Dissecting the diverse phenotypes and pathological impacts of neutrophils in colitis-to-CRC progression

Bui, Triet M

doi:https://doi.org/10.21985/n2-h3y3-xc12

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Dissecting the diverse phenotypes and pathological impacts of neutrophils in colitis-to-CRC progression

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Tissue-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of inflammatory bowel disease (IBD) and colorectal cancer (CRC), where these immune cells can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this thesis dissertation, I aim to (1) establish whether in inflamed colon, PMNs shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy, and (2) define the changes in transcriptomic landscape of these tumor-infiltrating PMNs during the transition from inflammatory ulceration to CRC. For the first component of the dissertation, we leveraged human sporadic CRC biopsies, The Cancer Genome Atlas (TCGA) gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. Here we reveal that PMNs exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, PMN-mediated genotoxicity due to accumulation of DSBs led to the induction of Non-Homologous End-Joining (NHEJ), allowing for survival and growth of advanced CRC. The findings resulting from the first half of this dissertation therefore identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. The work further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibitors of NHEJ. For the second component of the dissertation, bulk transcriptomic analyses of enriched peripheral blood and tissue-infiltrating PMNs effectively stratified them into three distinct clusters representing three spatially segregated anatomical compartments, including peripheral blood, inflamed colon tissue and the CRC tumor niche. Importantly, combinatory analyses of pathway overrepresentation, protein-network mapping, and gene-ontology scoring revealed a robust enrichment of biological pathways involved in vasculature development and angiogenesis uniquely in PMNs associated with the advanced CRC niche. These findings were validated in vivo as we found that temporary PMN depletion profoundly reduced tumor vascularity, vessel branching, and penetration depth of the tumor vessels. Moreover, we identified Spp1 (Osteopontin/OPN) and Mmp14 (MT1-MMP) to be highly induced both at transcript and protein levels in PMNs they left the circulation and entered the tumor niche, where OPN and MMP14 promoted tumor angiogenesis by respectively controlling endothelial cell chemotaxis and formation of branching structures. TCGA data mining and validation in UC/CRC patient cohorts further confirmed significant upregulation of Spp1 and Mmp14 in high-grade CRC, but not in UC patients. Importantly, in vivo inhibition studies using small-molecule allosteric inhibitor of MMP14 in AOM/DSS-driven models led to tumor vasculature disruption and profound CRC regression. In conclusion, the latter component of this dissertation identified a CRC niche-specific compartmentalization of PMN transcriptional programing, highlighting the novel PMN plasticity. Our findings further established the contribution of CRC-associated PMNs via OPN/MMP14 activity to the formation of a complex tumor vasculature and subsequent neoplastic progression from inflammatory colitis to advanced CRC.

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