Jackie E. Bader

Summary: Obesity is a leading risk factor for progression and metastasis of many cancers ^1,2 , yet can also promote improved survival for some cancers ^3-5 and enhance responses to some immune checkpoint blockade therapies ^6-8 . The role of the immune system in the obesity-cancer connection and how obesity influences immunotherapy, however, remain unclear. While PD-1 expression by macrophages has been described ^9-12 , we found that obesity selectively induced PD-1 on macrophages and that PD-1 directly impaired macrophage function. Single cell RNA sequencing of murine colorectal carcinoma tumors showed obesity remodeled myeloid and T cell populations, with fewer clonally expanded effector T cells and increased abundance of PD-1 ⁺ tumor-associated macrophages (TAM). Cytokines and molecules associated with obesity, including IL-6, leptin, and insulin, and the unsaturated fatty acid palmitate, induced PD-1 expression on macrophages in a glycolysis-dependent manner. PD-1 ⁺ TAMs had increased mitochondrial respiration and expression of genes regulating oxidative phosphorylation, lipid uptake and cell cycle while PD-1 ^- TAMs showed greater signatures of phagocytosis and antigen presentation to T cells. These patterns were directly regulated by PD-1, as recombinant PD-L1 reduced macrophage glycolysis and phagocytic capacity, and this was reversed with blocking PD-1 antibody. Conversely, PD-1-deficient Pdcd1 ^-/- TAMs had high rates of glycolysis, phagocytosis, and expression of MHC-II. Myeloid-specific PD-1 deficiency correlated with slower tumor growth, enhanced TAM antigen presentation capability, and increased CD8 T cell activation together with reduced markers of exhaustion. These findings show metabolic signaling in obesity induces PD-1-mediated suppression of TAM function and reveal a unique macrophage-specific mechanism to modulate immune tumor surveillance and checkpoint blockade. This may contribute to increased cancer risk yet improved response to PD-1 blockade in TAM-enriched tumors and obesity.

Cancer cells characteristically consume glucose through Warburg metabolism¹, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells^2,3,4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

The tumor microenvironment (TME) includes transformed cancer and infiltrating immune cells ^1,2 . Cancer cells can consume large quantities of glucose through Warburg metabolism ^3,4 that can be visualized with positron emission tomography (PET). While infiltrating immune cells also rely on glucose, disruptions to metabolism can contribute to tumor immunological evasion ^5–9 . How immune cell metabolism is programmed or restrained by competition with cancer cells for nutrients, remains uncertain. Here we used PET tracers to measure the accessibility of glucose and glutamine to cell subsets in the TME. Surprisingly, myeloid cells including macrophages were the greatest consumers of intra-tumoral glucose, followed by T cells and cancer cells. Cancer cells, in contrast, had the highest glutamine uptake. This distinct nutrient partitioning was programmed through selective mTORC1 signaling and glucose or glutamine-related gene expression. Inhibition of glutamine uptake enhanced glucose uptake across tumor resident cell types and shifted macrophage phenotype, demonstrating glucose is not limiting in the TME. Thus, cancer cells are not the only cells in tumors which exhibit high glucose uptake in vivo and instead preferentially utilize glutamine over other cell types. We observe that intrinsic cellular programs can play a major role in the use of some nutrients. Together, these data argue cell selective partitioning of glucose and glutamine can be exploited to develop therapies and imaging strategies to alter the metabolic programs of specific cell populations in the TME.

Objective: Depletion of macrophages is thought to be a therapeutic option for obesity-induced inflammation and metabolic dysfunction. However, whether the therapeutic effect is a direct result of reduced macrophage-derived inflammation or secondary to decreases in fat mass is controversial as macrophage depletion has been shown to disrupt energy homeostasis. This study was designed to determine if macrophage depletion via clodronate-liposome (CLD) treatment could serve as an effective intervention to reduce obesity-driven inflammatory and metabolic impairments independent of changes in energy intake. Methods: Following 16 weeks of high-fat diet (HFD) or AIN-76A control diet (LFD) (n=30/diet treatment), male C57BL/6J mice were assigned to either a CLD or PBS-liposome-treatment (n=15/group) for 4 weeks. PBS-liposome-treated groups were pair-fed to their CLD-treated dietary counterparts. Metabolic function was assessed pre- and post- liposome treatment. Adipose tissue as well as liver were investigated for macrophage infiltration and the presence of inflammatory mediators. Additionally, a complete blood count was performed. Results: CLD treatment reduced energy intake. When controlling for energy intake, CLD treatment was unable to rescue metabolic dysfunction or Non-Alcoholic-Fatty-Liver Disease development, and impaired adipose tissue insulin action. Moreover, CLD treatment induced neutrophilia, anemia, increased adipose tissue mRNA expression of the pro-inflammatory cytokines, IL-6 and IL-1β, and augmented circulating IL-6 and MCP-1 concentrations (p<0.05). Conclusion: This study suggests that the use of CLD treatment to deplete macrophages attenuates obesity by limiting energy intake. Moreover, after controlling for the benefits of weight loss, the accompanying detrimental side effects limit CLD treatment as an effective therapeutic strategy.

Clinical studies provide strong evidence that obesity and associated adipose tissue (AT) inflammation are risk factors for breast cancer (BrCA); however, mechanistic knowledge of the interaction of obesity, BrCA, and menopausal status has proven to be not only lacking, but contradictory. Obesity-induced inflammation and elevated biosynthesis of estrogens, through aromatase-mediated metabolism of precursors, have been linked with hormone receptor positive (HP) postmenopausal BrCA but not previously associated with premenopausal BrCA risk. Thus, further delineation of the interaction of obesity, inflammation, and aromatase is required for the development of therapeutic treatment options. The purpose of this study was to examine the effect of high fat diet (HFD)-induced inflammation on tumorigenesis in a model of pre and postmenopausal HP BrCA. Female PyMT/MMTV ovary intact and ovariectomized mice were fed low and HFD diets to examine the role of obesity-induced inflammation and hormone production in the development of HP BrCA. Tumor statistics for number, volume, weight, histopathology scoring and gene expression of macrophage and inflammatory mediators were measured in the AT and mammary gland at sacrifice. HFD feedings of ovary intact mice resulted in increased adiposity and tumorigenesis, indicated by increased primary tumor volume, multiplicity, tumor burden, and increased tumor progression represented by histopathological scoring. HFD-induced obesity significantly upregulated aromatase and macrophage marker expression in the AT (F4/80 and CD11c) and mammary gland (Mertk) in a premenopausal model of BrCA. Conversely, HFD feedings had no significant effect on tumorigenesis in a postmenopausal model of BrCA despite large increases in adiposity in ovariectomized mice; however, limitations within the model may have precluded any significant findings. This data suggests that obesity-induced increases in inflammation and hormone production, via aromatase expression, is associated with increases in tumorigenesis in a model of premenopausal HP BrCA in the PyMT/MMTV strain.

Irritation reactions are a frequently reported occupational illness. The potential adverse effects of pharmaceutical compounds (PCs) on eye and skin can now be assessed using validated in vitro methods. Our overall aim is to reduce animal testing by replacing the historically utilized in vivo test methods with validated in vitro test methods which accurately determine the ocular and dermal irritation/corrosion potential of PCs to inform worker safety within the pharmaceutical space. Bristol-Myers Squibb (BMS) and the Institute for In Vitro Sciences (IIVS) have therefore conceptualized and internally qualified a tiered in vitro testing strategy to inform occupational hazards regarding eye and skin irritation and corrosivity of PCs. For the small scale pre-qualification phase, we paired historical in vivo and newly generated in vitro data for 15 PCs to determine the predictive capacity of in vitro assays already validated for the eye and skin irritation/corrosion endpoints and accepted for certain regulatory submissions. During the post-qualification phase, a group of 24 PCs were subjected exclusively to the developed tiered testing strategy, which is based on three Organisation for Economic Co-operation and Development (OECD) in vitro methods. The qualified in vitro testing strategy utilizes the Corrositex® assay for the corrosivity (OECD TG 435), the Bovine Corneal Opacity and Permeability (BCOP) assay for ocular irritation (OECD TG 437), and the EpiDerm™ tissue model-based Skin Irritation Test (SIT) for dermal irritation (OECD TG 439). In the first step, the pH of each PC was determined. For compounds with pH extremes ≥11 or ≤2, the Corrositex® assay was generally conducted first. For compound(s) that were incompatible with or were negative in the Corrositex® assay or had pH values between 2 and 11, the BCOP assay and SIT were performed first. The results of the tiered testing strategy's qualification phase demonstrated that the BCOP assay is sensitive enough to identify a wide range of eye irritation/corrosion potentials and its over-prediction rate was considered acceptable to inform occupational hazards and ensure the proper handling practices of PCs. The SIT correctly predicted the skin irritation potential of 14 out of the 15 PCs included in the qualification phase, only over-predicting one PC. In the post-qualification phase, four PCs out of four tested were predicted corrosive by the Corrositex® assay and thus no further testing was needed or conducted. The rest of the PCs were evaluated in the BCOP assay (both neat and as a 20% dilution), with the higher response being used for hazard classification. Four PCs were determined to be severe eye irritants, 1 a moderate irritant, 8 were mild irritants, and 8 were non-irritants. The same set of PCs was evaluated using the SIT and were classified as non-irritants to skin. These results are consistent with the BMS historical in vivo results showing a very low number of PCs as skin irritants. This tiered in vitro testing strategy, which replaces the use of animal studies, was found to be reasonably accurate in its predictive capacity when compared to historical in vivo results and represents a conservative and reliable platform that can be utilized for the prediction of ocular and dermal irritation/corrosion potential of PCs and for subsequent GHS classification and worker safety hazard communications.

We examined the role of macrophages in inflammation associated with colorectal cancer (CRC). Given the emerging evidence on immune-microbiota interactions in CRC, we also sought to examine the interaction between macrophages and gut microbiota. To induce CRC, male C57BL/6 mice (n = 32) received a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulfate (DSS)-supplemented water in weeks 1, 4, and 7. Prior to the final DSS cycle (week 7) and twice weekly until euthanasia, mice (n = 16/group) received either 200 μl ip of clodronate-filled liposomes (CLD) or phosphate-buffered saline (PBS) encapsulated liposomes to deplete macrophages. Colon tissue was analyzed for polyp burden, macrophage markers, transcription factors, and inflammatory mediators. Stool samples were collected, and DNA was isolated and subsequently sequenced for 16S rRNA. Clodronate liposomes decreased tumor number by ∼36% and specifically large (≥1 mm) tumors by ∼36% (P < 0.05). This was consistent with a decrease in gene expression of EMR1 in the colon tissue and polyp tissue as well as expression of select markers associated with M1 (IL-6) and M2 macrophages (IL-13, IL-10, TGFβ, CCL17) in the colon tissue (P < 0.05). Similarly, there was a decrease in STAT3 and p38 MAPK and ERK signaling in colon tissue. Clodronate liposomes increased the relative abundance of the Firmicutes phylum (P < 0.05) and specifically Lactobacillaceae and Clostridiaceae families, which have been associated with reduced CRC risk. Overall, these data support the development of therapeutic strategies to target macrophages in CRC and provide support for further evaluation of immune-microbiota interactions in CRC. NEW & NOTEWORTHY We found that macrophage depletion during late-stage tumorigenesis is effective at reducing tumor growth. This was associated with a decrease in macrophage markers and chemokines in the colon tissue and a decrease in transcription factors that are linked to colorectal cancer. The macrophage-depleted group was found to have an increased abundance of Firmicutes, a phylum with documented anti-tumorigenic effects. Overall, these data support the development of therapeutic strategies to target macrophages in colorectal cancer.

Noncoding RNAs are emerging as regulators of inflammatory and metabolic processes. There is evidence to suggest that miRNA155 (miR155) may be linked to inflammation and processes associated with adipogenesis. We examined the impact of global miRNA‐155 deletion (miR155^−/−) on the development of high‐fat diet (HFD)‐induced obesity. We hypothesized that loss of miR155 would decrease adipose tissue inflammation and improve the metabolic profile following HFD feedings. Beginning at 4–5 weeks of age, male miR155^−/− and wild‐type (WT) mice (n = 13–14) on a C57BL/6 background were fed either a HFD or low‐fat diet for 20 weeks. Body weight was monitored throughout the study. Baseline and terminal body composition was assessed by DEXA analysis. Adipose tissue mRNA expression (RT‐qPCR) of macrophage markers (F4/80, CD11c, and CD206) and inflammatory mediators (MCP‐1 and TNF‐α) as well as adiponectin were measured along with activation of NFκB‐p65 and JNK and PPAR‐γ. Adipose tissue fibrosis was assessed by picrosirius red staining and western blot analysis of Collagen I, III, and VI. Glucose metabolism and insulin resistance were assessed by Homeostatic Model Assessment – Insulin Resistance (HOMA‐IR), and a glucose tolerance test. Compared to WT HFD mice, miR155^−/− HFD mice displayed similar body weights, yet reduced visceral adipose tissue accumulation. However, miR155^−/− HFD displayed exacerbated adipose tissue fibrosis and decreased PPAR‐γ protein content. The loss of miR155 did not affect adipose tissue inflammation or glucose metabolism. In conclusion, miR155 deletion did not attenuate the development of the obese phenotype, but adipose tissue fibrosis was exacerbated, possibly through changes to adipogenic processes.

Monocyte chemoattractant protein 1 (MCP-1) has been implicated as a major modulator in the progression of mammary tumorigenesis, largely due to its ability to recruit macrophages to the tumor microenvironment. Macrophages are key mediators in the connection between inflammation and cancer progression and have been shown to play an important role in tumorigenesis. Thus, MCP-1 may be a potential therapeutic target in inflammatory and difficult-to-treat cancers such as triple negative breast cancer (TNBC). We examined the effect of MCP-1 depletion on mammary tumorigenesis in a model of TNBC. Tumor measurements were conducted weekly (until 22 weeks of age) and at sacrifice (23 weeks of age) in female C3(1)/SV40Tag and C3(1)/SV40Tag MCP-1 deficient mice to determine tumor numbers and tumorvolumes. Histopathological scoring was performed at 12 weeks of age and 23 weeks of age. Gene expression of macrophage markers and inflammatory mediators were measured in the mammary gland and tumor microenvironment at sacrifice. As expected, MCP-1 depletion resulted in decreased tumorigenesis, indicated by reduced primary tumor volume and multiplicity, and a delay in tumor progression represented by histopathological scoring (12 weeks of age). Deficiency in MCP-1 significantly downregulated expression of macrophage markers in the mammary gland (Mertk and CD64) and the tumor microenvironment (CD64), and also reduced expression of inflammatory cytokines in the mammary gland (TNFα and IL-1β) and the tumor microenvironment (IL-6). These data support the hypothesis that MCP-1 expression contributes to increased tumorigenesis in a model of TNBC via recruitment of macrophages and subsequent increase in inflammatory mediators.

Obesity presents a significant public health concern given its association with increased cancer incidence, unfavorable prognosis, and metastasis. However, there is very little literature on the effects of weight loss, following obesity, on risk for colon cancer or liver cancer. Therefore, we sought to study whether intentional weight loss through diet manipulation was capable of mitigating colon and liver cancer in mice. We fed mice with a high-fat diet (HFD) comprised of 47% carbohydrates, 40% fat, and 13% protein for 20 wk to mimic human obesity. Subsequently, azoxymethane (AOM) was used to promote colon and liver carcinogenesis. A subset of obese mice was then switched to a low-fat diet (LFD) containing 67.5% carbohydrate, 12.2% fat, and 20% protein to promote intentional weight loss. Body weight loss and excess fat reduction did not protect mice from colon cancer progression and liver dysplastic lesion in the AOM-chemical-cancer model even though these mice had improved blood glucose and leptin levels. Intentional weight loss in AOM-treated mice actually produced histological changes that resemble dysplastic alterations in the liver and presented a higher percentage of F4/80⁺CD206⁺ macrophages and activated T cells (CD4⁺CD69⁺) in the spleen and lymph nodes, respectively. In addition, the liver of AOM-treated mice exposed to a HFD during the entire period of the experiment exhibited a marked increase in proliferation and pNF-κB activation. Altogether, these data suggest that intentional weight loss following chemical-induced carcinogenesis does not affect colon tumorigenesis but may in fact negatively impact liver repair mechanisms.