Spatial transcriptomic analysis of colorectal cancer (CRC) tissues has revealed that the organization of the tumor-stroma boundary plays a critical role in determining the efficacy of anti-PD1 immunotherapy. The study, published in Nature Communications, integrates single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (Stereo-seq) data from CRC patients to map the spatial architecture of the tumor microenvironment (TME) and its impact on immunotherapy response.
The research team analyzed surgical tissues from 23 CRC patients, including both mismatch repair-deficient (dMMR) and proficient (pMMR) cases, some of whom received neoadjuvant anti-PD1 antibody treatment. The analysis identified 15 spatial clusters, representing major anatomical structures such as normal epithelia/tumor, smooth muscle, tumor-stroma boundary, stroma, immune aggregates, and low mRNA-enriched regions.
Distinct Tumor-Stroma Boundary Features in dMMR and pMMR CRC
Further investigation revealed that dMMR and pMMR tumors exhibit distinct cellular components and spatial distributions within the tumor-stroma boundary. In pMMR CRC, the tumor-stroma boundary displayed a well-organized, barrier-like structure that clearly separated the tumor region from immune aggregates and stromal regions. Conversely, dMMR tumors showed a less organized structure.
Interestingly, dMMR patients who experienced stable disease (dSD) after anti-PD1 therapy exhibited a well-organized tumor-stroma boundary structure similar to that observed in pMMR tumors, which are generally insensitive to immunotherapy. This suggests that the spatial organization of the tumor-stroma boundary may contribute to immunotherapy resistance in dMMR CRC.
Immune Cell Accumulation in Immunotherapy Responders
In treatment-naïve dMMR patients, immune cell clusters, particularly myeloid cells and T/NK cells, accumulated in close proximity to the tumor-stroma boundary. In contrast, immune cells in pMMR tumors showed a discontinuous distribution, with a tendency to accumulate in the stromal region but not the tumor region. Notably, dMMR patients who achieved partial or complete response (dPR/dCR) to anti-PD1 therapy exhibited higher proportions of cDC1, cDC2, DC_LAMP3, Monocyte_S100A8, CD4_CXCL13, CD4_Tcm, CD4_Treg, CD8_Teff, CD8_Tem and CD8_CXCL13 in the boundary region, compared to pMMR patients.
Further analysis revealed that LAMP3+ dendritic cells (DCs) and CXCL13-expressing T cells tend to accumulate in the tumor-stroma boundary of immunotherapy responders. These immune cells were found to be in close proximity, suggesting potential interactions that promote T cell activation and anti-tumor immunity. Specifically, the expression profiling of PD1-PD-L1 axis confirmed that CD274 was predominantly expressed on DC_LAMP3, while CD4_CXCL13 and CD8_CXCL13 subsets showed higher PDCD1 expression, indicating the potential interactions of DC_LAMP3 towards CD4_CXCL13 and CD8_CXCL13.
Role of CXCL14+ CAFs in Immunotherapy Resistance
To understand the mechanisms underlying immunotherapy resistance in pMMR CRC, the researchers investigated the spatial transcriptomic features of treatment-naïve pMMR and dSD tumors. They identified three distinct immune states within the tumor-stroma boundary, with state 0 being more prevalent in pMMR and dSD tumors. State 0 was characterized by a higher TIDE score, indicating immune dysfunction and exclusion.
Interestingly, fibroblasts were the predominant cell type in state 0. Further analysis identified five cancer-associated fibroblast (CAF) subsets, with CXCL14+ CAFs being more abundant in pMMR and dSD tumors. Functional analysis revealed that CXCL14+ CAFs are involved in extracellular matrix (ECM) organization and structure, suggesting that they contribute to the formation of a physical barrier that prevents T cell infiltration.
IHH/PTCH1 Axis Promotes CXCL14+ CAF Differentiation
Finally, the researchers explored the potential mechanisms driving CAF differentiation into CXCL14+ CAFs. They identified the IHH/PTCH1 signaling axis as a key regulator of CXCL14 expression in CAFs. Tumor cells may promote CXCL14+CAFs via IHH/PTCH1 axis to constrain ICB efficacy in pMMR CRC patients. Treatment of CXCL14+CAFs with IHH recombinant protein resulted in a notable increase in MMP11 release. Furthermore, exposure of CXCL14+CAFs to conditional medium (CM) from pMMR CRC HT29 cells also let to a similar upregulation of MMP11, which could be suppressed by IHH inbitior Vismodegib.
Clinical Implications
These findings highlight the importance of the spatial organization of the tumor-stroma boundary in determining immunotherapy response in CRC. Targeting CXCL14+ CAFs to disrupt the structural barrier in the tumor-stroma boundary may enhance immunotherapy efficacy, particularly in pMMR CRC. Further research is needed to validate these findings and develop novel therapeutic strategies that target the tumor-stroma boundary to improve immunotherapy outcomes in CRC patients.
