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Colorectal

Colorectal Poster Presentations 1

P13: Neutrophil extracellular traps are an immunotherapeutic target in colorectal cancer

Thursday, March 21, 2024
9:50am – 9:56am ET
Location: C110
Introduction: Necrosis is a known predictor of adverse outcomes in colorectal cancer (CRC), and is typically thought to be a passive, non-targetable biologic feature. In mouse breast and lung cancer models, neutrophils (polymorphonuclear cells [PMNs]) have been shown to directly cause necrosis via formation of neutrophil extracellular traps (NETs). We evaluated the pathogenic role of NETs in inducing necrosis in the context of CRC.


Methods: Tissue samples were collected from patients undergoing resection of CRC and colorectal liver metastases (CRLM). AKPS (APCKO KRASG12D P53KO SMAD4KO) organoids generated from murine colon epithelium were implanted into the colon of C57BL/6 mice via colonoscopic guidance. Resulting tumors and metastases to liver and lung recapitulate the histopathological progression of human CRC. Immune cells in the blood, tumor, and bone marrow were longitudinally analyzed during tumor progression using flow cytometry. T-cells, PMNs, and NETs were analyzed in tumors by immunofluorescence (IF) and tissue clearing. T-cells were quantified by a pathologist. Tumor-bearing mice were treated with disulfiram to block NET formation. H&E-stained slides were scored for percentage of tumor necrosis. Finally, single cell RNA-sequencing was performed on human CRLM.


Results: Extent of necrosis in human CRC (n=24) significantly correlated with stage (p=0.05) and microsatellite stable disease (p=0.03), independent of tumor size. Notably, PMNs and NETs were abundant in necrotic regions (Fig 1a-b). Necrotic regions had clear disruption of microvasculature integrity and abundance of intravascular NETs on tissue clearing (Fig 1c). In contrast, T-cell abundance inversely correlated with necrosis. Compared to non-necrotic tumors, necrotic tumors demonstrated gene expression signatures known to induce myelopoesis (e.g. CSF1, CXCL2, CXCL12) and pathways related to hypoxia, epithelial-mesenchymal transition, and migration. Tumor-bearing mice demonstrated a shift toward myelopoesis in the bone marrow and higher levels of circulating PMNs during tumor progression, with abundant NETs in necrotic regions of tumors. Blocking NETs decreased necrosis in primary tumors vs. vehicle-treated mice.


Conclusions: Our work suggests that necrosis is associated with increased NET production and adverse clinical predictors in CRC, while blocking NET formation mitigated necrosis. Future work will focus on elucidating the mechanistic and therapeutic impact of NET-targeting strategies, including in combination with immune checkpoint blockade.

Learning Objectives:

  • Upon completion, participant will be able to define what a neutrophil extracellular trap is.
  • Upon completion, participant will be able to describe the pathogenic role of NETs in necrosis.
  • Upon completion, participant will be able to describe the potential role of NETs as an immunotherapuetic target in colorectal cancer.