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

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.

The Underexplored Landscape of Hypoxia-Inducible Factor 2 Alpha and Potential Roles in Tumor Macrophages: A Review.

Low tissue oxygenation, termed hypoxia, is a characteristic of solid tumors with negative consequences. Tumor-associated macrophages (TAMs) accumulate in hypoxic tumor regions and correlate with worse outcomes in cancer patients across several tumor types. Thus, the molecular mechanism in which macrophages respond to low oxygen tension has been increasingly investigated in the last decade. Hypoxia stabilizes a group of hypoxia-inducible transcription factors (HIFs) reported to drive transcriptional programs involved in cell survival, metabolism, and angiogenesis. Though both tumor macrophage HIF-1α and HIF-2α correlate with unfavorable tumor microenvironments, most research focuses on HIF-1α as the master regulator of hypoxia signaling, because HIF-1α expression was originally identified in several cancer types and correlates with worse outcome in cancer patients. The relative contribution of each HIFα subunit to cell phenotypes is poorly understood especially in TAMs. Once thought to have overlapping roles, recent investigation of macrophage HIF-2α has demonstrated a diverse function from HIF-1α. Little work has been published on the differential role of hypoxia-dependent macrophage HIF-2α when compared to HIF-1α in the context of tumor biology. This review highlights cellular HIF-2α functions and emphasizes the gap in research investigating oxygen-dependent functions of tumor macrophage HIF-2α.

Macrophage and Neutrophil Interactions in the Pancreatic Tumor Microenvironment Drive the Pathogenesis of Pancreatic Cancer.

Despite modest improvements in survival in recent years, pancreatic adenocarcinoma remains a deadly disease with a 5-year survival rate of only 9%. These poor outcomes are driven by failure of early detection, treatment resistance, and propensity for early metastatic spread. Uncovering innovative therapeutic modalities to target the resistance mechanisms that make pancreatic cancer largely incurable are urgently needed. In this review, we discuss the immune composition of pancreatic tumors, including the counterintuitive fact that there is a significant inflammatory immune infiltrate in pancreatic cancer yet anti-tumor mechanisms are subverted and immune behaviors are suppressed. Here, we emphasize how immune cell interactions generate tumor progression and treatment resistance. We narrow in on tumor macrophage (TAM) spatial arrangement, polarity/function, recruitment, and origin to introduce a concept where interactions with tumor neutrophils (TAN) perpetuate the microenvironment. The sequelae of macrophage and neutrophil activities contributes to tumor remodeling, fibrosis, hypoxia, and progression. We also discuss immune mechanisms driving resistance to standard of care modalities. Finally, we describe a cadre of treatment targets, including those intended to overcome TAM and TAN recruitment and function, to circumvent barriers presented by immune infiltration in pancreatic adenocarcinoma.

Hypoxia-Inducible Factor α Subunits Regulate Tie2-Expressing Macrophages That Influence Tumor Oxygen and Perfusion in Murine Breast Cancer.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Stress-induced Norepinephrine Downregulates CCL2 in Macrophages to Suppress Tumor Growth in a Model of Malignant Melanoma.

Psychological stressors have been implicated in the progression of various tumor types. We investigated a role for stress in tumor immune cell chemotaxis in the B16F10 mouse model of malignant melanoma. We exposed female mice to 6-hour periods of restraint stress (RST) for 7 days, then implanted B16F10 malignant melanoma tumor cells and continued the RST paradigm for 14 additional days. We determined serum corticosterone and liver catecholamine concentrations in these mice. To evaluate the tumor microenvironment, we performed IHC and examined cytokine expression profiles using ELISA-based analysis of tumor homogenates. We found that tumors in mice subjected to RST grew significantly slower, had reduced tumor C-C motif ligand 2 (CCL2), and contained fewer F4/80-positive macrophages than tumors from unstressed mice. We observed a concomitant increase in norepinephrine among the RST mice. An in vitro assay confirmed that norepinephrine downregulates CCL2 production in both mouse and human macrophages, and that pretreatment with the pan-ß-adrenergic receptor inhibitor nadolol rescues this activity. Furthermore, RST had no effect on tumor growth in transgenic CCL2-deficient mice. This study suggests that stress reduces malignant melanoma by reducing recruitment of tumor-promoting macrophages by CCL2.

MTI-101 treatment inducing activation of Stim1 and TRPC1 expression is a determinant of response in multiple myeloma.

The emergence of drug resistance continues to be a major hurdle towards improving patient outcomes for the treatment of Multiple Myeloma. MTI-101 is a first-in-class peptidomimetic that binds a CD44/ITGA4 containing complex and triggers necrotic cell death in multiple myeloma cell lines. In this report, we show that acquisition of resistance to MTI-101 correlates with changes in expression of genes predicted to attenuate Ca2+ flux. Consistent with the acquired resistant genotype, MTI-101 treatment induces a rapid and robust increase in intracellular Ca2+ levels in the parental cells; a finding that was attenuated in the acquired drug resistant cell line. Mechanistically, we show that pharmacological inhibition of store operated channels or reduction in the expression of a component of the store operated Ca2+ channel, TRPC1 blocks MTI-101 induced cell death. Importantly, MTI-101 is more potent in specimens obtained from relapsed myeloma patients, suggesting that relapse may occur at a cost for increased sensitivity to Ca2+ overload mediated cell death. Finally, we demonstrate that MTI-101 is synergistic when combined with bortezomib, using both myeloma cell lines and primary myeloma patient specimens. Together, these data continue to support the development of this novel class of compounds for the treatment of relapsed myeloma.