Inhibiting VIP Receptor Signaling Promotes T Cell Mediated Immune Response in Murine Melanoma Models Öffentlichkeit
Dhamsania, Rohan Ketan (Spring 2020)
Abstract
The most commonly diagnosed cancer in the US is skin cancer. Melanoma accounts for the vast majority of all skin cancer deaths. Current treatment for aggressive and metastatic melanomas includes targeted therapies including BRAF and MEK kinase inhibitors and immune checkpoint blockade therapies including anti-CTLA-4 and anti-PD-1 therapies. However, just 40% of the patients respond to these therapies and 25%-40% of these patients relapse within 3 years of treatment. As a result, it is imperative to explore more rigorous cancer therapies.
Vasoactive Intestinal Peptide (VIP) is a 28 amino acid immunosuppressive neuropeptide. Its potent immunosuppressive effects have been shown in leukemia and CMV mouse models, in which antagonism to VIP (ANT-08) has downregulated expression of PD-1 and PD-L1 in activated T cells and dendritic cells. In addition, ANT-08 has shown to significantly promote T cell proliferation in vitro. Interestingly, according to The Cancer Genome Atlas (TCGA), metastatic melanoma patients with low VIP mRNA expression has better survival then patients with high VIP mRNA expression in the tumor. Thus, we hypothesize that antagonizing VIP receptor signaling would activate the adaptive immune system. We describe herein that T cells from VIP-KO mice have dramatically increased levels of TNF-a and IL-6 and ANT-08 stimulates secretion of these proinflammatory cytokines in wildtype mice. In addition, ANT-08 promotes infiltration of proliferating CD4+ T cells in the murine melanoma tumor microenvironment.
Along with this, VIP causes phosphorylation of CREB, which is downstream to BRAF kinases – a target of several melanoma targeted therapies. It has been shown that VIP receptor antagonists are able to block phosphorylation of CREB. Thus, we hypothesize that antagonizing VIP receptor signaling would activate the adaptive immune system and block phosphorylation of CREB. We describe herein the direct cytostatic effects of ANT-08 on D4M, BRAFv600E mutation positive melanoma model.
Cumulatively, using ANT-08 to activate the adaptive immune system and block phosphorylation of CREB, we hypothesize that ANT-08 will elicit an anti-cancer effects against murine melanoma models. Thus, blocking VIP receptor signaling elicits a new strategy to activate the adaptive immune system and to develop novel immuno-oncology therapy.
Table of Contents
Introduction and Background Information________________________________________1
What is melanoma………………………………………………………………………...2
Staging and Progression of melanoma……………………………………………………3
Current Treatments for melanoma………………………………………………………..5
T Cell Mediated Apoptosis of melanoma cells…………………………………………...7
Melanoma immune surveillance escape…………………………………………………..9
Melanoma current immunotherapies…………………………………………………….11
Challenges for melanoma immunotherapies……………………………………………..14
Current targeted therapies for melanoma and its challenges…………………………….16
Scope of Thesis…………………………………………………………………………..18
Antagonizing VIP receptor signaling pathway..…………………………………………22
Experimental model……………………………………………………………………...23
Methods and Materials________________________________________________________25
Cell lines………………………………………………………………………………….26
Cytostatic assay…………………………………………………………………………..26
Splenocyte Harvestation………………………………………………………………….27
T cell isolation assay..……………………………………………………………………27
T cell proliferation assay…………………………………………………………………28
Bioluminescent imaging of mice…………………………………………………………29
Animal care………………………………………………………………………………29
Tumor transplantation model…………………………………………………………….30
Anti-mouse PD1 treatment……………………………………………………………….30
Murine tissue harvestation………………………………………………………………..31
Immunohistochemistry…………………………………………………………………..31
Co-culture assay………………………………………………………………………….31
Western Blotting…………………………………………………………………………32
VIP Enzyme Immunoassay (EIA) ……………...………………………………………..32
V-PLEX………………………………………………………………………………….33
Flow Cytometry………………………………………………………………………….33
Result______________________________________________________________________35
Murine Melanoma cell lines and tumor tissue does not endogenously secrete VIP……....36
Elevated levels of VIP in serum of B16F10 tumor bearing mice…………………………38
ANT-08 promotes in-vitro proliferation of murine T cells……………………………….40
VIP is secreted by activated T cells……………………………………………………….42
ANT-08 inhibits T cells autocrine VIP receptor signaling……………..…………………43
ANT-08 promotes secretion of TNF-a in enriched T cells from VIP-WT mice…………..45
ANT-08 did not promote secretion of TNF-a in B16F10 tumor bearing mice serum…….47
ANT-08 promotes secretion of IL-6 in enriched T cells from VIP-WT mice……………..48
ANT-08 promotes secretion of IL-6 in VIP-WT mice serum…………………………….50
ANT-08 caused significantly higher proportion of proliferating CD4+ cells in
TILs from B16F10 tumors………………………………………………………………..51
ANT-08 as a monotherapy agent did not yield significant survival
differences in B16F10 melanoma model…………………………………………………52
Anti-PD1 did not improve survival rates in VIP-KO mice bearing B16F10 tumors……...55
D4M and B16F10 cells express VIP receptors…………………………………………...58
ANT-08 has direct cytostatic effects on the growth of D4M cells in vitro…………………59
ANT-08 does not have mono-therapeutic or synergistic effect with anti-PD1
on tumor growth and survival rates against the D4M inoculated tumors in mice…………61
Discussions _________________________________________________________________64
References__________________________________________________________________ 73
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