Background: At the invasive front of primary colorectal cancer (CRC), the tumor growth pattern (TGP) holds a critical role in patient outcome. An infiltrative growth pattern (IGP) is a well-established marker of poor prognosis, in contrast to the more favorable pushing growth pattern (PGP). To elucidate the molecular mechanisms underlying this key clinical observation, we integrated transcriptomics and spatial single-cell proteomics investigations of the tumor microenvironment (TME).
Methods: Large-scale bulk RNA sequencing (n = 154) was applied, followed by validation (n = 104) using multiplex immunofluorescence (mIF). An unsupervised machine learning was applied to the cellular neighbourhood clustering analysis.
Results: The transcriptomic analysis revealed that IGP had a significantly higher abundance of Cancer-Associated Fibroblasts (CAFs) and enrichment of Epithelial–Mesenchymal Transition (EMT)- and Extracellular Matrix (ECM)-related pathways. At the spatial single-cell protein level of PGP, we observed a fibrous wall at the tumor boundary in interface CN, constructed by co-localized clusters of CAF-PDPN and CAF-PDPN-SMA-FAP. This barrier likely limits local invasion and suppresses tumor budding, correlating with a more favorable prognosis. In contrast, IGP revealed that CAF-SMA-FAP created a potent immune-exclusive niche, a key mechanism of immune evasion. This was evidenced by a significantly larger spatial exclusion distance from CD3+ T-cells compared to PGP, suggesting a potential mechanism for resistance to immunotherapies.
Conclusion: Different CAF subtypes govern distinct architectures at the tumor invasive front. In PGP, they form a barrier associated with tumor limitation, while in the more aggressive IGP, they establish an immune-exclusive niche that facilitates tumor progression by shielding the tumor from immune attack. This study identifies CAF-SMA-FAP as a potential therapeutic target to disrupt this niche. Further investigations using spatial transcriptomics are in progress to unravel the precise molecular pathways that can be targeted to overcome immune evasion and improve patient outcomes in CRC.
