Date of Award

January 2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

David D. Bradley

Abstract

CCL2, a chemokine also referred to as monocyte chemoattractant protein 1 (MCP-1) and small inducible cytokine A, is chemotactic for monocytes. CCL2-induced recruitment of CCR2+Ly6C(hi) monocytes increases vascular permeability CCR2+ endothelium aiding the escape and migration of tumor cells. CCL2 is produced by cancers of varied immunogenicity and augments tumor proliferation and metastasis. Elevated CCL2 expression by tumor cells is linked to poor prognosis.We have previously shown that two bacterially derived superantigens, Staphylococcal enterotoxin G and I (SEG/SEI) stimulate large numbers of T cells in an antigen-independent fashion and promote enhanced survival in the poorly immunogenic B16-F10 melanoma model. Conversely, the poorly immunogenic Lewis Lung Carcinoma (LLC) was not responsive to SEG/SEI and SEG/SEI stimulation did not change the time to death in LLC-implanted mice. All these studies were performed in humanized HLA-DQ8 (DQA1*0301 and DQB1*0302) transgenic mice. Here we provide the background mechanistic differences between LLC and B16-F10 that may explain the disparate outcomes. Interestingly, the LLC cells secrete high levels of CCL2 in vitro whereas B16-F10 cells do not, which we analyzed via Flowcytometric Bead Array. We also show elevated levels of CCL2 in MC-38, an immunogenic murine colorectal cancer cell line. We hypothesized that the elevated CCL2 present in the LLC model, but lacking in the B16-F10 model, resulted in the influx of CCR2+Ly6C(hi) monocytes. We have used Crispr Cas9 as our genome editing tool to inhibit the expression of CCL2 and thereby investigate the effects of CCL2 inhibition on LLC tumor proliferation and metastasis. We also provide data to support the successful creation of LLC MCP-1 KO’s using CRISPR-Cas9. These findings, taken in toto, suggest that combinatorial therapies inhibiting CCL2 with the addition of treatments that enhance the tumor-specific response, e.g. anti PD-1 antibodies, may alter the tumor environment of treatment refractory cancers that induce high CCL2 production, like LLC and MC-38, thus allowing a potent anti-tumor and anti-metastatic response and increased survival.

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