Transforming growth factor-beta-induced protein as a novel secreted immune check-point in colorectal cancer

We set up a LC/Mass-spectometry (Orbitrap)-based platform to identify the secreted proteome (secretome) in conditioned medium (CM) from fresh tumor and non-tumor surgery samples. By this approach, we selected a multitude of secreted proteins that were upregulated in colorectal cancer (CRC) secretome as compared to the non-tumor, in order to identify those potentially acting as secreted immune check-points (sICs). Their discovery may represent a tremendous resource for tumor specific drug targets, potentially acting as sIC inhibitors in both cold and hot tumors, unlike current IC inhibitors (e.g., IpilumumAb and NivolumAb) causing a partial or no remission in the majority of cold tumors. The transforming growth factor-beta-induced (TGFBI) protein (previously called BIG-H3) was found significantly upregulated in CRC secretome (as compared with the non-tumor). TGFBI is an RGD-containing extracellular matrix protein that binds to type I, II and IV collagens, serves as a ligand recognition sequence for several integrins, and inhibits cell adhesion. Release of TGFBI from primary tumors has been associated with increased tumor proliferation/migration/metastasis, indirectly inhibits adhesion of mononuclear cells by occupancy of various integrins on endothelial cells, but its role as sIC has not been fully investigated. We first validated by Elisa that TGFBI was overexpressed in CM from CRC tissue samples (as compared with non-tumor CM), in serum from CRC patients (as compared with HD sera), and positively correlated with the tumor stage (according to the TNM classification). Interestingly, tissue-IHC and confocal microscopy revealed that TGFBI was overexpressed by tumor cells, T cells, monocytes and plasma cells in tumors in a significantly higher extent than in non-tumor, suggesting a massive involvement of the tumor microenvironment (TME) in secreting it. These data are also confirming at the level of the same cell populations isolated from tumor tissues. Importantly, the recombinant form of TGFBI, as well as the tumor CM containing high levels of native TGFBI, significantly inhibited various functions (IFN- and TNF- production, GZB and T-bet expression…) of anti-CD3/CD28-activated CD4 and CD8 T cells, which could be restored by the addition of the neutralizing anti-TGFBI mAb in vitro. Finally, we are validating that TGFBI can act as a sIC by using human 3D CRC organoids as a surrogate of animal models in vivo. Human 3D-organoids generated from various tumor tissues allow to determine the interaction between tumor and immune system, the response (activation, cytokine production, killing…) by autologous CD8 and CD4 T cells derived from cancer patients, the role of sICs in inhibiting anti-tumor T cell response, the role of related sIC inhibitors in unlashing the anti-tumor T cell response. Human-based models, such as human organoids, can offer effective ways “to accelerate transition to a research system that does not involve testing on animals”, as the European Parliament has recently declared (see go.nature.com/3hzprhj). Anti-tumor immune responses are often unable to clear stabilized tumors due to the presence of various T cell membrane immune checkpoints (mICs) delivering inhibitory signals into tumor-infiltrating lymphocytes (TILs) (i.e., T cell exhaustion). Monoclonal antibodies (mAbs) against mICs (acting as mIC inhibitors [mICIs]) restore anti-tumor responses by TILs leading to a dramatic reduction of several metastatic tumors. This occurs mainly with the so defined “hot tumors” – including melanoma, non-small cell lung cancer, bladder, kidney, head and neck cancer – which are characterized by significant DNA instability, due to the lack of mismatching repair mechanisms, very high mutational burden and thus generating a huge repertoire of mutated (passenger) neoantigens, and a high number of TILs. However, current mICIs (e.g., IpilumumAb and NivolumAb) cause a partial or no remission in the majority of the so-called “cold tumors” expressing a low rate of somatic mutations and, as a consequence, showing low frequencies of TILs. Cold tumors are most breast, ovarian, prostate, pancreatic cancers, glioblastomas, but also the majority of colorectal or hepatocellular carcinomas (CRC or HCC), which are microsatellite stable (MSS) tumors with effective DNA mismatching repair system. In addition to poor T cell infiltration, tumor microenvironment (TEM) in “cold tumors” is characterized by low major histocompatibility complex (MHC) class I and PD-L1 expression, high infiltration of immunosuppressive (IS) cell populations (e.g., tumor-associated macrophages [TAMs], regulatory T cells [Tregs], myeloid-derived suppressor cells [MDSCs], cancer-associated fibroblasts [CAFs]), and high density of secreted IS molecules (e.g., TGF-, IL-10, IL-6, Arginase, VEGF, GM-CSF, Wnts)(3-5). Therefore, a main objective of the scientific community is the development of novel strategies addressed to restore anti-tumor immunosuppression in order to meet the medical need to cure cold tumors (e.g., to convert them into hot tumors).