In this study, we identified subpopulations of seven cell types and addressed their spatial map in three heterogeneous ESCC samples. We integrated scRNA-seq and ST analyses using MIA to evaluate the enrichment score of different cell populations across the cancer and stromal regions, revealing the comprehensive map of various cell types and subsets in the TME. In addition, we evaluated the correlation of distinct cell subpopulations with the stromal and cancer regions in ESCC. The findings revealed heterogeneity between the primary and metastatic sites, providing meaningful biological insights into the mechanisms of ESCC metastasis.
TME is a complex tissue environment for cancer development and progression, which contains various types of cells, including tumour, immune and stromal cells (25). Compared with tumour cells within the TME, stromal cells have genetic stability and are considered a promising therapeutic biomarker for cancer treatment (26). In addition, immune cells within the TME play a key role in tumorigenesis and metastasis and may be important for immunotherapy effectiveness as evidenced by the immunosuppressive mechanisms possibly associated with Treg–macrophage interactions in ESCC (27). Chen et al. used scRNA-seq to show that the TME of ESCC is heterogeneous, demonstrating substantial differences in stromal cells between tumour and normal tissues, which may contribute to carcinogenesis (28). However, the main restrictions for TME in cancer treatment are temporal and spatial differences, which largely contribute to the heterogeneity of cancer (29). To the best of our knowledge, this study is the first to present a spatial landscape of multiple cell subpopulations in ESCC, indicating its intra- and inter-tumoral heterogeneity, which may provide novel insights into the investigation of efficient therapies. MIA revealed that cell subpopulations were heterogeneous in the tumour and stromal subregions among all samples. The number and variety of cells were much greater in the cancer region of the T1 sample and stromal region of the T3 sample.
Stromal cells were more significantly enriched in the TME of ESCC, suggesting their more active role in tumorigenesis and metastasis. A recent study demonstrated that more epithelial cells and fibroblasts were found in ESCC tumour tissues and the adjacent normal tissues, respectively, which is consistent with the results of this study (30). EMT detaches epithelial cells and promotes metastasis and therapeutic resistance, indicating the potential value of stromal cells of TME in the development of novel therapies (31). The inflammatory and remodelling processes are regulated by the interactions between oesophageal epithelial cells and eosinophils, which may drive various tumorigenic phenotypes in ESCC (32, 33). In this study, most subtypes of epithelial cells were more abundant in cancer regions than in stromal regions. Two subsets of epithelial cells were substantially enriched in cancer regions and unrelated to stromal regions, which may be considered powerful indicators for ESCC. CAFs are important for tumorigenesis and are a heterogeneous component within the TME (34). In this study, two CAF subsets—iCAFs and myCAFs—were identified. iCAFs were mostly clustered in the stromal regions, whereas no difference was found in the distribution of myCAFs between the cancer and stromal regions. Fang et al. found that CXCL1 promotes the formation of iCAFs via the CXCR2–pSTAT3 pathway, contributing to the progression of ESCC (35). In addition, IL-6 regulates the interaction between fibroblasts and tumour cells within the TME in ESCC (36). We hypothesised that iCAFs interact with tumour cells through specific factors in the stromal region instead of direct interaction in the tumour region. In addition, some specific pathways enriched in iCAF subpopulations may be candidates for future research, such as ‘NTF3-activated NTRK3 signalling’ and ‘COX reactions’. Non-immune stromal and endothelial cells were heterogeneous among the three ESCC samples in this study and did not have a clear preference for tumour or stromal subregions.
Immune cells within the TME play a key role in cancer progression by interacting with tumour cells by secreting different chemokines, cytokines and other signalling molecules (37). In this study, compared with non-immune cells, immune cell subpopulations were heterogeneous in ESCC, and most subsets were enriched in the cancer region of the T1 sample. TME components can promote EMT, invasion and angiogenesis to facilitate metastasis in various cancers, such as oesophageal (10), pancreatic (38) and breast (39) cancers. In this study, T1 sample was obtained from the primary site of metastatic ESCC, whereas the other two samples were obtained from primary ESCC. The types and proportion of immune cells within the TME in metastatic ESCC were different from those in primary cancer, including naïve CD4 + T cells, neutrophils and B cells, and might affect cancer cells in different ways. Gu et al. showed that tumour-educated B cells secrete HSPA4-targeting IgG and subsequently facilitate the metastasis of breast cancer (40). In addition, neutrophils can interact with L-17-producing γδ T cells and promote metastasis in breast cancer (41). We conducted pseudotime analysis using T1 and N1 samples and presented the spatial features of cancer and stromal regions to determine DEGs and different pathways between the primary and metastatic regions. A recent study used scRNA-seq analysis and revealed differences in cells between the primary and metastatic sites of head and neck cancer (42). In this study, combined with scRNA-seq and spatial information, pathways related to metabolism and tumorigenesis were found to be enriched at metastatic sites, showing the dynamic activity of tumour and stromal cells during metastasis. In addition, patients with higher expression of DEGs had substantially unfavourable clinical outcomes, indicating that these metastatic genes were related to a poor prognosis of ESCC. Therefore, cells within the TME play a significant role in cancer metastasis, which may serve as prognostic predictors and provide new insights into the investigation of effective therapies for ESCC.
Although this study is the first to reveal the spatial features of various cell subsets in ESCC and provide a widely applicable method to comprehensively map an entire tissue using MIA, it has some limitations. First, the number of patients enrolled in this study was limited. Because this was an exploratory and the first study on ESCC in this field, it was inappropriate to enrol more patients. Based on the findings, we believe that more patients can be enrolled for further study, and more distinct TME features in ESCC can be identified. Second, the resolution of ST technology remains to be a shortcoming. The size of the ST array may not be sufficient to cover the whole tissue, and ST arrays do not achieve comparable resolution for every spot at the single-cell scale. In addition, the transcriptomic data can be only accessed within the cells of each spot and are lost at intervals between every two spots. With the development of ST technology, higher resolution and shorter interval distance may be achieved in the future.