A dual TGF-β-driven immunosuppressive mechanism controls T cell recruitment and expansion in metastatic colorectal cancer

Poor prognosis colorectal cancer (CRC) is characterized by an immune suppressive TGF-β-activated tumor microenvironment (TME). Pharmacological inhibition of TGF-β signaling promotes T cell infiltration and synergizes with immune checkpoint blockade (ICB) to eliminate metastatic disease in experimental models. Through genetic dissection in model systems and analysis of patient datasets, here we uncover a network of genetic programs regulated by TGF-β signaling in liver metastasis. We show that productive immune responses require both continuous recruitment of tumor-specific CD8+ T cells and subsequent clonal expansion in the TME. TGF-β signaling prevents the recruitment of memory CD8+ T cells by enforcing residence in the lymph nodes. In the absence of newly arrived T cells, the response of tumor-resident T cells to immunotherapy is insufficient to eradicate metastases. We further unveil a genetic program induced by TGF-β in macrophages, which characterizes CRC patients with a high risk of developing metastatic disease. TGF-β-activated macrophages prevent the clonal expansion of newly arrived T cells in the TME through the extracellular matrix protein osteopontin. Overall, our findings reveal how TGF-β signaling integrates immune-suppressive responses in the innate and adaptive immune systems to enable metastatic outgrowth. C57BL/6J mice, were purchased from Janvier Labs at six weeks of age, and injected at 7-8 weeks. Sex was matched with the origin of the tumor. Intrasplenic injections were used for liver colonization by the introduction of dissociated organoids (small 3-4 cell clusters) into the portal circulation. Galunisertib (also known as LY2157299) was synthesized in house and prepared as previously described (Tauriello et al, 2018). Galunisertib or vehicle control was administered by oral gavage in a 0.15 ml volume, twice per day, starting 12-15 days after cell injection for metastasis-initiation experiments, unless otherwise indicated. An 800 mg/kg dose per treatment was used for all experiments. For checkpoint immunotherapy or dual immunotherapy, we used rat anti-PD-L1 (10F.9G2; BioXCell BE0101) or rat anti-IgG2b (LTF-2; BioXCell BE0090) isotype-control antibodies. Whole individual metastases were excised from livers ex vivo and mechanically disaggregat¬ed in 1 ml of Trizol (Life Technologies, 15596018) using a tissue disruptor. Disaggregat¬ed tissues in Trizol were stored in -80ºC until processing. For RNA extraction, samples in Trizol were thawed. 200μl of chloroform (Sigma, 372978) was then added and samples were vigorously mixed for 15 seconds. Mixed samples were centrifuged at 12.000 g for 15 minutes at 4ºC, after which a colorless upper phase containing the RNA was transferred into a new Eppendorf, and mixed with one volume of 70% ethanol. The mix was then processed using a PureLink® RNA Mini Kit (Life Technologies, 12183025) following the manufacturer’s specifications. RNA was eluted using distilled water (Invitrogen, 10977-035) and the concentration was determined using the Nanodrop 1000 spectrophotometer (ThermoFisher).