Deciphering of intra-tumoural heterogeneity and the interplay between metastasis-associated meta-program and myofibroblasts in gastric cancer

Abstract

Background: Gastric cancer (GC) exhibits high heterogeneity that relies on the oncogenic properties of cancer cells and multicellular interactions in the tumour microenvironment. However, the heterogeneity of GC and their molecular characteristics are still largely unexplored.

Methods: We employed single-cell and spatial transcriptomics to comprehensively map the intra-tumoural heterogeneity within GC. Additionally, in vitro experiments, clinical sample analyses, and patient-derived organoid models (PDOs) were conducted to validate the key interaction patterns between tumor cells and stromal cells.

Results: Seven robust meta-programs (MP1–MP7) in GC were defined with distinct biological significance and spatial distributions. MP3 and MP4 were intimately associated with distinct CD8 T cells skewed toward a cytotoxic or exhaustion state, while MP7, characterised by the highest degree of malignancy, harboured an immune lockdown microenvironment around it and spatially associated with myofibroblasts (myCAFs). Notably, we clarified the interplay between the MP7 and myCAFs, where MP7 induces the chemotactic migration of fibroblasts and promoting their transformation into myCAFs via GDF15/TGFBR2, and in turn, myCAFs-derived RSPO3 up-regulates EGR1 to promote the transformation to MP7 in GC cells and human PDOs. Ultimately, the accumulation of myCAFs around MP7 led to fewer infiltration of CD8 T cells, resulting an immune-deprived microenvironment and the diminished efficacy of immunotherapy. Additionally, based on the gene expression signatures of MP7 GC cells, we predicted specific drugs and verified more potent inhibitory effects of Taselisib and Lapatinib for MP7 GC cells than conventional drugs at the same concentration.

Conclusion: Taken together, these results deepened the understanding of GC heterogeneity and paved the way for novel therapeutic strategies by targeting MP7 GC cells and their interaction loop with myCAFs in GC treatment.

Keywords

gastric cancer / intra-tumoural heterogeneity / myofibroblasts / tumour immune microenvironment

References

[1] Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: gLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3): 209-249.

[2] Smyth EC, Nilsson M, Grabsch HI, et al. Gastric cancer. Lancet. 2020; 396(10251): 635-648.

[3] Joshi SS, Badgwell BD. Current treatment and recent progress in gastric cancer. CA Cancer J Clin. 2021; 71(3): 264-279.

[4] Li S, Yu W, Xie F, et al. Neoadjuvant therapy with immune checkpoint blockade, antiangiogenesis, and chemotherapy for locally advanced gastric cancer. Nat Commun. 2023; 14(1): 8.

[5] Li C, Tian Y, Zheng Y, et al. Pathologic response of phase III study: perioperative camrelizumab plus rivoceranib and chemotherapy versus chemotherapy for locally advanced gastric cancer (DRAGON IV/CAP 05). J Clin Oncol. 2025; 43(4): 464-474.

[6] Al-Batran SE, Homann N, Pauligk C, et al. Perioperative chemotherapy with fluorouracil plus leucovorin, oxaliplatin, and docetaxel versus fluorouracil or capecitabine plus cisplatin and epirubicin for locally advanced, resectable gastric or gastro-oesophageal junction adenocarcinoma (FLOT4): a randomised. Lancet. 2019; 393(10184): 1948-1957.

[7] Kang YK, Chin K, Chung HC, et al. S-1 plus leucovorin and oxaliplatin versus S-1 plus cisplatin as first-line therapy in patients with advanced gastric cancer (SOLAR): a randomised, open-label, phase 3 trial. Lancet Oncol. 2020; 21(8): 1045-1056.

[8] Wang R, Dang M, Harada K, et al. Single-cell dissection of intratumoral heterogeneity and lineage diversity in metastatic gastric adenocarcinoma. Nat Med. 2021; 27(1): 141-151.

[9] Chen P, Zhao D, Li J, et al. Symbiotic macrophage-glioma cell interactions reveal synthetic lethality in PTEN-null glioma. Cancer Cell. 2019; 35(6): 868-884.

[10] Liao W, Overman MJ, Boutin AT, et al. KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer. Cancer Cell. 2019; 35(4): 559-572.e7.

[11] Ye Z, Zeng D, Zhou R, et al. Tumor microenvironment evaluation for gastrointestinal cancer in the era of immunotherapy and machine learning. Front Immunol. 2022; 13: 819807.

[12] Marusyk A, Almendro V, Polyak K. Intra-tumour heterogeneity: a looking glass for cancer?. Nat Rev Cancer. 2012; 12(5): 323-334.

[13] Gonzalez H, Hagerling C, Werb Z. Roles of the immune system in cancer: from tumor initiation to metastatic progression. Genes Dev. 2018; 32(19-20): 1267-1284.

[14] Sahai E, Astsaturov I, Cukierman E, et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer. 2020; 20(3): 174-186.

[15] Ma Q, Long W, Xing C, et al. Cancer stem cells and immunosuppressive microenvironment in glioma. Front Immunol. 2018; 9: 2924.

[16] Raghavan S, Mehta P, Xie Y, et al. Ovarian cancer stem cells and macrophages reciprocally interact through the WNT pathway to promote pro-tumoral and malignant phenotypes in 3D engineered microenvironments. J Immunother Cancer. 2019; 7(1): 190.

[17] Caputo S, Grioni M, Brambillasca CS, et al. Galectin-3 in prostate cancer stem-like cells is immunosuppressive and drives early metastasis. Front Immunol. 2020; 11: 1820.

[18] Park DJ, Sung PS, Kim JH, et al. EpCAM-high liver cancer stem cells resist natural killer cell-mediated cytotoxicity by upregulating CEACAM1. J Immunother Cancer. 2020; 8(1): e000301.

[19] Bayik D, Lathia JD. Cancer stem cell-immune cell crosstalk in tumour progression. Nat Rev Cancer. 2021; 21(8): 526.

[20] Yuan S, Norgard RJ, Stanger BZ. Cellular plasticity in cancer. Cancer Discov. 2019; 9(7): 837.

[21] Wang G, Lu X, Dey P, et al. Targeting YAP-dependent MDSC infiltration impairs tumor progression. Cancer Discov. 2015; 6(1): 80.

[22] Fu K, Hui B, Wang Q, et al. Single-cell RNA sequencing of immune cells in gastric cancer patients. Aging. 2020; 12(3): 2747-2763.

[23] Zhang M, Hu S, Min M, et al. Dissecting transcriptional heterogeneity in primary gastric adenocarcinoma by single cell RNA sequencing. Gut. 2021; 70(3): 464-475.

[24] Zhang P, Yang M, Zhang Y, et al. Dissecting the single-cell transcriptome network underlying gastric premalignant lesions and early gastric cancer. Cell Rep. 2019; 27(6): 1934-1947.

[25] Suvà ML, Tirosh I. Single-cell RNA sequencing in cancer: lessons learned and emerging challenges. Mol Cell. 2019; 75(1): 7-12.

[26] Gavish A, Tyler M, Greenwald AC, et al. Hallmarks of transcriptional intratumour heterogeneity across a thousand tumours. Nature. 2023; 618(7965): 598-606.

[27] Fan J, Lu F, Qin T, et al. Multiomic analysis of cervical squamous cell carcinoma identifies cellular ecosystems with biological and clinical relevance. Nat Genet. 2023; 55(12): 2175-2188.

[28] Liu B, Li S, Cheng Y, et al. Distinctive multicellular immunosuppressive hubs confer different intervention strategies for left- and right-sided colon cancers. Cell Rep Med. 2024; 5(6): 101589.

[29] Guo D, Sheng K, Zhang Q, et al. Single-cell transcriptomic analysis reveals the landscape of epithelial-mesenchymal transition molecular heterogeneity in esophageal squamous cell carcinoma. Cancer Lett. 2024; 587: 216723.

[30] Guo DZ, Zhang X, Zhang SQ, et al. Single-cell tumor heterogeneity landscape of hepatocellular carcinoma: unraveling the pro-metastatic subtype and its interaction loop with fibroblasts. Mol Cancer. 2024; 23(1): 157.

[31] Tirosh I, Izar B, Prakadan SM, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016; 352(6282): 189-196.

[32] Rambow F, Rogiers A, Marin-Bejar O, et al. Toward minimal residual disease-directed therapy in melanoma. Cell. 2018; 174(4): 843-855.

[33] Jiang Y, Yang J, Liang R, et al. Single-cell RNA sequencing highlights intratumor heterogeneity and intercellular network featured in adamantinomatous craniopharyngioma. Sci Adv. 2023; 9(15): eadc8933.

[34] Li R, Ferdinand JR, Loudon KW, et al. Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer. Cancer Cell. 2022; 40(12): 1583-1599.e10.

[35] Baran Y, Bercovich A, Sebe-Pedros A, et al. MetaCell: analysis of single-cell RNA-seq data using K-nn graph partitions. Genome Biol. 2019; 20(1): 206.

[36] Cable DM, Murray E, Zou LS, et al. Robust decomposition of cell type mixtures in spatial transcriptomics. Nat Biotechnol. 2022; 40(4): 517-526.

[37] Chu T, Wang Z, Pe'er D, Danko CG. Cell type and gene expression deconvolution with BayesPrism enables bayesian integrative analysis across bulk and single-cell RNA sequencing in oncology. Nat Cancer. 2022; 3(4): 505.

[38] Fustero-Torre C, Jiménez-Santos MJ, García-Martín S, et al. Beyondcell: targeting cancer therapeutic heterogeneity in single-cell RNA-seq data. Genome Med. 2021; 13(1): 187.

[39] Sathe A, Grimes SM, Lau BT, et al. Single cell genomic characterization reveals the cellular reprogramming of the gastric tumor microenvironment. Clin Cancer Res. 2020; 26(11): 2640.

[40] Tan Z, Pan K, Sun M, et al. CCKBR+ cancer cells contribute to the intratumor heterogeneity of gastric cancer and confer sensitivity to FOXO inhibition. Cell Death Differ. 2024; 31(10): 1302-1317.

[41] Cheng X, Dai E, Wu J, et al. Atlas of metastatic gastric cancer links ferroptosis to disease progression and immunotherapy response. Gastroenterology. 2024; 167(7): 1345-1357.

[42] Wang H, Deng G, Ai M, et al. Hsp90ab1 stabilizes LRP5 to promote epithelial-mesenchymal transition via activating of AKT and wnt/β-catenin signaling pathways in gastric cancer progression. Oncogene. 2019; 38(9): 1489-1507.

[43] Li Y, Liu C, Zhang X, et al. CCT5 induces epithelial-mesenchymal transition to promote gastric cancer lymph node metastasis by activating the wnt/β-catenin signalling pathway. Br J Cancer. 2022; 126(12): 1684-1694.

[44] Tseng YC, Tsai YH, Tseng MJ, et al. Notch2-induced COX-2 expression enhancing gastric cancer progression. Mol Carcinog. 2012; 51(12): 939-951.

[45] Zhong F, Lin Y, Zhao L, et al. Reshaping the tumour immune microenvironment in solid tumours via tumour cell and immune cell DNA methylation: from mechanisms to therapeutics. Br J Cancer. 2023; 129(1): 24-37.

[46] Yang J, Zhang N, Luo T, et al. TCellSI: a novel method for T cell state assessment and its applications in immune environment prediction. iMeta. 2024; 3(5): e231.

[47] Grinshpun A, Ren S, Graham N, et al. Phase ib dose-escalation trial of taselisib (GDC-0032) in combination with HER2-directed therapies in patients with advanced HER2+ breast cancer. ESMO Open. 2024; 9(6): 103465.

[48] Krop IE, Jegede OA, Grilley-Olson JE, et al. Phase II study of taselisib in PIK3CA -mutated solid tumors other than breast and squamous lung cancer: results from the NCI-MATCH ECOG-ACRIN trial (EAY131) subprotocol I. JCO Precis Oncol. 2022(6): e2100424.

[49] Smyth EC, Rowley S, Cafferty FH, et al. Safety and efficacy of the addition of lapatinib to perioperative chemotherapy for resectable HER2-positive gastroesophageal adenocarcinoma: a randomized phase 2 clinical trial. JAMA Oncol. 2019; 5(8): 1181.

[50] Kim ST, Banks KC, Pectasides E, et al. Impact of genomic alterations on lapatinib treatment outcome and cell-free genomic landscape during HER2 therapy in HER2+ gastric cancer patients. Ann Oncol. 2018; 29(4): 1037-1048.

[51] Xu J, Yu B, Wang F, Yang J. Single-cell RNA sequencing to map tumor heterogeneity in gastric carcinogenesis paving roads to individualized therapy. Cancer Immunol Immunother. 2024; 73(11): 233.

[52] Yasuda T, Wang YA. Gastric cancer immunosuppressive microenvironment heterogeneity: implications for therapy development. Trends Cancer. 2024; 10(7): 627-642.

[53] Luo J, Cheng K, Ji X, et al. Anlotinib enhanced CD8+ T cell infiltration via induction of CCL5 improves the efficacy of PD-1/PD-L1 blockade therapy in lung cancer. Cancer Lett. 2024; 591: 216892.

[54] Jacobs C, Shah S, Lu WC, et al. HSF1 inhibits antitumor immune activity in breast cancer by suppressing CCL5 to block CD8+ T-cell recruitment. Cancer Res. 2024; 84(2): 276-290.

[55] Wang T, Wei Y, Tian L, et al. C-C motif chemokine ligand 5 (CCL5) levels in gastric cancer patient sera predict occult peritoneal metastasis and a poorer prognosis. Int J Surg. 2016; 32: 136-142.

[56] Sugasawa H, Ichikura T, Kinoshita M, et al. Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination. Int J Cancer. 2008; 122(11): 2535-2541.

[57] Sima AR, Sima HR, Rafatpanah H, et al. Serum chemokine ligand 5 (CCL5/RANTES) level might be utilized as a predictive marker of tumor behavior and disease prognosis in patients with gastric adenocarcinoma. J Gastrointest Cancer. 2014; 45(4): 476-480.

[58] Bademler S, Kılıç B, Üçüncü M, et al. The role of biomarkers in the early diagnosis of gastric cancer: a study on CCR5, CCL5, PDGF, and EphA7. Curr Issues Mol Biol. 2024; 46(9): 10651-10661.

[59] Li Y, Wang H, Wang J, et al. Chemokine receptor 4 expression is correlated with the occurrence and prognosis of gastric cancer. FEBS Open Bio. 2020; 10(6): 1149-1161.

[60] Bertolini G, D'Amico L, Moro M, et al. Microenvironment-modulated metastatic CD133+/CXCR4+/EpCAM- lung cancer-initiating cells sustain tumor dissemination and correlate with poor prognosis. Cancer Res. 2015; 75(17): 3636-3649.

[61] Hermann PC, Huber SL, Herrler T, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 2007; 1(3): 313-323.

[62] Bockorny B, Semenisty V, Macarulla T, et al. BL-8040, a CXCR4 antagonist, in combination with pembrolizumab and chemotherapy for pancreatic cancer: the COMBAT trial. Nat Med. 2020; 26(6): 878-885.

[63] Li T, Liu M, Pei D. Friend or foe, the role of EGR-1 in cancer. Med Oncol. 2020; 37(1): 7.

[64] Zhao J, Li H, Yuan M. EGR1 promotes stemness and predicts a poor outcome of uterine cervical cancer by inducing SOX9 expression. Genes Genomics. 2021; 43(5): 459-470.

[65] Ma Z, Gao X, Shuai Y, et al. EGR1-mediated linc01503 promotes cell cycle progression and tumorigenesis in gastric cancer. Cell Prolif. 2021; 54(1): e12922.

[66] Satoh T, Xu RH, Chung HC, et al. Lapatinib plus paclitaxel versus paclitaxel alone in the second-line treatment of HER2 -amplified advanced gastric cancer in asian populations: tyTAN-a randomized, phase III study. J Clin Oncol. 2014; 32(19): 2039-2049.

[67] Hecht JR, Bang YJ, Qin SK, et al. Lapatinib in combination with capecitabine plus oxaliplatin in human epidermal growth factor receptor 2-positive advanced or metastatic gastric, esophageal, or gastroesophageal adenocarcinoma: tRIO-013/LOGiC-a randomized phase III trial. J Clin Oncol. 2016; 34(5): 443-451.

[68] Dickler MN, Saura C, Richards DA, et al. Phase II study of taselisib (GDC-0032) in combination with fulvestrant in patients with HER2-negative, hormone receptor-positive advanced breast cancer. Clin Cancer Res. 2018; 24(18): 4380-4387.

[69] Dent S, Cortés J, Im YH, et al. Phase III randomized study of taselisib or placebo with fulvestrant in estrogen receptor-positive, PIK3CA-mutant, HER2-negative, advanced breast cancer: the SANDPIPER trial. Ann Oncol. 2021; 32(2): 197-207.

[70] Lopez S, Schwab CL, Cocco E, et al. Taselisib, a selective inhibitor of PIK3CA, is highly effective on PIK3CA-mutated and HER2/neu amplified uterine serous carcinoma in vitro and in vivo. Gynecol Oncol. 2014; 135(2): 312-317.

[71] Xiao Y, Wang Z, Gu M, et al. Cancer-associated fibroblasts: heterogeneity and their role in the tumor immune response. Clin Exp Med. 2024; 24(1): 126.

[72] Fischer AS, Müllerke S, Arnold A, et al. R-spondin/YAP axis promotes gastric oxyntic gland regeneration and helicobacter pylori-associated metaplasia in mice. J Clin Invest. 2022; 132(21): e151363.

[73] Siddiqui JA, Seshacharyulu P, Muniyan S, et al. GDF15 promotes prostate cancer bone metastasis and colonization through osteoblastic CCL2 and RANKL activation. Bone Res. 2022; 10(1): 6.

[74] Melero I, De Miguel Luken M, De Velasco G, et al. Neutralizing GDF-15 can overcome anti-PD-1 and anti-PD-L1 resistance in solid tumours. Nature. 2025; 637(8048): 1218-1227.

RIGHTS & PERMISSIONS

2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.