Background: Microsatellite unstable colorectal cancers (MSI+ CRCs) expressing PD-L1, respond to anti-PD-1 or anti-PD-L1 checkpoint blockade, whereas microsatellite-stable tumors do not respond the same. Our aim was to examine how the immune landscape relates to different aspects of the CRC's biology, including neoepitope burden. Methods: We used TCGA data to stratify patients based on a cytolytic T-cell activity expression index and correlated immune cytolytic activity (CYT) with mutational, structural, and neoepitope features of each tumor sample. The expression of several immune checkpoints was verified in an independent cohort of 72 CRC patients, relative to their MSI status, using immunohistochemistry and RT-qPCR. Results: CRC exhibits a range of intertumoral cytolytic T-cell activity, with lower cytolytic levels in the tumor, compared to the normal tissue. We separated CRC patients into CYT-high and CYT-low subgroups. High cytolytic activity correlated with increased mutational load in colon tumors, the count of MHC-I/−II classically defined and alternatively defined neoepitopes, high microsatellite instability and deregulated expression of several inhibitory immune checkpoints (VISTA, TIGIT, PD-1, IDO1, CTLA-4, and PD-L1, among others). Many immune checkpoint molecules (IDO1, LAG3, TIGIT, VISTA, PD-1, PD-L1 and CTLA-4) expressed significantly higher in MSI+ CRCs compared to MSS tumors. The expression of Treg markers was also significantly higher in CYT-high tumors. Both individual and simultaneous high levels of CTLA-4 and PD-L1 had a positive effect on the patients' overall survival. On the reverse, simultaneous low expression of both genes led to a significant shift towards negative effect. Assessed globally, CYT-low CRCs contained more recurrent somatic copy number alterations. PD-L1 protein was absent in most samples in the independent cohort and stained lowly in 33% of MSI CRCs. PD-L1+ CRCs stained moderately for CD8 and weakly for FOXP3. CYT-high colon tumors had higher TIL load, whereas CYT-high rectum tumors had higher TAN load compared to their CYT-low counterparts. Conclusions: Overall, we highlight the link between different genetic events and the immune microenvironment in CRC, taking into consideration the status of microsatellite instability. Our data provide further evidence that MSI+ and CYT-high tumors are better candidates for combinatorial checkpoint inhibition.

BackgroundRecently, immune-checkpoint blockade has shown striking clinical results in different cancer patients. However, a significant inter-individual and inter-tumor variability exists among different cancers. The expression of the toxins granzyme A (GZMA) and perforin 1 (PRF1), secreted by effector cytotoxic T cells and natural killer (NK) cells, were recently used as a denominator of the intratumoral immune cytolytic activity (CYT). These levels are significantly elevated upon CD8+ T-cell activation as well as during a productive clinical response against immune-checkpoint blockade therapies. Still, it is not completely understood how different tumors induce and adapt to immune responses.MethodsHere, we calculated the CYT across different cancer types and focused on differences between primary and metastatic tumors. Using data from 10,355, primary tumor resection samples and 2,787 normal samples that we extracted from The Cancer Genome Atlas and Genotype-Tissue Expression project databases, we screened the variation of CYT across 32 different cancer types and 28 different normal tissue types. We correlated the cytolytic levels in each cancer type with the corresponding patient group’s overall survival, the expression of several immune-checkpoint molecules, as well as with the load of tumor-infiltrating lymphocytes (TILs), and tumor-associated neutrophils (TANs) in these tumors.ResultsWe found diverse levels of CYT across different cancer types, with highest levels in kidney, lung, and cervical cancers, and lowest levels in glioma, adrenocortical carcinoma (ACC), and uveal melanoma. GZMA protein was either lowly expressed or absent in at least half of these tumors; whereas PRF1 protein was not detected in almost any of the different tumor types, analyzing tissue microarrays from 20 different tumor types. CYT was significantly higher in metastatic skin melanoma and correlated significantly to the TIL load. In TCGA-ACC, skin melanoma, and bladder cancer, CYT was associated with an improved patient outcome and high levels of both GZMA and PRF1 synergistically affected patient survival in these cancers. In bladder, breast, colon, esophageal, kidney, ovarian, pancreatic, testicular, and thyroid cancers, high CYT was accompanied by upregulation of at least one immune-checkpoint molecule, indicating that similar to melanoma and prostate cancer, immune responses in cytolytic-high tumors elicit immune suppression in the tumor microenvironment.ConclusionOverall, our data highlight the existence of diverse levels of CYT across different cancer types and suggest that along with the existence of complicated associations among various tumor-infiltrated immune cells, it is capable to promote or inhibit the establishment of a permissive tumor microenvironment, depending on the cancer type. High levels of immunosuppression seem to exist in several tumor types.

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.

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