Spatially Resolved Tumor Ecosystem Niches and Cell State Evolution in Gastric Adenocarcinoma

Authors
Haoran Ma^1#, Supriya Srivastava^2#, Taotao Sheng³, Benedict Shi Xiang Lian¹, Shamaine Wei Ting Ho³, Xuewen Ong¹, Su Ting Tay¹, Craig Ryan Cecil Joseph⁴, Jeffrey Huey Yew Lum⁵, Chang Xu¹, Kie Kyon Huang¹, Angie Lay Keng Tan¹, Yeek Teck Goh³, Michelle NG³, Joseph J Zhao^6,7, Joe Yeong^4,8, Wei Peng Yong^7,9,10, Jimmy Bok Yan So^7,9,11,12,13, Raghav Sundar^{1,2,6,7,9,14*}, Patrick Tan^{1,3,9,10,15,16,17*}

# Joint first author
*Joint corresponding and senior author

Affiliations

1. Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore;
2. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;
3. Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore;
4. Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore;
5. Department of Pathology, National University Hospital, Singapore;
6. Yong Loo Lin School of Medicine, National University of Singapore, Singapore;
7. Department of Haematology-Oncology, National University Cancer Institute, Singapore;
8. Bioinformatics Institute, Agency for Science, Technology and Research, Singapore;
9. Singapore Gastric Cancer Consortium, Singapore;
10. Cancer Science Institute of Singapore, National University of Singapore, Singapore;
11. Department of Surgery, University Surgical Cluster, National University Health System, Singapore;
12. Division of Surgical Oncology, National University Cancer Institute, Singapore;
13. NUS Centre for Cancer Research, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;
14. The N.1 Institute for Health, National University of Singapore, Singapore;
15. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;
16. Cellular and Molecular Research, National Cancer Centre, Singapore;
17. Singhealth/Duke-NUS Institute of Precision Medicine, National Heart Centre Singapore, Singapore

Corresponding Authors
Patrick Tan (gmstanp@duke-nus.edu.sg)
Raghav Sundar (mdcragh@nus.edu.sg)

Background
Intra-tumoral heterogeneity (ITH) poses a significant challenge to the prognosis and treatment of Gastric Cancer (GC). However, there exist limited data on spatial ITH in GC.

Materials and Methods
We integrated 1,563 NanoString Digital Spatial Profiler (DSP) regions of interest (ROIs) and 152,423 Single-Cell RNA Sequencing (scRNAseq) profiles across 130 GC samples from 70 individual patients. ITH discovery was based on DSP and scRNAseq data, and then validated on an independent tissue microarray dataset.

Results
Our analysis revealed spatial intratumor subgroups (G1 and G2) within each GC sample, indicative of spatially resolved RNA-ITH. G2 regions exhibited upregulation of tumor pathways associated with aggressive clinical behaviour and an immunosuppressed tumor microenvironment (TME). Immune-exhaustion in G2 regions correlated with increased aneuploidy patterns. Trajectory analysis in DSP and single-cell copy number variation (sCNV) calling identified two distinct within-tumor evolution patterns: branched evolution and diaspora evolution. Diaspora GC displayed a poorer prognosis compared with branched GC and exhibited upregulation of genes related to epithelial-mesenchymal transition (EMT) and hypoxia pathways. SOX9 was identified as a driver of the G2 RNA-ITH subgroup in diaspora GC. Additionally, diaspora GCs showed a unique TME composition, characterized by cell types promoting tumor growth and metastasis, such as VWF+ ACKR1+ endothelial cells and SPP1+ FN1+ TAM cells. Analysis of the tumor-stroma interface (TSI) revealed its unique ecological state.

Conclusion
This study enhances our understanding of the spatial and molecular complexity of GC ITH, providing a valuable resource for future exploration and discovery.