ABSTRACT: Background: Intratumoral heterogeneity (ITH) limits the accurate diagnosis and treatment of colorectal cancer (CRC). Currently, diagnoses, treatment decisions, and prognoses for CRC are determined through multiple superficial endoscopic biopsies. However, there is still a lack of understanding regarding the immune microenvironment in samples obtained from multiple biopsy sites. Additionally, CRC cases with positive tumor deposition (TD) are associated with a higher risk of distant metastasis and a worse prognosis. Methods: A total of 11 tissue specimens from three TD-positive CRC patients were annotated to identify the primary tumor (T), the mucosal side (Ti) of primary loci, the serosal side (To) of primary loci, TD, and lymph nodes (LN). For each subregion, we analyzed the transcriptome profile using single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing, Hotspot gene module analysis, and validated our findings with the NanoString ‘PanCancer Progression Panel’. Immunohistochemistry (IHC), Western blotting, Transwell functional experiments, and data from public cohort databases were employed to verify. Results: Cancercells 1/2/4/5, mainly composed of stem-like cells, had a higher risk of metastasis than CancerCells 6/8. CancerCells 6/8 with weak metastatic ability were located only in the Ti side, while CancerCells 1/2/4/5 tumor cells with strong metastatic ability were located in To and metastasis. Bulk sequencing revealed that immune and metastasis-related genes such as SPP1, CD44, and FXYD5 were significantly upregulated in To and/or metastasis than Ti side. Notably, the immune suppression characteristics are more pronounced in To than Ti, which is reflected in the exhaustion of T cell function, more recruitment of tumor-associated macrophages, and reduction of plasma cells. The TD sample had fewer T cells than T and LNm, which present an "immune desert" state. Thus, multiple endoscopic superficial biopsies are limited, and endoscopists and pathologists must consider investigating deeper sites of T, TD, or metastatic LN for the assessment of patient treatment. Furthermore, considering that FXYD5 promotes the expression/stability of the membrane protein CD44, we identified that CancerCells 1/2/4/5 co-expressing FXYD5 and CD44 were more likely to metastasize. Conclusions: This study provides new insights and references for the understanding of the heterogeneity of CRC, as well as for the clinical value of precise diagnosis and future precise treatment provided by in-depth sampling.
