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. Author manuscript; available in PMC: 2021 Oct 15.
Published in final edited form as: J Immunol. 2020 Sep 16;205(8):2301–2311. doi: 10.4049/jimmunol.2000185

Hypoxia-Inducible Factor alpha subunits regulate Tie2-expressing macrophages that influence tumor oxygen and perfusion in murine breast cancer

Kayla J Steinberger 1,2,4, Mary A Forget 6,7, Andrey A Bobko 2,3, Nicole E Mihalik 2,3, Marieta Gencheva 1,2, Julie M Roda 6, Sara L Cole 6,8, Xiaokui Mo 6,9, E Hannah Hoblitzell 1,2, Randall Evans 6, Amy C Gross 6, Leni Moldovan 6, Clay B Marsh 2, Valery V Khramtsov 1,2,3,5, Timothy D Eubank 1,2,3,4,#
PMCID: PMC7596922  NIHMSID: NIHMS1622290  PMID: 32938724

Abstract

Tie2-expressing monocytes (TEMs) are a distinct subset of pro-angiogenic monocytes selectively recruited to tumors in breast cancer. Due to 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 fackmacrophages among the mouse groups. In contrast, HIF-1αfl/fl/LysMcre mice had a significantly smaller percentage of tumor TEMs compared to control and HIF-2αfl/fl/LysMcre mice. Pro-angiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of pro-angiogenic 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 EPR 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 pro-hypoxia role for pro-angiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Keywords: angiogenesis, breast cancer, hypoxia, tumor macrophage, Tie2

Introduction

Hypoxia, a hallmark of tumor progression, occurs when the physiological demand for oxygen outweighs supply (1). During hypoxic events, hypoxia-inducible transcription factors alpha (HIF-α) increase expression of genes required for angiogenesis. In the presence of oxygen, the HIF-α subunits are hydroxylated by prolyl hydroxylases (PHD1, −2, and −3) and subsequently tagged for proteasome degradation (2,3). During hypoxia, hydroxylation does not occur and HIF-α stability is maintained allowing enhanced transcription of genes containing hypoxia response elements (HRE) within their promoters (4).

As the master regulator of oxygen homeostasis, HIF-1α drives the expression of genes associated with survival, metabolism and angiogenesis (5,6). Once thought as redundant to HIF-1α function, the understanding of macrophage HIF-2α function is evolving. For example, HIF-2α governs macrophage polarization towards an M2 phenotype (7). We previously reported distinct roles of macrophage HIF-1α and HIF-2α in tumor angiogenesis: macrophage HIF-1α drove VEGF production and angiogenesis, while HIF-2α regulated sVEGFR-1 expression hindering VEGF bioactivity (8–10). Despite the fact that macrophage HIF-2α correlates with poor prognosis in multiple cancer types, little work has been published on the differential role of hypoxia-dependent macrophage HIF-2α in the context of tumor biology (11–14).

Tumor-associated macrophages (TAMs) accumulate in tumors within hypoxic regions15. These TAMs promote angiogenesis and drive tumor progression and metastasis (16,17). In human breast, ovarian, cervical, prostate, and bladder cancers, increased numbers of macrophages correlate with tumor progression and poor prognosis (18). A subpopulation of these mononuclear phagocytes that express an endothelial cell receptor, TIE2, demonstrate enhanced pro-angiogenic activity when compared to TAMs (19). TIE2-expressing monocytes/macrophages (TEMs) facilitate tumor angiogenesis via expression of pro-angiogenic factors as well as enabling the maturation of new vessel sprouts through cell-to-cell interaction (20,21).

Hypoxia augments TIE2 receptor expression on human monocytes in vitro (19), and recently, a correlation has been shown between Tie2 mRNA expression in murine tumor macrophages and HIF1-α stabilization (22). The hypoxic tumor microenvironment (TME) is also believed to drive a more invasive and therapeutic-resistant phenotype in cancer cells (23), indicating oxygenation as a significant parameter of tumor pathophysiology. Magnetic resonance techniques can be used for noninvasive oxygen concentration assessment due to considerable depth of penetration of magnetic fields in living tissue (24). Electron paramagnetic resonance (EPR)-based spectroscopy has naturally high sensitivity to O2 (25) and provides longitudinal, noninvasive measurements of transient tumor oxygen concentration which can be correlated to functional angiogenesis in pre-clinical models.

Because we observed contrasting roles for macrophage HIF-α subunits in tumor angiogenesis, we hypothesized that HIF-1α and HIF-2α regulate Tie2 receptor expression differently on mononuclear phagocyte populations and the hypoxic tumor microenvironment would expand TEMs while presenting different outcomes in mice with macrophage HIF-1α or HIF-2α deficiency. These data support a previously known pro-angiogenic role for tumor TEMs and introduces a previously-unknown and counterintuitive pro-hypoxia role for TEMs in breast cancer that is regulated, in part, by HIF-2α suppression on HIF-1α.

Methods

Chemicals

Lithium octa-n-butoxy 2,3-naphthalocyanine (LiNc-BuO) microcrystals) (4 mg/20 mL), sonicated until homogenous, were incubated with Py8119 cells (PyMT) murine breast cancer cells (ATCC®, CRL-3278™) in F-12K Medium (ATCC®, 30–2004) containing 5% fetal bovine serum (FBS) (HyClone, SH30066.03).

PyMT Tumor Model

All in vivo experiments were done in strict accordance with protocols approved by the Institutional Animal Care and Use Committees at The Ohio State University and West Virginia University. PyMT tumor cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 1% PSA, 10% FBS, 10 μg/ml human insulin, and 5 μg/ml rmEGF. 1×106 cells were injected orthotopically into the number 4 mammary fat pads of 8–12 week-old female C57Bl/6J LysMcre control, HIF-1αfl/flLysMcre, or HIF-2αfl/flLysMcre mice. Alternatively, Py8119 (also C57Bl/6J background PyMT) mesenchymal murine adenocarcinoma cells (ATCC®, CRL-3278™) were cultured in F12 Kaighn’s Modified media and 5% FBS with or without LiNc-BuO crystals and injected as described above.

Electron Paramagnetic Resonance

Real-time tumor oxygen measurements were performed using L-band in vivo EPR spectrometer (L-band, Magnettech, Germany). Mice were anesthetized using air-isoflurane mixture. The surface coil resonator placed onto mammary tumor and the spectrometer tuned. EPR spectra were acquired as single 30-second scans. The instrument settings were: attenuation 15 dB; modulation amplitude, 10 mG; sweep field, 25 mG. The peak-to-peak width of the EPR spectrum was determined by fitting the spectra to the Lorentzian function and used to calculate pO2 using a standard calibration curve. Calibration procedure was performed as previously published (25,26).

Fluorescence Immunohistochemistry (IHC)

Four weeks post-implantation, tumors were sectioned and immunostained with CD31 antibody and imaged by fluorescent microscopy using a 20X objective lens. Five random images per tumor per group were captured in a blinded manner, analyzed for CD31-positivity (red pixels) and quantified using Adobe Photoshop CS2 (Adobe Systems) histogram analysis as previously reported (26).

Five minutes prior to euthanasia, tumor-bearing mice were injected retro-orbitally with Texas red-conjugated dextran (TRD, molecular weight 70,000, ThermoFisher) (20 μg dextran/g mouse weight) or Dylight 594 labeled tomato lectin (Vector Laboratories) (1mg/1mL). After 5 min mice were perfused with PBS intracardially. Upon harvest, tumors were weighed, fixed in 4% paraformaldehyde for 6 hrs, cryoprotected with 20% sucrose, frozen, and sectioned at 20 μm. Tissue sections were stained with the following antibodies: CD31 (1:50, FITC rat anti-mouse, BD Pharmingen 561813), F4/80 (1:100, rabbit anti-mouse, Abcam, ab111101), NG2 (1:100, rabbit anti-mouse, EMD Millipore AB5320) and Tie2 (1:100, mouse anti-human, BD Pharmingen 557039). Detection of Tie2, F4/80, and NG2 was accomplished using Alexa Fluor 488 goat anti-mouse and Alexa Fluor 647 secondary antibodies (1:5000, Invitrogen A11006 and A21245). Images were captured using a Nikon A1R Confocal system with Plan Fluor 40X Oil objective lens. Five to ten images were collected per section with Hamamatsu Orca Flash 4.0 monochrome cMOS camera and processed using ImageJ by thresholding using Triangle function.

Tumor Necrosis

Autofluorescent necrotic tissue within the tumors was evaluated as described (27). Frozen sections were imaged using a MIF Olympus VS120 Slide Scanner at 4X objective using the FITC filter set (Ex 494/Em 519). Images were collected using a Pike 505C VS50 camera then analyzed with Image J by thresholding using the Intermodes function.

Ex vivo Micro-computed Tomography (μCT)

Tumor-bearing mice were intracardially perfused with Microfil injection compounds MV-112 and MV-Diluent (Flow Tech, Carver, MA). Before Microfil perfusion, mice were anesthetized, perfused with PBS then 4% paraformaldehyde. Four days after Microfil perfusion, tumors were excised, embedded in 1% agarose, and scanned using a non–gantry-based SkyScan 1172 μCT system (SkyScan, Kontich, Belgium). Tumors were scanned 360° around the vertical axis, in rotation steps of 0.1° with an AU filter of 0.25 μm. Generated isotropic pixel sizes were 6.6 μm. Stacks were re-constructed using ring reduction and 30% beam hardening correction. Images were thresholded using the Yen function in Image J then visualized using Imaris software.

Docetaxel treatment

5×105 Py8119 tumor cells were implanted into the abdominal mammary fat pads of 8–12-week-old female C57Bl/6J LysMcre control, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice. After one week, the mice were randomized into treatment groups and treated with either vehicle (isotonic saline) or docetaxel (30 mg/kg body weight) (NDC 0409–0201-02, Hospira) one time per week for four weeks. Twice a week, mice were weighed, and tumor growth was determined using calipers and the equation: volume=0.5(shortest dimension2 x longest dimension). After four treatment weeks, the mice were sacrificed, tumors resected and weighed for burden.

Microarray analysis of gene expression

Gene expression changes were analyzed in bone marrow-derived macrophages (BMDMs) (10) from female C57Bl/6J LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice subjected to normoxia (21% O2) and hypoxia (0.5% O2) for 24 hours using Mouse430_2 Affymetrix GeneChips. The mRNA (100 ng) was processed using the Ovation RNA Amplification System V2 and the Encore Biotin Module V2 (both from NuGEN Technologies, Inc). The quantity and quality of the preparations were controlled using Nanodrop (NanoDrop Technologies) and Experion Automated Electrophoresis System (Bio-Rad Laboratories). The arrays were scanned by the GeneChip Scanner 3000 (Affymetrix) using the GeneChip Operating Software (GCOS) Version 1.4.0.036. Partek Genomic Suite was used for data analysis by ANOVA on .cel files imported using robust multichip average with GC content adjustment (GC-RMA) for background correction and normalized by quantile normalization. The Venn diagram represents differentially expressed genes (≤0.5 or ≥2 fold change) among comparisons.

Statistical Analyses

Analysis of variance (ANOVA) with Post-hoc Tukey’s test was used for experiments without repeated measures while repeated measures ANOVA was used for experiments with longitudinal measurements and IHC experiments in which non-consecutive sections from the same tumor were used as an n. Statistical outliers were determined by Grubb’s test. The p-values were adjusted using Holm’s procedure to conserve the family-wise type I error rate at 0.05. Data was analyzed by SAS 9.3 software (SASInc, Cary, NC).

Data Sharing Statement

For original data, please contact tdeubank@hsc.wvu.edu.

Results

HIF-1α-deficiency reduces the TEM population

To examine Tie2 receptor expression in response to tumor hypoxia, we depleted HIF-1α or HIF-2α in mononuclear phagocytes via lysozyme M expression of cre recombinase and loxP sites surrounding HIF-1α or HIF-2α in both alleles (9). We orthotopically injected C57Bl/6J PyMT tumor cells into LysMcre control, HIF-1αfl/fl/LysMcre or HIF-2αfl/fl/LysMcre mice. After four weeks, the tumors were weighed for burden. Tumors from HIF-2αfl/fl/LysMcre mice were significantly larger than tumors from control mice (Figure 1A) (p=0.001). In comparison, tumor burden from macrophage HIF-1α-deficient mice were not significantly different (p=0.1842) when compared to control mice. These data emphasize the impact of HIF-2α in host immune cells, and not tumor cells, as a suppressive force regulating tumor burden in this model.

Figure 1. HIF-1α-deficiency reduces the TEM population.

Figure 1.

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(A) Py8119 tumor cells were injected into a mammary fat pad of female LysMcre control, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice. Tumors from control LysMcre and HIF-1αfl/fl/LysMcre mice were similar in weight at sacrifice while tumors from HIF-2αfl/fl/LysMcre mice were significantly larger (p=0.001). Circles (●) represent each mouse and bar represents the mean tumor volume in each group (n=24,22,24). (B) PyMT tumors were removed, digested to single cell suspension, immunostained for Tie2 receptor and F4/80 (left). Flow cytometric analysis revealed no statistical difference in the percent of total F4/80+ cells (black box) in the tumors. However, there was a significant reduction in the percent of F4/80+/Tie2+ cells (shaded box) in the tumors from the HIF-1αfl/fl/LysMcre mice (p<0.0001). Results represent the mean ± SEM percent total F4/80+ cells and percent F4/80+/Tie2+ cells in the tumors (n=6 for LysMcre control, n=9 for HIF-1αfl/fl/LysMcre, and n=9 for HIF-2αfl/fl/LysMcre).

We next asked if HIF-α-deficiency would alter tumor TEM number. PyMT tumors were homogenized, separated by Ficoll gradient, and co-immunolabeled with antibodies specific for F4/80 and Tie2 receptor then analyzed by flow cytometry. While the percent of total F4/80+ cells in the tumors were unchanged across all mouse groups, the percent of tumor F4/80+/Tie2+ cells from the HIF-1αfl/fl/LysMcre mice were statistically reduced compared to tumors from the control and HIF-2αfl/fl/LysMcre mice (p<0.0001) (Figure 1B).

HIF-α subunit deficiency differentially affects angiogenic gene expression in tumor macrophages

Because myeloid HIF-1α deficiency decreased tumor TEM numbers (Figure 1), we investigated if HIF-2α deficiency may have also altered macrophage angiogenic phenotype. BMDMs were differentiated from bone marrow of LysMcre (control), HIF-1αfl/fl/LysMcre or HIF-2αfl/fl/LysMcre mice then exposed to hypoxia for 24 h. Microarray analysis on angiogenic genes revealed differential expression (defined as ≥2 or ≤0.5 fold change) upon HIF-α deficiency (Figure 2B). Hypoxia WT macrophages upregulated several genes (Vegfa, Serpine1, Il1b, and Ptgs1) in comparison to macrophages exposed to normoxia. These differentially upregulated genes were downregulated when either HIF-α subunits were knocked out reflecting the overlapping role of both subunits in angiogenic gene expression. To understand their differential roles we compared fold change in hypoxic HIF-2αfl/fl/LysMcre macrophages and hypoxic HIF-1αfl/fl/LysMcre macrophages to control hypoxic macrophages. Three genes had higher expression in hypoxic HIF-1αfl/fl/LysMcre macrophages (Itgb3, Plau, and Vegfb) while two genes were upregulated in hypoxic HIF-2αfl/fl/LysMcre macrophages (Mmp2 and Egf). Many angiogenic genes were downregulated in both hypoxic HIF-1αfl/fl/LysMcre macrophages (36 genes) and hypoxic HIF-2αfl/fl/LysMcre macrophages (45 genes). Thirty-two of these genes overlapped suggesting redundancy in those targets (Figure 2B, right). However, hypoxic HIF-1αfl/fl/LysMcre macrophages had downregulation of 4 different genes (Efna1, Hlf, Egf, and Angpt1) while HIF-2αfl/fl/LysMcre macrophages had downregulation of 14 different genes (Flt1, Col18a1, Pgf, Ccl2, Plau, Fgfr3, Anpep, Ptk2, Ecgf1, Col4a3bp, Kdr, Igf1, Angpt2, and Jag1). These data suggest that the HIFα subunits differentially regulate angiogenic profiles in hypoxic macrophages.

Figure 2. HIF-α subunit deficiency differentially affects angiogenic gene expression in tumor macrophages.

Figure 2.

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Gene expression changes were analyzed in BMDMs from female C57Bl/6J LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice subjected to normoxia (21% O2) or hypoxia (0.5% O2) for 24 hours. Microarray analysis on angiogenic genes (left) demonstrates differential increases (green) and decreases (red) in expression among the groups. The Venn diagram represents differentially expressed genes (≤0.5 or ≥2 fold change) among comparisons.

Suppressed TEM population decreases microvessel density but increases tumor oxygenation

Because tumors from HIF-1αfl/fl/LysMcre mice had a decreased TEM population, we predicted a decrease in tumor endothelial cell density in HIF-1αfl/fl/LysMcre mice. We implanted PyMT tumor cells into control, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice. After four weeks, tumors were sectioned and immunolabeled to determine microvessel density (Figure 3A). We observed a 57% reduction in CD31-positivity in tumors from HIF-1αfl/fl/LysMcre mice compared to tumors from control mice (p=0.003) and a 79% reduction compared to HIF-2αfl/fl/LysMcre mice (p<0.0001) (Figure 3A, right). We observed a significant increase (2.3-fold) in microvessel density in the tumors from HIF-2αfl/fl/LysMcre mice relative to tumors from control mice (p=0.0324) (Figure 3A, right). Not only do these data support the role of HIF-1α and TEMs in the regulation of blood vessel formation, but also suggest that the presence of HIF-2α in wild type macrophages containing both HIF-1α and HIF-2α may act to suppress HIF-1α-dependent TEM differentiation, leading to reduced microvessel density.

Figure 3. Less tumor TEMs reduces endothelial cell density but increases tumor oxygen.

Figure 3.

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(A) Four weeks post-implantation, tumors were removed from control, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and sectioned and immunostained with a CD31 antibody (red) (left). Tumors from HIF-1αfl/fl/LysMcre mice had less angiogenesis (CD31+ pixels) than tumors from control or HIF-2αfl/fl/LysMcre mice (p=0.003 and p<.0001, respectively) (right). Results represent the mean ± SEM percent of CD31+ pixels. (B) EPR was used to measure tumor oxygen concentration longitudinally once per week over four weeks post-injection of LiNc-BuO crystal-containing Py8119 cells. (C) LiNcBuO crystals were used to measure tumor oxygen by placing the resonator directly on the tumor. Mean tumor oxygen concentrations were measured across mouse groups over three weeks post-injection. On week 3 post-injection, tumors from HIF-1αfl/fl/LysMcre mice had significantly higher oxygen concentrations than control LysMcre or HIF-2αfl/fl/LysMcre groups (p=0.044 and 0.041, respectively). No other comparisons were significant. Results represent the mean oxygen concentration ± SEM (n=6 per group except on Week 2 post-injection n=5 for control group). * represents p<0.01.

To investigate the implications of the changing CD31+ vessel density on the oxygen concentration in the tumor, we used Electron Paramagnetic Resonance (EPR) to measure the partial pressure of oxygen. We incubated an oxygen-sensitive nanoprobe with PyMT tumor cells until the probe was internalized, and injected these cells into the mammary fat pads of LysMcre control, HIF-1αfl/fl/LysMcre or HIF-2αfl/fl/LysMcre mice. We used L-band EPR to measure oxygen beginning 1 week after implantation and continued once each week until sacrifice (Figure 3B). At three weeks post-implantation, tumors from HIF-1αfl/fl/LysMcre mice had significantly higher oxygen concentration when compared to tumors from control or HIF-2αfl/fl/LysMcre mice (p=0.044 and 0.041, respectively, Figure 3C) even though tumors from HIF-1αfl/fl/LysMcre mice had less CD31+ vessels at the same time point (Figure 3A). In contrast, tumors from LysMcre control mice became more hypoxic over time while tumors from HIF-2αfl/fl/LysMcre mice remained at unchanging hypoxia during tumor progression. Taken together, these results demonstrate that HIF-1α deficiency in macrophages partially rescues tumor hypoxia.

Long-term oxygen deprivation causes tissue necrosis

Histological evaluation suggests HIF-2αfl/fl/LysMcre tumors had more tissue necrosis (reflected by enhanced autofluorescence, top row represents original image while the bottom row is thresholded to remove background using the Intermodes function in ImageJ) when compared to those of HIF-1αfl/fl/LysMcre or control mice (p=0.041 and p=0.026, respectively, Figure 4). These data suggest that prolonged tumor hypoxia throughout progression (Figure 3C) caused by macrophage HIF-2α-deficiency augments necrosis in PyMT tumors.

Figure 4. Myeloid-specific HIF-2α subunit deficiency exacerbates tumor necrosis.

Figure 4.

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To investigate necrosis, four weeks after Py8119 injections, tumors from control, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre female mice were removed, sectioned then imaged directly under a green filter to visualize auto-fluorescent necrotic tissue (top). Tumors from HIF-2αfl/fl/LysMcre mice had significantly more necrosis than tumors from control LysMcre or HIF-1αfl/fl/LysMcre mice (p=0.041 and p=0.026, respectively) (n=6,6,7 per group, respectively). Results represent mean pixel count ± SEM. Scale bars= 5mm.

Myeloid HIF-1α deficiency enhances vasculature perfusion

During embryonic development, increases in vasculature density implies an increase of oxygen delivery into the tissue. However, neovascularization in tumors is often abnormal in architecture resulting in poorly aligned endothelial cells and increased permeability (34). Based on the results from the EPR data, we hypothesized that tumors from HIF-1αfl/fl/LysMcre had vessels that are more highly perfused than the more hypoxic tumors of the other two groups. We injected tumor-bearing female LysMcre control, HIF-1αfl/fl/LysMcre and HIF-2αfl/fl/LysMcre mice with Dylight 594-labelled tomato lectin which binds endothelium prior to euthanasia. Tumors sections were then stained for CD31 and analyzed by confocal microscopy.

There were no differences in the number of CD31+ pixels when compared to lectin+ pixels in tumors from HIF-1αfl/fl/LysMcre mice (p=0.415) (Figure 5A). However, both HIF-2αfl/fl/LysMcre and control mice had significantly less lectin+ pixels than CD31+ vessels (p<0.001) suggesting that there are more vessels in these tumors that are not being perfused. As another measure, functional vasculature is associated with pericyte coverage (35) (NG2 positivity). In breast tumors, there is a seemingly negative correlation between pericytes and angiogenesis as pericyte coverage in low vascular density areas of breast tumors is significantly higher than in areas with high vascular density (36). We hypothesized that TEM blood vessel association decreases pericyte density. To investigate, we immunostained tumor sections with NG2 and CD31 antibodies then analyzed by confocal microscopy. There were no differences in NG2+ pixels among the three mouse groups (Figure 5B). However, tumors from HIF-1αfl/fl/LysMcre mice had significantly less NG2+ and CD31+ that co-localized (p<0.001) while tumors from HIF-2αfl/fl/LysMcre mice had significantly more NG2+ and CD31+ that co-localized (p<0.001) when compared to tumors from control mice. While it has been shown that TEM populations may depend on pericyte coverage based on evidence demonstrating NG2 null mice with MMTV-PyMT tumors lose substantial amounts of TEMs(37), this data suggests the reverse may be true as well, namely a dependency of pericyte interactions with endothelium on TEMs.

Figure 5. Myeloid-specific HIF-1α subunit deficiency increases vessel perfusion.

Figure 5.

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(A) Four weeks post-implantation, tumor-bearing control, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice were given retro-orbital injections of lectin (magenta) which labels perfused blood vessels (left). Tumors were harvested, sectioned, and immunostained with CD31 antibody (green) to visualize perfused vessels (white in the bottom overlay row). Both HIF-2αfl/fl/LysMcre and control mice had significantly less lectin+ pixels than CD31+ vessels (p<0.001) (27 images analyzed per n for 3n’s per group). Results represent mean pixel count ± SEM and were analyzed using repeated measures ANOVA. (B) Four weeks post-implantation, tumors were removed from control, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and sectioned and immunostained with CD31 (green) and NG2 (magenta) antibodies to stain endothelial cells and pericytes, respectively. Tumors from HIF-1αfl/fl/LysMcre mice had significantly less co-localized NG2+ and CD31+ cells (p<0.001) while tumors from HIF-2αfl/fl/LysMcre mice had significantly more co-localized NG2+ and CD31+ cells (p<0.001) when compared to tumors from control mice (15 images analyzed per n for 3 n’s per group). Results represent the mean pixel count ± SEM and were calculated using repeated measures ANOVA. Scale bars= 50μm.

We next investigated vessel architecture throughout whole tumors as it was once thought that large, dilated blood vessels inside tumors were more efficient at delivering blood (34). However, the field has recognized that these vessels are thin-walled, leaky, tortuous and as a result, less efficient in delivering blood (34). Tumor-bearing mice were intra-cardially perfused with Microfil, a CT contrast agent that polymerizes to create a three-dimensional cast of the vasculature (38), and tumors scanned using a μCT system (Figure 6A). Images of tumor vasculature from control and HIF-2αfl/fl/LysMcre mice contained large, tortuous vessels (Figure 6B) while tumors from macrophage HIF-1a KO mice had qualitatively less hyper-dilated vessels than the other tumors. While this methodology does not visualize the microvasculature at which oxygen exchange takes place, it does reveal the architecture of the tumor vasculature and how macrophage HIF-1α deficiency alters this architecture. Together, these data suggest that macrophage HIF-1α deficiency enhances vascular perfusion by influencing the vessel architecture.

Figure 6. Macrophage HIF-1α deficiency alters the vessel architecture.

Figure 6.

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(A) Four weeks post-implantation, tumor-bearing control, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice were anesthetized and blood was exsanguinated. The vasculature was washed through intracardiac injection of PBS followed by fixation with 4% PFA. Lastly, Microfil was intracardially injected then allowed to polymerize over 48 hrs. The tumors were harvested and embedded in 1% agarose. Tumors were imaged using the Skyscan 1272 MicroCT system at high resolution (6.6 μm) and perfused vessels (red) were (B) visualized and analyzed using Imaris software. Scale bars= 500 μm.

HIF-1α deficiency in myeloid cells augments docetaxel effectiveness

Dysfunctional tumor architecture leading to intermittent fluid flux and high tissue interstitial pressures contribute to a failure in systemic chemotherapy effectiveness in cancer patients (39). Because we observed that mice with myeloid cell HIF-1α deficiency had better perfusion and increased oxygen into their tumors compared to LysMcre wild type or mice with HIF-2α deficiency, we asked if this increased perfusion would augment the treatment efficacy of a standard breast cancer chemotherapy, docetaxel. We again implanted Py8119 tumor cells into the fat pads of LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice. Starting one week after implantation (all mice had palpable tumors), we treated the mice with docetaxel or vehicle once a week over four weeks. Tumor growth was measured twice a week using calipers. The growth curves for each mouse in each group are shown in Figure 7A. We found no statistical difference in tumor growth between the vehicle- and docetaxel-treated LysMcre or HIF-2αfl/fl/LysMcre mice, while the docetaxel significantly inhibited tumor growth in the HIF-1αfl/fl/LysMcre mice (Figure 7B) over the four weeks (p<0.001). After, to confirm the tumor growth data, we collected the tumors and weighed them for burden determination since some tumors grow into the peritoneum and cannot be accurately measured using calipers. The tumor burden data validated the growth data as while the average tumor mass from the LysMcre and HIF-2αfl/fl/LysMcre mice were not significantly different when comparing vehicle to docetaxel treatment, average tumor mass from the HIF-1αfl/fl/LysMcre mice were significantly smaller in response to docetaxel (p=0.0068) (Figure 7C).

Figure 7. Macrophage HIF-1α deficiency augments docetaxel effectiveness.

Figure 7.

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(A) Wild type control, myeloid cell HIF-1α-deficient or HIF-2α-deficient mice were implanted with 5×105 Py8119 tumor cells. After one week, the mice were randomized into treatment groups and subjected to 30mg/kg body weight docetaxel or vehicle by intraperitoneal injection one time per week for a total of four weeks. Tumor growth from LysMcre control mice (vehicle, light gray; docetaxel, dark grey), HIF-1αfl/fl/LysMcre mice (vehicle, light blue; docetaxel, dark blue), and HIF-2αfl/fl/LysMcre mice (vehicle, light green; docetaxel, dark green) was monitored using calipers to determine tumor volumes at each measure day twice a week. Dotted line indicates an outlier value for that measure point for that animal. (B) Tumor growth rates were compared for docetaxel effectiveness. Docetaxel treatment had no significant effect on tumor growth compared to vehicle in either control or myeloid cell HIF-2α-deficient mice, while it did have a significant effect on tumor growth relative to vehicle in the mice with myeloid cell HIF-1α-deficiency (p<0.001). Results represent the mean tumor growth rate ± SD and were calculated using repeated measures ANOVA. (C) After four weeks, the tumors were resected and weighed for tumor burden. Docetaxel treatment had no significant effect on tumor mass compared to vehicle in either control or myeloid cell HIF-2α-deficient mice, while it did have a significant effect on tumor mass relative to vehicle in the mice with myeloid cell HIF-1α-deficiency (p=0.0068). LysMcre vehicle: n=4; LysMcre docetaxel, n=4; HIF-1αfl/fl/LysMcre vehicle: n=6; HIF-1αfl/fl/LysMcre docetaxel: n=6; HIF-2αfl/fl/LysMcre vehicle: n=6; HIF-2αfl/fl/LysMcre docetaxel: n=6. Boxes represent the median (line) and mean (X) of tumor mass in grams ± SD and were calculated using one-way ANOVA (p=0.0019) with Tukey’s post-hoc testing for differences between groups.

Discussion

This study establishes a novel role for HIF-2α in suppressing HIF-1α upregulation of Tie2 receptor expression on a subpopulation of monocytes/macrophages. The impetus of this study originated from reports that hypoxia increases TIE2 expression on human monocytes in vitro (19). Hypoxia induced-expression of Tie2 on endothelial cells has been attributed to both HIF-1α and HIF-2α (42,43). Given these seemingly cell-dependent, incongruent roles for the HIF-α subunits, we set out to determine the role for the HIF-α subunits in Tie2 receptor expression on macrophages.

Hypoxia-stabilized HIF-2α has been implicated in monocyte recruitment (44). In murine hepatocellular and colitis-associated colon carcinoma models, macrophage HIF-2α regulates tumor macrophage recruitment by regulating M-CSFR and CXCR4 receptor expression under hypoxic conditions (45). Decreases in the HIF-1α-deficient F4/80+Tie2+ population with no change in the F4/80 population within the tumors illustrates a possible role for the HIF-1α in TEM differentiation within the tumor.

In vitro HIF-1α depletion affects the differentiation of myeloid suppressor (CD11+ and GR-1+) cells into F4/80 positive macrophages (46). In our model, the percentage of total F4/80 cells in the PyMT tumors from all three mouse groups was not different while the percent of those F4/80 cells with Tie2 positivity decreased significantly with HIF-1α knockout. These data raise the possibility that the number of F4/80+ cells correlating with poor prognosis in patients with breast cancer are derived and recruited from a smaller, more specific subpopulation of F4/80+ cells, namely the F4/80+/Tie2+ TEMs which express high levels of tumor promoting MRC-1 (mannose receptor), SDF-1/CXCL12, and IL-10 (28–33,46,47).

TEM function in the PyMT tumor model was highlighted as our knockout of HIF-2α increased the microvessel density, decreased perfusion, and exacerbated tumor hypoxia and necrosis. “These data reflect a study in which knocking out VEGF in myeloid cells via a LysMcre transgene resulted in decreased vascular density but increased perfusion and response to chemotherapy in PyMT tumors (50). However, we found no significant differences in Vegf mRNA expression among tumor CD11b+Tie2- or CD11bTie2+ cell in this model (Supplemental Figure 1). Though no differences were seen in the myeloid cells, it is possible that VEGF may still have an unexplored, myeloid-independent role in the presentation of these phenotypes.” Our observation supports data on TEMs in tumors where co-injection of Tie2+ monocytes with glioblastoma cells increased vascular density (48). However, we have found that these TEM-dependent increases in vessel density confer reduced vessel perfusion and increased tumor hypoxia which is in accord with data reporting that increased TEM numbers in ANG2 over-expressing mice exacerbates breast tumor hypoxia (46).

Most importantly, a recent study reported myeloid HIF-2α-deficiency accelerated tumor development in a fibrosarcoma model, and the authors were the first to suggest a tumor-repressing ability of HIF-2α (49). “Though our manuscript focuses on the function of macrophages, lysozyme M expression is also found in neutrophils. Thus, our model can also induce neutrophil HIF deficiency. Because macrophages are the primary myeloid cells in Py8119 tumors (51, 52), we attribute the phenotypes discussed to their function. However, neutrophils make up a small population in these tumors (51, 52) and may contribute to the observed phenotype here and remains unexplored.”

To our knowledge, this is the first study demonstrating not only the opposing roles of HIF-1α and HIF-2α on the function of TEMs in breast tumors, but also the suppressive role that HIF-2α plays in the differentiation to pro-angiogenic and M2-like TEMs. Lastly, these data introduce an undescribed and counterintuitive pro-hypoxia role for pro-angiogenic TEMs in breast cancer which may contribute to hypoxia-induced treatment resistance in breast tumors.

Supplementary Material

1

Key Points.

HIFα subunits differentially regulate Tie2 expression on macrophages

Myeloid HIF-1α-deficiency enhances tumor oxygenation and chemotherapeutic response

Acknowledgements

Microcrystals were provided by Dr. Valery Khramtsov, Director of the West Virginia University In vivo Multifunctional Magnetic Resonance (IMMR) center at West Virginia University. We would also like to acknowledge the WVU IMMR center for functional EPR analyses and each Dr. Mark and Oxana Tseytlin for their valuable input.

This work was supported by National Cancer Institute Grant R00 CA131552 (T.D.E), WVCTSI grant (GM104942; West Virginia State Startup Funds) (T.D.E), NIH R01 CA194013 (T.D.E and V.V.K.), NIH R01 HL067167 and NHLBI R01HL109481 (C.B.M), NIH R01 CA192064 (V.V.K), NIH P20GM121322 (A.A.B), and Ruby Distinguished Fellowship to (K.J.S). Imaging experiments and image analysis were performed in the West Virginia University Animal Models & Imaging Facility, which has been supported by the WVU Cancer Institute and NIH grants P20RR016440, P30RR032138/GM103488, P20GM103434, and U54GM104942 and the WVU Flow Cytometry & Single Cell Core, WVCTS grant GM104942 and WV-INBRE grant GM103434.

Footnotes

Conflict of Interest Disclosures

There are no conflicts of interest to disclose.

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