapeutic targets using CXCL12/CXCR4 signaling activated by CAFs are not fully understood. Experimental Design: We first analyzed CXCL12 expression in resected GC tissues of 110 patients by immunohistochemistry (IHC). We next established primary normal fibroblasts (NFs) and CAFs from GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. Furthermore, we evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. Results: We determined that high CXCL12 expression was significantly associated with larger tumor size, deeper tumor depth, lymphatic invasion and poor prognosis in GC. We established new primary CAFs and their adjacent NFs from resected stomach tissue in GC patients. We evaluated the change in mobility during the direct co-culture of GC cells and fibroblasts using real-time imaging. The motility of GC cells significantly increased upon direct co-culture with CAFs more than it did in cells with NFs compared with control RPMI during observation for 24 hours. The motility of GC cells with CAFs was effectively suppressed by AMD 3100 compared to GC cells with NFs. CXCL12/CXCR4 activation by CAFs mediated the clustering of integrin β1 on the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling compared with PF-573,228. Conclusions: These results suggest that CXCL12 derived from CAFs promotes invasive ability by enhancing the clustering of integrin β1 in GC cells and results in GC progression. The current data provide evidence that CXCL12/CXCR4 inhibition by AMD3100 is more efficient in suppressing GC cell invasion by not only inhibiting intracellular signaling but also by attenuating the interaction between cancer cells and the extracellular matrix compared with PF-573,228. Our present study represents the rationale for establishing a therapeutic strategy targeting CXCL12/CXCR4 signaling. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hidetaka Sugihara, Eto Kojiro, Hiroshi Sawayama, Keisuke Miyake, Yuki Kiyozumi, Keisuke Kosumi, Ryuma Tokunaga, Kazuto Harada, Junji Kurashige, Masaaki Iwatsuki, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 423. doi:10.1158/1538-7445.AM2015-423

Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, but its clinical benefit is limited in advanced gastric cancer (GC). Cancer-associated fibroblasts (CAFs) have been reported to be associated with ICB resistance, but the underlying mechanism has not been fully elucidated. Our previous single-cell RNA-seq analysis of GC revealed that POSTN+FAP+ extracellular matrix CAFs (eCAFs) communicate with macrophages. Here, we evaluated the correlation between eCAFs and ICB response in TCGA-STAD and real-world cohorts. Immune infiltration analysis and correlation analysis were performed to assess the relationship between eCAFs and macrophages. We first confirmed a negative correlation between the abundance of eCAFs and the overall response rate (ORR) to anti-PD-1 treatment in TCGA-STAD and real-world GC cohorts. Overexpression of POSTN in CAFs enhanced macrophage chemotaxis, while POSTN interference showed the opposite effect in vitro and in vivo. Furthermore, the cell density of POSTN+ CAFs was positively correlated with the infiltration level of CD163+ macrophages in GC patient tissues. The results demonstrated that POSTN secreted by CAFs enhances macrophage chemotaxis by activating the Akt signaling pathway in macrophages. Additionally, we found that POSTN+FAP+ eCAFs may exist in multiple solid tumors and are associated with ICB resistance. eCAFs promote macrophage chemotaxis through the secretion of POSTN, thereby leading to ICB resistance. High expression of POSTN is likely to predict a poor response to ICB. POSTN downregulation may be considered as a candidate therapeutic strategy to improve ICB efficacy.

Regulation of Gastric Carcinogenesis by InflammatoryCytokines.

Cancer-associated fibroblasts (CAFs) are an important part of tumour microenvironment, but its role in immunotherapy of gastric cancer (GC) is still needed to further study. In this study, we firstly distinguish the GC related CAFs via single cell sequencing dataset. CAFs in deep layers of GC tissues gain more developmental potential. Moreover, we found Glypican-3 (GPC3) is up-regulated in the CAFs subgroups of the advanced GC and correlated with poor prognosis in GC patients. In addition, higher GPC3 expression GC patients have higher TIDE (Tumour Immune Dysfunction and Exclusion) score, dysfunction and exclusion score. independent GC cohort also show GC patients with GPC3high CAFs have lower response rate to PD-1 therapy. GPC3 secreted from CAFs up-regulated PD-L1, TIM3, CD24, CYCLIN D1, cMYC and PDK mRNA expression level in HGC-27 cells. At last, in vivo model demonstrate that targeting GPC3high CAFs sensitizing the PD-1 blockage therapy in GC. In conclusion, GPC3 expression in CAFs is a critical prognostic biomarker, and targeting GPC3high cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in GC. Key messages Glypican-3 (GPC3) is up-regulated in the CAFs subgroups of the advanced gastric cancer. Gastric cancer patients with GPC3high CAFs have lower response rate to PD-1 therapy. Targeting GPC3high CAFs sensitizing the PD-1 blockage therapy in gastric cancer.

Gastric cancer (GC) remains a significant cause of cancer-related mortality worldwide. Despite the transformative impact of immune checkpoint blockade (ICB) therapies across various cancers, only a minority of patients with metastatic GC (mGC) benefit, emphasizing the urgent need for precise biomarkers to predict therapeutic responses and optimize patient selection. In this multi-omics study, we conducted whole exome and transcriptome sequencing on 12 tumors from mGC patients treated with nivolumab as first-line therapy. To validate our findings, we performed whole transcriptome sequencing on 17 additional tumors and analyzed 45 tumors from public dataset (PRJEB25780) of patients who received ICB therapy as second or third-line treatment. Comprehensive multi-omics analyses were conducted using single-cell RNA sequencing (n = 5, GSE167297) and spatial transcriptome sequencing (n = 2, independent internal dataset). ICB-sensitive tumors exhibited robust activation of the interferon response pathway, while ICB-resistant tumors displayed epithelial-mesenchymal transition signatures. Intriguingly, at the single-cell level, genes associated with ICB sensitivity were predominantly expressed in immune cells, whereas genes associated with resistance were primarily found in cancer-associated fibroblasts (CAFs), particularly the desmoplastic CAF (dCAF) subtype. We identified DCN as a hallmark dCAF marker, and high DCN expression was inversely correlated with PD-L1 levels, ICB resistance, and poor prognosis in mGC (log-rank p = 0.027). This study elucidates the critical influence of the tumor microenvironment, specifically dCAFs, in mediating ICB resistance in mGC. Our findings highlight DCN as a representative marker for dCAF and a promising negative predictive biomarker for ICB response. These findings highlight the complex stromal-immune interactions and open avenues for personalized treatment for mGC.

Background: Cancer-associated fibroblasts (CAFs) are principal constituents of the tumor microenvironment (TME) and play a critical role in tumor progression. The CXCL12/CXCR4 axis regulates multiple facets of the TME. The aim of this study was to determine the relationship between CXCL12 expression in CAFs and the malignant progression of gastric cancer (GC).Methods: In the GEO (Gene Expression Omnibus) database, we performed transcriptome analysis on paired gastric cancer RNA sequencing samples, and scRNA analysis was performed on advanced malignant GC samples from the scRNA sequencing data set. Fibroblast cells were co-cultured with GC cells, and invasion, migration, epithelial-mesenchymal transformation (EMT) were determined. After blocking the expression of fibroblast CXCL12, cells were co-cultured with a GC cell line. Detection of GC cell line invasion, migration, EMT and CXCR4, Wnt5a and β-Catenin expression levels was performed. Primary CAFs and gastric normal fibroblasts were isolated and CXCL12 mRNA and protein expression were determined. In addition, a cohort of 285 GC cases was established, protein expression was evaluated immunohistochemically, and prognostic results were analyzed.Results: GC transcriptome analysis suggested that cytokine-cytokine receptor interaction and the Wnt signaling pathway in GC tissues were significantly up-regulated. scRNA analysis of advanced malignant GC samples showed that severe intestinal metaplasia (SIM) in GC specimens of different malignant grades had obvious fibroblast clusters compared to non-atrophic gastritis (NAG) and early gastric cancer (EGC). In the SIM group, fibroblast cluster, CXCL12, CXCR4, and Wnt5a were overexpressed. Co-culturing with fibroblast cells significantly increased the invasion, migration, and EMT of GC cells, and blocking CXCL12 in CAFs disturbed the expression of Wnt5a and β-catenin. In our cohort of GC patients, high CXCL12 expression in CAFs significantly correlated with histological grade (P = 0.012) and TNM stage (P = 0.014), as well as with poor overall survival (p = 0.0107).Conclusion: High expression of CXCL12 in CAFs in a GC microenvironment can affect the migration, invasion, and EMT of GC cells. Furthermore, it can cause poor prognosis in patients with GC.

Objectives: Gastric cancer is one of the most threatening malignancies with a high incidence and metastasis rate. Many patients are diagnosed in an advanced stage sometimes with multi-organ metastasis, which leads a poor prognosis of gastric tumors. Chemokine and their receptors have been originally demonstrated as essential mediators of leukocyte directional migration, particularly during infection and inflammation, and have further emerged as crucial players in all stages of tumor development. It has been reported that an important CXCL12-CXCR4 signaling is involved in tumor development and metastasis in several types of cancer including gastric cancer. Cancer associated fibroblasts (CAF) are reported to communicate microenvironment-derived signals through chemokine/chemokine receptor interaction such as CXCL12-CXCR4 signaling. The aim of our study was to evaluate the role of CXCL12-CXCR4 signaling in crosstalk between gastric cancer and CAF. Methods and Results: We evaluated CXCL12 and CXCR4 expression status by immunohistochemistry using a nonbiased database of 89 curatively resected gastric cancers. As a result, CXCL12/ CXCR4 expression are significant correlation with metastasis. Furthermore, we performed Western blotting analysis for CXCL12 and CXCR4 in human gastric cancer cell lines. CXCR4 expression was detectable in all gastric cancer cell lines. We isolated CAF from human gastric cancer tissue resected in our institute. We performed Western blotting analysis for alpha-SMA and CXCL12 in CAF, and confirmed higher expression in CAF than normal fibroblast (NF) isolated from normal gastric mucosa of same patients. Then we established stable AGS gastric cancer cell line expressing GFP. Invasion assay revealed that AGS (gastric cancer cell line) cells with CAF showed more invasive phenotype than that without CAF. We also found the motility of GFP-expressing AGS was elevated during direct co-culture of GFP-expressing AGS cells and CAF compared with NF. Conclusions: These findings revealed that cancer associated fibroblasts were implicated in cancer cell invasion and metastasis through CXCL12-CXCR4 signaling in gastric cancer. The therapeutic blockade of this pathway with CXCR4 antagonist might abrogate gastric cancer cell invasion and metastasis. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hietaka Sugihara, Hiroshi Sawayama, Ryuichi Karashima, Satoshi Ida, Yu Imamura, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Cancer associated fibroblasts stimulates cancer cell invasion through CXCL12-CXCR4 signaling in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 185. doi:10.1158/1538-7445.AM2014-185

Peritoneal metastasis (PM) is most frequent in gastric cancer (GC) and cancer-associated fibroblasts (CAFs) play a critical role in this process. However, the concrete mechanism of crosstalk between CAFs and cancer cells in PM of GC remains unclear. Microarray sequencing of GC focus and PM lesions was performed, and biglycan (BGN) was screened for further study. Clinically, BGN expression was higher in GC tissues than adjacent normal tissues, and high expression correlated with poor prognosis. In vitro experiments demonstrated that BGN promoted tumor progression and the transformation of mesothelial cells (MCs) into cancer-associated fibroblasts like cells (CAFLCs). In turn, CAFLCs-derived fibroblast activation protein (FAP) facilitated the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of GC cells. GC-derived BGN combined with toll like receptor 2 (TLR2)/TLR4 on MCs to activate the NF-κB pathway and promote the transformation of MCs into CAFLCs by the recovery experiment, coimmunoprecipitation assay, nuclear and cytoplasmic protein extraction assay. CAFLCs-derived FAP could activate the JAK2/STAT3 signaling pathway in GC. Finally, activated STAT3 promoted BGN transcription in GC, resulting in a BGN/FAP-STAT3 positive feedback loop. Taken together, mutual interaction between tumor cells and activated MCs mediated by a BGN/FAP-STAT3 positive feedback loop facilitates PM of GC and provides a potential biomarker and therapeutic target for GC metastasis.

Background: Cancer-associated fibroblasts (CAFs) are important modifiers of tumor progression, which can actively communicate with cancer cells through soluble factors. Interleukin-33 (IL-33) is a regulator of immune and inflammatory responses and has also been associated with certain epithelial tumors. Although the importance of CAFs in gastric cancer has been documented in experimental studies, the mechanisms by which CAFs and IL-33 promote gastric cancer remain unclear. Objectives: The aim of this study was to determine the role of IL-33 in the epithelial-stromal interaction in gastric cancer and if CAFs can promote the invasion of gastric cancer cells via ERK pathway through IL-33. Methods: CAFs and normal fibroblasts (NFs) were isolated from gastric cancer tissues and adjacent non-cancerous tissues respectively. The identification of CAFs was detected by immunofluorescence (IF) and qRT-PCR. IL-33 expression in CAFs and gastric cancer cells was detected by qRT-PCR. CAFs was co-cultured with gastric cancer cells in the presence of exogenous TNF-α, IL-1β and/or their corresponding neutralizing antibodies, the expression of IL-33 in CAFs was detected using qRT-PCR. EMT changes and activation of ERK pathway in gastric cancer cells after co-cultured with CAF-conditioned media were detected by qRT-PCR and western blot, and cell invasion assays were performed by using 8 μm transwell chambers. Results: Gastric CAFs expressed more α-SMA, a marker of myofibroblasts, and more fibroblast activation protein (FAP) when compared with patient-matched NFs. In comparison to gastric cancer cells and NFs, IL-33 was highly expressed in CAFs. Upregulation of IL-33 in CAFs can be induced by co-culture with gastric cancer cells or in the presence of TNF-α and IL-1β, while deprivation of TNF-α and IL-1β using their neutralizing antibodies inhibited the upregulation of IL-33. CAFs induced EMT changes of gastric cancer cells, which was demonstrated by decreased expression of E-cadherin and increased expression of Twist1, MMP2, ZEB1 and ZEB2. However, deprivation of IL-33 using a neutralizing antibody impaired the EMT changes of gastric cancer cells. Gastric cancer cells co-cultured with CAFs or CAFs conditioned media showed enhanced ability of invasion than gastric cancer cells alone. However, adding neutralizing IL-33 antibody or IL-33-siRNA-CAFs conditioned media or U0126 into the culture system led to significantly decreased invasion (P < 0.05) of gastric cancer cells. Moreover, IL-33 secreted from CAFs activated ERK pathway in gastric cancer cells, however, the invasion of gastric cancer cells was attenuated when IL-33-ERK pathway was blocked. Conclusions: TNF-α and IL-1β derived from gastric cancer cells can significantly enhance the expression of IL-33 in CAFs, and IL-33 secreted by CAFs promotes gastric cancer invasion via the ERK signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Liping Su, Quan Zhou, Chenchen Wang, Xiongyan Wu, Xiaofeng Wang, Jianfang Li, Zhenggang Zhu, Bingya Liu. IL-33 secreted by cancer-associated fibroblasts mediates epithelial-stromal interactions and promotes gastric cancer invasion via ERK signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5069. doi:10.1158/1538-7445.AM2015-5069

Chemoresistance induced by cancer-associated fibroblasts (CAFs) is well recognized, yet its mechanisms remain unclear and no CAF-targeted therapies are available. Gastric cancer (GC) with extensive CAF infiltration correlates with poor prognosis, and emerging evidence highlights the GAS6/AXL axis in CAF-GC interactions. This study targets GAS6/AXL axis to overcome CAF-mediated chemoresistance and enhance chemotherapy efficacy. We investigated the effect of 9im, a selective AXL inhibitor, on CAF-induced chemoresistance in GC using transwell migration, western blotting, viability assays, flow cytometry, drug efflux assays and in vivo experiments. Transcriptome analysis identified chemoresistance-related genes, and luciferase assays assessed STAT3 binding to the ABCG1 promoter. RNA-ISH analyzed GAS6 + CAFs in GC samples, while public data evaluated the prognostic significance of ABCG1 and CAF markers. GAS6 expression was elevated in CAF. CAF-derived GAS6 promoted GC cell migration and chemoresistance via AXL activation, effects reversed by 9im. STAT3 bound to the ABCG1 promoter, enhancing expression, while STAT3 inhibition reduced ABCG1 levels. 9im restored chemosensitivity in GC models. Clinically, ABCG1 and CAF marker co-expression correlated with poor prognosis. CAF-derived GAS6 induces GC chemoresistance via the AXL/STAT3/ABCG1 pathway. 9im restores chemosensitivity by inhibiting AXL and ABCG1-mediated efflux. AXL inhibitors with chemotherapy may improve GC treatment.

BackgroundEpstein-Barr virus (EBV)-associated gastric cancer (GC) (EBVaGC) is a distinct molecular subtype of GC with a favorable prognosis. However, the exact effects and potential mechanisms of EBV infection on immune...

Gastric cancer (GC) is one of the main causes of cancer death. The tumor microenvironment has a profound effect on inducing tumor growth, metastasis, and immunosuppression. Fibroblast activation protein-α (FAP) is a protein that is usually expressed in fibroblasts, such as cancer-associated fibroblasts, which are major components of the tumor microenvironment. However, the role of FAP in GC progression and treatment is still unknown. In this study, we explored these problems based on GC patient samples and experimental models. We found that high FAP expression was an independent prognosticator of poor survival in GC patients. FAP+ cancer-associated fibroblasts (CAFs) promoted the survival, proliferation, and migration of GC cell lines in vitro. Moreover, they also induced drug resistance of the GC cell lines and inhibited the antitumor functions of T cells in the GC tumor microenvironment. More importantly, we found that targeting FAP+ CAFs substantially enhanced the antitumor effects of immune checkpoint blockades in GC xenograft models. This evidence highly suggested that FAP is a potential prognosticator of GC patients and a target for synergizing with other treatments, especially immune checkpoint blockades in GC.

ObjectiveA significant proportion of patients can not benefit from neoadjuvant chemotherapy (NCT) due to drug resistance. Cancer-associated fibroblasts (CAFs) influence many biological behaviours of tumors, including chemo-resistance. This study aims to explore whether CAFs expressing FAP, CD10, and GPR77 affect the efficacy of NCT and the prognosis of patients with gastric cancer, and its mechanism.MethodsOne hundred seventy-one patients with locally progressive gastric adenocarcinoma who had undergone NCT and radical surgery were collected. Immunohistochemistry was used to detect the expression of FAP, CD10, and GPR77 in CAFs; the EMT markers (N-cadherin, Snail1, and Twist1) and the CSC markers (ALDH1, CD44, and LGR5) in gastric cancer cells. The χ2 test was used to analyze the relationship between the expression of CAF, EMT, and CSC markers and the clinicopathological factors, as well as the relationship between CAF markers and EMT, and CSC markers. Logistic regression and Cox risk regression were used to analyze the relationship between the expression of CAF, EMT, and CSC markers and TRG grading and OS; Kaplan-Meier analysis was used for survival analysis and plotting the curves.ResultsThe expression of CAF markers FAP, CD10, and GPR77 was closely associated with that of EMT markers; FAP and CD10 were closely related to CSC markers. In the univariate analysis of pathological response, CAF markers (FAP, CD10, GPR77), EMT markers (N-cadherin, Snail1, Twist1), and CSC markers (ALDH1, LGR5, CD44), were all closely associated with pathological response (all p < 0.05). Only Twist1 was an independent factor affecting pathological response in multifactorial analysis (p = 0.001). In a univariate analysis of OS, expression of FAP and CD10 in CAF, as well as expression of EMT biomarkers (N-cadherin, Snail1), were significant factors influencing patient prognosis (all p < 0.05). Multifactorial analysis revealed N-cadherin (p = 0.032) and Snail1 (p = 0.028), as independent prognostic factors affecting OS.ConclusionFAP, CD10, and GPR77 labeled CAF subgroup may lead to NCT resistance and poor prognosis by inducing EMT and CSC of gastric cancer cells in locally advanced gastric cancer patients.

Cancer-associated fibroblast (CAF) has emerged as a key contributor to the remodeling of tumor microenvironment through the expression and secretion of extracellular matrix (ECM) proteins, thereby promoting carcinogenesis. However, the precise contribution of ECM proteins from CAFs to gastric carcinogenesis remains poorly understood. In this study, we find that matrilin-3 (MATN3), an upregulated ECM protein associated with poorer prognosis in gastric cancer patients, originates from CAFs in gastric cancer tissues. Ectopic expression of MATN3 in CAFs significantly promotes the invasion of gastric cancer cells, which can be attenuated by neutralizing MATN3 with its antibody. Notably, a portion of MATN3 protein is found to form puncta in gastric cancer tissues ECM. MATN3 undergoes phase separation, which is mediated by its low complexity (LC) and coiled-coil (CC) domains. Moreover, overexpression of MATN3 deleted with either LC or CC in CAFs is unable to promote the invasion of gastric cancer cells, suggesting that LC or CC domain is required for the effect of CAF-secreted MATN3 in gastric cancer cell invasion. Additionally, orthotopic co-injection of gastric cancer cells and CAFs expressing MATN3, but not its ΔLC and ΔCC mutants, leads to enhanced gastric cancer cell invasion in mouse models. Collectively, our works suggest that MATN3 is secreted by CAFs and undergoes phase separation, which promotes gastric cancer invasion.