Dose-Specific Intratumoral GM-CSF Modulates Breast Tumor Oxygenation and Antitumor Immunity

Abstract

GM-CSF has been employed as an adjuvant to cancer immunotherapy with mixed results based on dosage. We previously showed that GM-CSF regulated tumor angiogenesis by stimulating soluble vascular endothelial growth factor (VEGF) receptor-1 from monocytes/macrophages in a dose-dependent manner that neutralized free VEGF, and intratumoral injections of high-dose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic polyoma middle T Ag (PyMT) triple-negative breast cancer (TNBC). In this study, we assessed both immunoregulatory and oxygen-regulatory components of low-dose versus high-dose GM-CSF to compare effects on tumor oxygen, vasculature, and antitumor immunity. We performed intratumoral injections of low-dose GM-CSF or saline controls for 3 wk in FVB/N PyMT TNBC. Low-dose GM-CSF uniquely reduced tumor hypoxia and normalized tumor vasculature by increasing NG2+ pericyte coverage on CD31+ endothelial cells. Priming of "cold," anti-PD1-resistant PyMT tumors with low-dose GM-CSF (hypoxia reduced) sensitized tumors to anti-PD1, whereas high-dose GM-CSF (hypoxia exacerbated) did not. Low-dose GM-CSF reduced hypoxic and inflammatory tumor-associated macrophage (TAM) transcriptional profiles; however, no phenotypic modulation of TAMs or tumor-infiltrating lymphocytes were observed by flow cytometry. In contrast, high-dose GM-CSF priming increased infiltration of TAMs lacking the MHC class IIhi phenotype or immunostimulatory marker expression, indicating an immunosuppressive phenotype under hypoxia. However, in anti-PD1 (programmed cell death 1)-susceptible BALB/c 4T1 tumors (considered hot versus PyMT), high-dose GM-CSF increased MHC class IIhi TAMs and immunostimulatory molecules, suggesting disparate effects of high-dose GM-CSF across PyMT versus 4T1 TNBC models. Our data demonstrate a (to our knowledge) novel role for low-dose GM-CSF in reducing tumor hypoxia for synergy with anti-PD1 and highlight why dosage and setting of GM-CSF in cancer immunotherapy regimens require careful consideration.