Simple SummaryThe identification of markers of metastatic disease is critical to improving colorectal cancer survival rates. Activin A plays a critical role in tumor and immune cells in colorectal cancer. Here, we aimed to expand on our previous work to determine how activin A influences tumor cell signaling and immune activation status. Our findings suggest that activin A inhibits the activity of immune cells found in the tumors of colorectal cancer patients while promoting metastasis. Furthermore, we provide data suggesting activin A stimulates a targetable pathway in tumor cells and immune cells leading to metastatic outcomes. This work highlights the application of novel, commercially available imaging techniques to colorectal cancer patient samples to confirm signaling networks driving disease progression. The identification of activin-induced pathways driving metastasis may provide an early target for colorectal cancer to inhibit progression.AbstractWe have shown that activin A (activin), a TGF-β superfamily member, has pro-metastatic effects in colorectal cancer (CRC). In lung cancer, activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. Here, we hypothesized that activin exerts cell-specific effects in the tumor microenvironment (TME) of CRC to promote anti-tumoral activity of immune cells and the pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. We generated an Smad4 epithelial cell specific knockout (Smad4−/−) which was crossed with TS4-Cre mice to identify SMAD-specific changes in CRC. We also performed IHC and digital spatial profiling (DSP) of tissue microarrays (TMAs) obtained from 1055 stage II and III CRC patients in the QUASAR 2 clinical trial. We transfected the CRC cells to reduce their activin production and injected them into mice with intermittent tumor measurements to determine how cancer-derived activin alters tumor growth in vivo. In vivo, Smad4−/− mice displayed elevated colonic activin and pAKT expression and increased mortality. IHC analysis of the TMA samples revealed increased activin was required for TGF-β-associated improved outcomes in CRC. DSP analysis identified that activin co-localization in the stroma was coupled with increases in T-cell exhaustion markers, activation markers of antigen presenting cells (APCs), and effectors of the PI3K/AKT pathway. Activin-stimulated PI3K-dependent CRC transwell migration, and the in vivo loss of activin lead to smaller CRC tumors. Taken together, activin is a targetable, highly context-dependent molecule with effects on CRC growth, migration, and TME immune plasticity.