Single-cell RNA sequencing reveals cellular heterogeneity in lineage specification of MC38 subcutaneous tumor cells with either SPP1 overexpression or vector control

SPP1 (secreted phosphoprotein 1), also known as osteopontin, has emerged as a pivotal factor in the tumor microenvironment (TME) across various cancers. It has been reported that SPP1-positive macrophages are associated with immunosuppressive activity and poor prognosis in colorectal cancer (CRC). The increasing proportion of SPP1-positive macrophages contributes to immune suppression and immune desert by inhibiting T cell function in liver metastatic lesions. However, the role of SPP1 in colorectal cancer liver metastasis (CRLM) remains incompletely understood. Therefore, the impact of tumor-secreted SPP1 on the CRC microenvironment warrants further investigation. To explore the effect of SPP1 on diverse TME cell populations, we conducted single-cell RNA sequencing on MC38 xenografts. Our results revealed an increase in fibroblasts and a concurrent decrease in T cells following SPP1 overexpression, suggesting that SPP1 promotes cancer-associated fibroblast (CAF) infiltration into the CRC TME. Overall design: First, a stable MC38 cell pool expressing SPP1 or vector was established using lentiviral transduction, and Western blotting was performed to verify the successful expression of SPP1. These cells were then subcutaneously inoculated into C57BL/6 mice for tumor formation. Seven days later, mice in the OE-SPP1 and OE-vector groups were intraperitoneally injected with anti-PD1 (10 mg/kg, twice a week). After two weeks of treatment, the mice were sacrificed, and subcutaneous tumors from one mouse in each group were selected for single-cell RNA sequencing. Finally, single-cell sequencing data analysis was conducted to identify the specific cell populations within the tumor microenvironment whose proportions were altered by OE-SPP1.