Author ORCID Identifier
Semester
Summer
Date of Graduation
2023
Document Type
Dissertation
Degree Type
PhD
College
School of Medicine
Department
Microbiology, Immunology, and Cell Biology
Committee Chair
Timothy D. Eubank
Committee Member
Benoit Driesschaert
Committee Member
Edwin Wan
Committee Member
Rosana Schafer
Committee Member
Mark Olfert
Committee Member
Werner Geldenhuys
Abstract
GM-CSF has been employed as an adjuvant to cancer immunotherapies in solid tumors due to its ability to drive immune stimulatory macrophages and other myeloid cells that promote anti-tumor responses. However, GM-CSF does not always exert immune stimulatory effects on myeloid cells, and sometimes even drives immune suppressive phenotypes. GM-CSF effects on myeloid cells and the tumor microenvironment (TME) appear to vary across dosages and even cancer types. However, an optimal dosing regimen for GM-CSF has yet to be determined to-date, and this is further complicated by the fact that GM-CSF is often administered via oncolytic viruses or tumor vaccines engineered to overexpress GM-CSF, which makes dosing difficult. One possibility is that GM-CSF effects on macrophages other than immunoregulatory properties has not been considered. Our laboratory previously showed a novel but under-appreciated function for GM-CSF in modulating tumor-associated macrophages (TAM)-mediated angiogenesis and oxygenation which may impact anti-tumor immunity. During times of hypoxia, TAMs, along with tumor cells, are normally producers of pro-angiogenic molecules such as vascular endothelial growth factor (VEGF) that drive dysfunctional tumor angiogenesis. We found that GM-CSF stimulated soluble VEGFR-1 (sVEGFR-1), an anti-angiogenic molecule that binds to and neutralizes VEGF from bioactivity on endothelial cells, from monocytes/macrophages in a dose-dependent manner. Intratumor (IT) injections of highdose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic PyMT triple-negative breast cancer (TNBC) during the “anti-angiogenesis” era. However, it is now known that tumor hypoxia portends poor prognosis in breast and other solid tumors by driving immune suppression and therapeutic resistance. Current efforts in the field focus on reducing tumor hypoxia to improve therapeutic responses. Vessel normalization is an emerging strategy in the field which aims to repair dysfunctional tumor vasculature and enhance oxygen delivery to overcome hypoxia. Thus, we posited that low-dose GM-CSF may stimulate lower levels of TAM sVEGFR-1 that might bind excess VEGF to normalize the tumor vasculature and reduce hypoxia, while also driving immunostimulatory macrophages that promote anti-tumor immune responses for a novel “double-punch” effect. We also wondered whether high-dose GM-CSF may drive immune suppressive macrophage phenotype and reduced treatment efficacy due to exacerbation of hypoxia at this dose. Thus, the goal of this study was to assess and compare the oxygen-regulatory and immune-regulatory components of low-dose vs. high-dose GM-CSF in a mouse model of TNBC to better understand effects of GM-CSF and dosing on the tumor microenvironment. Using orthotopic PyMT tumors, we determined that low-dose IT GM-CSF did in fact reduce tumor hypoxia in vivo and normalized tumor vasculature by increasing pericyte coverage on the tumor vasculature. Priming of PyMT tumors with low-dose GM-CSF (hypoxia reduced) sensitized tumors to anti-programmed cell death-1 (anti-PD1) immune checkpoint blockade (ICB) immunotherapy. These effects were significant because PyMT tumors are notoriously immunologically “cold” (immune deserts) and ICB-resistant. Immunologically, while low-dose GM-CSF reduced hypoxic and inflammatory TAM transcriptional profiles, no phenotypic modulation of TAM or tumor-infiltrating lymphocytes (TIL) were observed by flow cytometry to suggest the primary effect of low-dose GM-CSF was reduction of hypoxia. On the other hand, high-dose GM-CSF primed PyMT tumors (hypoxia exacerbated) did not respond to anti-PD1. We also observed that high-dose GM-CSF priming increased infiltration of TAMs, however they lacked MHCIIhi phenotype or immune stimulatory marker expression despite the association of GM-CSF with driving immune stimulatory myeloid cells. Both the presence of TAM and hypoxia correlate with poor prognosis in breast cancer patients, and our results suggest that high dose GM-CSF drives increased TAM under hypoxia which may negatively affect immunotherapy. We next studied the effects of GM-CSF in the immunologically “hot” (considered “hot” vs. PyMT), anti-PD1-susceptible 4T1 model of TNBC to assess whether GM-CSF immunomodulation differed between tumor types. Interestingly, high-dose GM-CSF increased MHCIIhi TAM and immune stimulatory molecules in 4T1 tumors, suggesting disparate effects of high-dose GM-CSF across PyMT vs. 4T1 TNBC models which may contribute to confounding results across cancer types. These data demonstrate a novel role for low-dose GM-CSF in reducing tumor hypoxia for synergy with anti-PD1 and highlight that both oxygen-regulatory and immune-regulatory effects of GM-CSF require careful consideration in dosage and setting of GMCSF in cancer immunotherapy regimens. Lastly, IT injections of GM-CSF are not always clinically feasible, especially for less-accessible tumors. Here, we also formulated, characterized, and optimized GM-CSF-loaded polymeric nanoparticles intended for systemic but targeted delivery of dose-specific GM-CSF to tumors. Collectively, these data characterize novel effects and delivery modalities for GM-CSF that may improve use and efficacy in cancer immunotherapy.
Recommended Citation
Mihalik, Nicole E., "Localized delivery of GM-CSF in the regulation of breast tumor oxygen and anti-tumor immunity through macrophages" (2023). Graduate Theses, Dissertations, and Problem Reports. 12083.
https://researchrepository.wvu.edu/etd/12083
Embargo Reason
Publication Pending
Movie S2. In vivo tumor oxygen in a low-dose GM-CSF-treated tumor (from Page 77).
MovieS1_SalineTumor_RSEPRIMovie.mov (569 kB)
Movie S1. In vivo tumor oxygen in a saline-treated tumor (from page 76).