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Stress correlates with reduced tumor size and burden, VEGF and CCL2 levels, and infiltrating macrophages compared to unstressed mice.

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-β-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.

Neither norepinephrine nor recombinant CCL2 have proliferative effects on B16F10 cells.

Restraint stress increases tumor CD3+/CD4+ T cell infiltration

Neither norepinephrine nor recombinant CCL2 have toxic effects on B16F10 cells.

Stress confers survival in a murine model of melanoma.

Supplementary Table for qPCR sequences

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-β-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.

Simple Summary The survival rates for patients with pancreatic adenocarcinoma are very low. This dismal prognosis is due in part to late detection and early development of metastases, and successful treatments for pancreatic adenocarcinoma are also lacking. One potential method of treatment is immunotherapy, which has been successfully implemented in several cancers. Despite success in other cancer types, there has been little progress in pancreatic adenocarcinoma. To understand these shortcomings, we explore the roles of macrophages and neutrophils, two prominent immune cell types in the pancreatic tumor environment. In this review, we discuss how macrophages and neutrophils lead to the harsh environment that is unique to pancreatic adenocarcinoma. We further explore how these immune cells can impact standard of care therapies and decrease their effectiveness. Macrophages and neutrophils could ultimately be targeted to improve outcomes for patients with pancreatic adenocarcinoma. Abstract 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.

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-1αfl/fl/LysMcre, or HIF-2αfl/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-1αfl/fl/LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2αfl/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.

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.

Tissue oxygenation in the tumor microenvironment serves as a significant parameter in tumor pathophysiology. In fact, normalization of tumor oxygen is associated with inhibition of tumor growth and metastatic capacity. Irregular tumor vessels produced during tumor angiogenesis have low blood perfusion; thus, oxygen transport is decreased in tumors. Tie2-expressing monocytes (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer patients. Due to the hypoxic nature of the tumor microenvironment, we investigated if oxygen regulates the trafficking of these cells into tumors or if oxygen regulates monocyte differentiation into TEMs once inside the tumor proper. To understand the differentiation of F4/80+/Tie2- cells to Tie2-positivity, we orthotopically implanted PyMT breast tumor cells containing particulate lithium octa-n-butoxy-naphthalocyanine (LiNcBuO) into the mammary fat pads of LysM-Cre control and HIF-1αfl/fl/LysM-Cre mice and evaluated tissue oxygenation by electron paramagnetic resonance (EPR), TEM infiltration, tumor angiogenesis, and metastatic potential. Longitudinal monitoring of physiologic oxygen in tumors using EPR demonstrates the significant role of oxygen-driven TEM regulation in tumor angiogenesis and progression. Citation Format: Kayla J. Steinberger, Mary Forget, Xiaokui Mo, Randall Evans, Amy Gross, Leni Moldovan, Andrey Bobko, Valery Khramstov, Clay B. Marsh, Timothy D. Eubank. HIF-1α regulates the Tie2 receptor on Tie2-expressing monocytes in PyMT breast tumors and augments angiogenic function and metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5207.