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Due to advances in diagnosis and treatment, cancer–related mortality has decreased, and by the year 2030, there will be 22 million cancer survivors in the United States. This success comes with an increased incidence of serious adverse effects, mainly in the cardiovascular system. While new treatment modalities are emerging for Triple-negative breast cancer (TNBC), most current strategies include anthracycline-based regimens to manage disease. Therefore, novel strategies are needed to overcome anthracycline-induced cardiotoxicities in this patient population. Activation of the TSP1/CD47 signaling axis is implicated in the progression of heart failure, with reported increases in TSP1 levels following myocardial infarction. Therefore, we examined the potential of CD47 blockade as a strategy to prevent cardiac injury as a consequence of cancer chemotherapy. Our data in a syngeneic orthotopic breast cancer model shows that blockade of CD47 using an in vivo anti-sense phosphodiesterase morpholino (PMO) preserved ejection fraction, fractional shortening, and cardiac output when compared to DOX treatment while preserving oncologic efficacy of chemotherapy. To determine a potential mechanism of cardioprotection, hearts of control and CD47 PMO-treated mice were subjected to RNA sequencing. Gene set enrichment analysis (GSEA) showed significant positive enrichment for metabolic pathways including pyruvate metabolism (NES= 2.3 , p< 0.002), and oxidative phosphorylation (NES=2.0, p< 0.01). During cardiac insult, metabolic flexibility of cardiomyocytes results in metabolic reprogramming from fatty acid oxidation to a glycolytic mechanism to overcome injury. Thus, DOX-associated cardiotoxicity may be mediated by an increase in TSP1 and a decrease in glycolysis, leading to the inability to overcome acute cellular stress. In vitro cellular bioenergetics, analysis revealed that TSP1 caused a dose-dependent reduction in glycolytic flux and glycolytic capacity in cardiac myoblast. This, coupled with preserved cardiac viability of cardiac cells treated with CD47 PMO in the presence of DOX, suggests that TSP1 may act through CD47 to prevent cardiac cell metabolic reprogramming needed to overcome injury. Furthermore, anti-sense experiments with siRNAs to Glut-4 and Hexokinase-II showed that the protection conferred by CD47 is mediated by activating these proteins. Therefore our studies suggest that the TSP1/CD47 axis may be central to the interplay of metabolism to preserve cardiac tissue integrity; thus, targeting this pathway may prevent the onset of chronic cardiac disease due to chemotherapy in cancer patients. Citation Format: Steven M. Bronson, Jessica D. Mackert, Mitra Kooshki, Adam S. Wilson, Nildris Cruz-Diaz, Pierre L Triozzi, Alexandra Thomas, Katherine L. Cook, David R. Soto-Pantoja. Blockade of CD47/Thrombospondin-1 signaling increases glycolytic metabolism as a protective mechanism against chemotherapy-associated cardiac injury in a model of Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-08-14.

Breast tumors have their own specific microbiota, distinct from normal mammary gland tissue. Patients with breast cancer that present with locally advanced disease often undergo neoadjuvant chemotherapy to reduce tumor size prior to surgery to allow breast conservation or limit axillary lymph node dissection. The purpose of our study was to evaluate whether neoadjuvant chemotherapy modulates the tumor microbiome and the potential impact of microbes on breast cancer signaling. Using snap-frozen aseptically collected breast tumor tissue from women who underwent neoadjuvant chemotherapy (n = 15) or women with no prior therapy at time of surgery (n = 18), we performed 16S rRNA-sequencing to identify tumoral bacterial populations. We also stained breast tumor microarrays to confirm presence of identified microbiota. Using bacteria-conditioned media, we determined the effect of bacterial metabolites on breast cancer cell proliferation and doxorubicin therapy responsiveness. We show chemotherapy administration significantly increased breast tumor Pseudomonas spp. Primary breast tumors from patients who developed distant metastases displayed increased tumoral abundance of Brevundimonas and Staphylococcus. We confirmed presence of Pseudomonas in breast tumor tissue by IHC staining. Treatment of breast cancer cells with Pseudomonas aeruginosa conditioned media differentially effected proliferation in a dose-dependent manner and modulated doxorubicin-mediated cell death. Our results indicate chemotherapy shifts the breast tumor microbiome and specific microbes correlate with tumor recurrence. Further studies with a larger patient cohort may provide greater insights into the role of microbiota in therapeutic outcome and develop novel bacterial biomarkers that could predict distant metastases.Implications:Breast tumor microbiota are modified by therapy and affects molecular signaling.

Obesity influences breast tumor microbiota populations. Breast tumor 16S sequencing results were stratified based upon patient body mass index (BMI). Tumor bacterial populations from non-obese patients with a BMI of 20-27 (n=9) were compared with bacterial populations from obese patients with a BMI of 31-48 (n=8). A. Relative abundance of bacterial genera in different tumor samples is visualized by bar plots. Each bar represents a single tumor and each colored box a bacterial taxon. The height of a color box represents the relative abundance of that organism within the sample. ''Other'' represents lower abundance taxa. B. Phylum level distribution in tumors from obese and non-obese patients. Primary breast tumors from obese patients have decreased proportional abundance of Comamonadaceae (C) and Ruminococcaceae (D) family level taxa. E. Obese patients' breast tumors display elevated abundance of Enterobacteriaceae_unclassified. *p

A. Uninfected 4T1 tumor volume from mice treated 1 x weekly control diet-derived fecal transplant. B. Uninfected 4T1 tumor volume from mice treated 1 x weekly lard diet-derived fecal transplant. C. Tumor volume over time of uninfected 4T1 cells mixed with control diet-derived fecal conditioned media infected RAW 264.7 (macrophage) cells. D. Tumor volume over time of uninfected 4T1 cells mixed with lard diet-derived fecal conditioned media infected RAW 264.7 (macrophage) cells. E. Tumor proliferation as determined by Ki67 immunoreactivity. F. Gram-positive bacteria content in 4T1 breast tumors. G. LPS positive bacteria content in 4T1 tumors. H. Infiltrating tumor-associated macrophage content in 4T1 tumors was determined by F4/80 immunoreactivity. n=5-6.*p

Obesity increases the risk of postmenopausal breast cancer. Obesity also influences the gut microbiome. The gut microbiome represents the totality of microorganisms living within the body and can influence the development of many different types of diseases, including breast cancer. Obesity is associated with an increase in the Firmicute-to-Bacteriodetes bacterial phyla ratio in the gut microbiome promoting dysbiosis and inflammation. We previously demonstrated that diet and obesity shift both the gut and breast tissue microbiome using a non-human primate model. To investigate the impact of omega-3 PUFA supplementation on the gut and breast microbiome, we used a combination of a preclinical murine model and human fecal samples collected from overweight and obese postmenopausal women enrolled in a weight loss clinical trial supplemented with placebo or high dose omega-3 PUFA (clinicaltrials.gov identifier: NCT02101970). Female C57BL/6 mice fed a Western (0.2% kcal from omega-3 PUFA) or a Western +omega-3 PUFA diet (1.3% kcal omega-3 PUFA) for 16 weeks demonstrate differences in intestinal permeability as measured by circulating plasma LPS, in vivo FITC-dextran permeability assay, and intestinal tight junction protein ZO-1 gene expression. Elevated LPS bioavailability associated with metabolic endotoxemia has been associated with breast cancer risk. Omega-3 PUFA supplementation shifts the gut and mammary gland (MG) microbiome. 16S sequencing of DNA isolated from matched fecal and MG samples demonstrate that omega-3 PUFA supplementation independently regulated both microbiomes. Moreover, omega-3 PUFA supplementation in a Western diet reduced Firmicute-to-Bacteriodetes ratio in the murine MG tissue. Obese and overweight postmenopausal women (n=46) were enrolled in a weight loss clinical trial (combination of calorie restriction and exercise) and were administered a placebo or 3.25 g/day combined eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids omega-3 PUFA supplements for 6 months. Bacterial sequencing of DNA isolated from fecal collections at baseline and after 6 months of intervention shows significant decrease in Firmicutes-to-Bacteriodetes ratio regardless of intervention group. However, when clustering subjects by relative weight loss and intervention (10% weight loss on placebo, or >10% weight loss on omega-3), women in the Citation Format: Katherine L. Cook, Adam S Wilson, David R Soto-Pantoja, Bruce F. Kimler, Shahid Umar, Carol J. Fabian. Omega-3 polyunsaturated fatty acid supplementation shifts the gut and breast microbiome to influence inflammation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-09-03.

Background: The microbiome consists of the totality of microorganisms (bacteria, fungi, protist, viruses, and phages) that live on and within the body. Studies implicate the gut bacterial microbiome as a risk factor for estrogen receptor-positive (ER+) breast cancer. While diet is the main contributor to the gut microbiome, medications also shift the bacterial microbiome. We currently do not know whether oral endocrine targeting therapies, such as aromatase inhibitors or tamoxifen shift the gut microbiome. Furthermore, we do not know whether gut microbiome populations can influence drug efficacy. Methods: Fecal samples from human donors were placed into ex-vivo colonic bioreactors for stabilization (n=3). Bioreactors were untreated or treated with letrozole or tamoxifen citrate for 48 hours. Samples were collected, DNA isolated, and metagenomic sequencing performed to determine direct drug-bug interactions. C57BL/6 mice were placed on a healthy control (HC; 21% kcal from fat derived from olive oil and fish oil) or a Western diet (45% kcal from fat derived from corn oil, lard, and milkfat) for 6 weeks. Mice within dietary patterns were randomized and administered control, tamoxifen (TAM; 37 ppm tamoxifen citrate), or an aromatase inhibitor (AI; 5 ppm letrozole) for 16 weeks. Metagenomics sequencing were performed on fecal DNA samples at study endpoint. Female BALB/c mice fed a HC or Western diet were injected with bone metastatic 4T1.2ER+ breast cancer cells. Mice were administered tamoxifen citrate, oral probiotics, or a combination of TAM + probiotics for 3-weeks. Tumor volume, tumor weight, and lung weight were recorded at the end of the study. Hindlimbs were analyzed for metastatic lesions. Results: Metagenomic sequencing from the ex-vivo colonic bioreactors treated with aromatase inhibitors or tamoxifen display differential shifts in several β-glucuronidase-expressing and obesity-associated bacterial species suggesting AI and selective estrogen receptor modulators have varying effects in the gut microbiome that may influence estrogen bioavailability and metabolic parameters. C57BL/6 mice on HC or Western diet treated with AI or TAM also display differences in the microbiota and phage populations with TAM elevating Lactobacillus johnsonii and letrozole increasing Lactococcus lactis bacterial proportional abundance. In the tumor-bearing model, combination of oral Lactobacillus probiotics and TAM significantly reduced tumor weight when compared with the tumor weight in control, TAM, or probiotic treated mice fed a Western diet. Combination of probiotics and TAM also prevented the development of ER+ bone metastatic lesions. Conclusions: Our study indicates oral endocrine therapies differentially affect the gut microbiome and these drug-bug interactions are sensitive to dietary-influenced baseline microbiota populations, which may influence drug efficacy and metabolic outcomes. Furthermore, our preclinical studies suggest oral probiotic supplements may enhance tamoxifen efficacy to reduce tumor growth and metastatic development. Further clinical studies are needed on this topic. Citation Format: Alana Arnone, Adam S. Wilson, Akiko Chiba, Bethany Kerr, David R. Soto-Pantoja, Alexandra Thomas, Katherine L. Cook. Endocrine-targeted therapies modify the gut microbiome affecting responsiveness in ER+ breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-02-05.

Triple-negative breast cancer (TNBC) is highly aggressive with a poor 5-year survival rate. Targeted therapy options are limited and most TNBC patients are treated with chemotherapy. This study aimed to determine whether doxorubicin (Dox) shifts the gut microbiome and whether gut microbiome populations influence chemotherapeutic responsiveness. Female BALB/c mice (n = 115) were injected with 4T1-luciferase cells (a murine syngeneic TNBC model) and treated with Dox and/or antibiotics, high-fat diet-derived fecal microbiota transplant (HFD-FMT), or exogenous lipopolysaccharide (LPS). Metagenomic sequencing was performed on fecal DNA samples. Mice that received Dox were stratified into Dox responders or Dox nonresponders. Mice from the Dox responders and antibiotics + Dox groups displayed reduced tumor weight and metastatic burden. Metagenomic analysis showed that Dox was associated with increased Akkermansia muciniphila proportional abundance. Moreover, Dox responders showed an elevated proportional abundance of Akkermansia muciniphila prior to Dox treatment. HFD-FMT potentiated tumor growth and decreased Dox responsiveness. Indeed, lipopolysaccharide, a structural component of Gram-negative bacteria, was increased in the plasma of Dox nonresponders and FMT + Dox mice. Treatment with exogenous LPS increases intestinal inflammation, reduces Dox responsiveness, and increases lung metastasis. Taken together, we show that modulating the gut microbiota through antibiotics, HFD-FMT, or by administering LPS influenced TNBC chemotherapy responsiveness, lung metastasis, and intestinal inflammation.

In utero dietary exposures are linked to the development of metabolic syndrome in adult offspring. These dietary exposures can potentially impact gut microbial composition and offspring metabolic health. Female BALB/c mice were administered a lard, lard + flaxseed oil, high sugar, or control diet 4 wk before mating, throughout mating, pregnancy, and lactation. Female offspring were offered low-fat control diet at weaning. Fecal 16S sequencing was performed. Untargeted metabolomics was performed on visceral adipose tissue (VAT) of adult female offspring. Immunohistochemistry was used to determine adipocyte size, VAT collagen deposition, and macrophage content. Hippurate was administered via weekly intraperitoneal injections to low-fat and high-fat diet-fed female mice and VAT fibrosis and collagen 1A (COL1A) were assessed by immunohistochemistry. Lard diet exposure was associated with elevated body and VAT weight and dysregulated glucose metabolism. Lard + flaxseed oil attenuated these effects. Lard diet exposures were associated with increased adipocyte diameter and VAT macrophage count. Lard + flaxseed oil reduced adipocyte diameter and fibrosis compared with the lard diet. Hippurate-associated bacteria were influenced by lard versus lard + flax exposures that persisted to adulthood. VAT hippurate was increased in lard + flaxseed oil compared with lard diet. Hippurate supplementation mitigated VAT fibrosis pathology. Maternal high-fat lard diet consumption resulted in long-term metabolic and gut microbiome programming in offspring, impacting VAT inflammation and fibrosis, and was associated with reduced VAT hippurate content. These traits were not observed in maternal high-fat lard + flaxseed oil diet-exposed offspring. Hippurate supplementation reduced VAT fibrosis. These data suggest that detrimental effects of early-life high-fat lard diet exposure can be attenuated by dietary omega-3 polyunsaturated fatty acid supplementation.

Abstract Background The objective of this study was to increase understanding of the complex interactions between diet, obesity, and the gut microbiome of adult female non-human primates (NHPs). Subjects consumed either a Western (n=15) or Mediterranean (n=14) diet designed to represent human dietary patterns for 31 months. Body composition was determined using CT, fecal samples were collected, and shotgun metagenomic sequencing was performed. Gut microbiome results were grouped by diet and adiposity. Results Diet was the main contributor to gut microbiome bacterial diversity. Adiposity within each diet was associated with subtle shifts in the proportional abundance of several taxa. Mediterranean diet-fed NHPs with lower body fat had a greater proportion of Lactobacillus animalis than their higher body fat counterparts. Higher body fat Western diet-fed NHPs had more Ruminococcus champaneliensis and less Bacteroides uniformis than their low body fat counterparts. Western diet-fed NHPs had significantly higher levels of Prevotella copri than Mediterranean diet NHPs. Western diet-fed subjects were stratified by P. copri abundance (P. copriHIGH versus P. copriLOW), which was not associated with adiposity. Overall, Western diet-fed animals in the P. copriHIGH group showed greater proportional abundance of B. ovatus, B. faecis, P. stercorea, P. brevis, and Faecalibacterium prausnitzii than those in the Western P. copriLOW group. Western diet P. copriLOW subjects had a greater proportion of Eubacterium siraeum. E. siraeum negatively correlated with P. copri proportional abundance regardless of dietary consumption. In the Western diet group, Shannon diversity was significantly higher in P. copriLOW when compared to P. copriHIGH subjects. Furthermore, gut E. siraeum abundance positively correlated with HDL plasma cholesterol indicating that those in the P. copriLOW population may represent a more metabolically healthy population. Untargeted metabolomics on urine and plasma from Western diet-fed P. copriHIGH and P. copriLOW subjects suggest early kidney dysfunction in Western diet-fed P. copriHIGH subjects. Conclusions In summary, the data indicate diet to be the major influencer of gut bacterial diversity. However, diet and adiposity must be considered together when analyzing changes in abundance of specific bacterial taxa. Interestingly, P. copri appears to mediate metabolic dysfunction in Western diet-fed NHPs.

Breast tumors have their own specific microbiota, distinct from normal mammary gland tissue. Patients with breast cancer that present with locally advanced disease often undergo neoadjuvant chemotherapy to reduce tumor size prior to surgery to allow breast conservation or limit axillary lymph node dissection. The purpose of our study was to evaluate whether neoadjuvant chemotherapy modulates the tumor microbiome and the potential impact of microbes on breast cancer signaling. Using snap-frozen aseptically collected breast tumor tissue from women who underwent neoadjuvant chemotherapy (n = 15) or women with no prior therapy at time of surgery (n = 18), we performed 16S rRNA-sequencing to identify tumoral bacterial populations. We also stained breast tumor microarrays to confirm presence of identified microbiota. Using bacteria-conditioned media, we determined the effect of bacterial metabolites on breast cancer cell proliferation and doxorubicin therapy responsiveness. We show chemotherapy administration significantly increased breast tumor Pseudomonas spp. Primary breast tumors from patients who developed distant metastases displayed increased tumoral abundance of Brevundimonas and Staphylococcus. We confirmed presence of Pseudomonas in breast tumor tissue by IHC staining. Treatment of breast cancer cells with Pseudomonas aeruginosa conditioned media differentially effected proliferation in a dose-dependent manner and modulated doxorubicin-mediated cell death. Our results indicate chemotherapy shifts the breast tumor microbiome and specific microbes correlate with tumor recurrence. Further studies with a larger patient cohort may provide greater insights into the role of microbiota in therapeutic outcome and develop novel bacterial biomarkers that could predict distant metastases. Implications: Breast tumor microbiota are modified by therapy and affects molecular signaling.

Anthracyclines are standard-of-care chemotherapy for the treatment of triple-negative breast cancer (TNBC). However, high anthracyclines cumulative doses increase heart failure risk. Designing therapeutic strategies that ameliorate cardiac toxicities without compromising oncologic efficacy are important to improve TNBC outcomes and survivorship. The purpose of this study was to determine the impact of diet on TNBC chemotherapeutic responsiveness and development of chemotherapy-induced cardiac damage. Female BALB/c mice fed a control, Western, Mediterranean, or Western + fish oil diet were injected with 1 × 10

BackgroundTriple-negative breast cancer(TNBC) lacks druggable targets and has high metastatic incidence. Immune checkpoint blockades (ICB) are FDA approved for TNBC treatment, but therapeutic response and biomarkers are limited. CD47 is an integral membrane protein overexpressed on cancer cells that alters anti-tumor immunosurveillance, resulting in tumor progression. CD47 is involved in metabolic reprogramming but whether CD47 is a marker of progression and its role in ICB response for TNBC remains unknown.MethodsHuman TNBC biopsies were subjected to immunohistochemical analysis to determine CD47 role in TNBC progression. To determine CD47 impact on tumor burden, a carcinogen-induced TNBC model was performed in female wild type(WT) and cd47 null(cd47-/-) C57Bl/6 mice. To evaluate immune infiltrate signaling, tumors underwent spatial tissue proteomics by multiplexing photo-cleavable antibodies in Formalin-Fixed Paraffin-Embedded samples. An orthotopic EMT-6 murine TNBC model was performed to investigate tumor burden for CD47 monotherapy or in combination with anti-PD-L1 therapy.ResultsHuman matched primary, and metastatic TNBC biopsies increased immunoreactivity to CD47, signifying a potential therapeutic target(n=24). CD47 deficiency in the carcinogen-induced DMBA model decreased tumor incidence, weight, and area compared to WT(n=8/group,*pConclusionsOur data indicates that CD47 may serve as a marker of anti-PD-L1 response, and targeting CD47 enhances immunogenicity and decreases immunosuppressive molecules, sensitizing TNBC tumors to anti-PD-L1 therapy to reduce tumor burden.AcknowledgementsDSP is supported by the NCI R21 (CA249349) and the American Cancer Society Research Scholar Grant (133727-RSG-19-150-01-LIB). ERS is supported by the NIAID Immunology and Pathogenesis T32 Training Grant (T32AI007401).Ethics ApprovalAnimal studies were approved by the Institutional Care and Use Committee, Wake Forest Health Sciences.

Most women diagnosed with breast cancer (BC) have estrogen receptor alpha positive (ER+) disease. ER+ BC preferentially metastasizes to bone; at which time it is considered incurable. Treatments for bone metastasis have not advanced in decades, in part due to a lack of appropriate ER+ BC bone metastasis models. We developed an immunocompetent ER+ BC murine model with spontaneous bone metastasis and antiestrogen responsiveness. To do this, we transduced triple-negative (TN) bone-tropic murine BC cell lines 4T1.2 and E0771/Bone to express ERα. These cells were then injected into the mammary fat pads of Balb/c (n=21) or C57Bl/6 (n=27), respectively. Once tumors established, mice were treated with either the selective estrogen receptor modulator (SERM) tamoxifen (TAM), the selective estrogen receptor degrader (SERD) ICI 182,780 (ICI, Faslodex, fulvestrant), or vehicle control for 21 days. Tumor volumes and weights significantly decreased in the ER+ groups treated with TAM and ICI compared with ER+ vehicle-treated groups. Staining for immune profiles and total RNA sequencing demonstrated modified immune cell infiltration between TN and ER-derived tumors. Approximately 25% of the mice with ER+ 4T1.2 tumors developed metastases to long bones while none of the mice with TN tumors developed metastases. This immunocompetent ER+ 4T1.2 BC model may allow for further exploration of ER+ BC bone metastasis mechanisms and for the development of new therapeutics for women diagnosed with bone metastasis from ER+ BC.Simple SummaryEstrogen receptor alpha positive (ER+) breast cancer is the most common subtype of breast cancer. When it metastasizes to bone, it becomes incurable. Little advancement has occurred in the treatment of bone metastasis from ER+ breast cancer, partly due to the lack of animal models. To establish an animal model of ER+ BC, we genetically modified two triple-negative breast cancer cell lines to express ERα and injected the cell lines into murine mammary glands. Mice were treated with standard antiestrogen therapies, the selective estrogen receptor modulator tamoxifen or the selective estrogen receptor degrader ICI 182,780. We found that compared to mice with triple-negative breast cancer, mice with ER+ breast cancer developed bone metastases and were responsive to antiestrogen therapy. This model allows for further exploration of bone metastasis mechanisms and for the development of new therapeutics, translating into improved clinical outcomes for women with bone metastasis from ER+ breast cancer.

BackgroundCD47 is an integral membrane protein that alters adaptive immunosurveillance when bound to the matricellular glycoprotein thrombospondin-1 (TSP1). We examined the impact of the CD47/TSP1 signaling axis on melanoma patient response to anti-PD-1 therapy due to alterations in T cell activation, proliferation, effector function, and bioenergetics.MethodsA syngeneic B16 mouse melanoma model was performed to determine if targeting CD47 as monotherapy or in combination with anti-PD-1 impacted tumor burden. Cytotoxic (CD8+) T cells from Pmel-1 transgenic mice were used for T cell activation, cytotoxic T lymphocyte, and cellular bioenergetic assays. Single-cell RNA-sequencing, ELISA, and flow cytometry was performed on peripheral blood mononuclear cells and plasma of melanoma patients receiving anti-PD-1 therapy to examine CD47/TSP1 expression.ResultsHuman malignant melanoma tissue had increased CD47 and TSP1 expression within the tumor microenvironment compared with benign tissue. Due to the negative implications CD47/TSP1 can have on antitumor immune responses, we targeted CD47 in a melanoma model and observed a decrease in tumor burden due to increased tumor oxygen saturation and granzyme B secreting CD8+ T cells compared with wild-type tumors. Additionally, Pmel-1 CD8+ T cells exposed to TSP1 had reduced activation, proliferation, and effector function against B16 melanoma cells. Targeting CD47 allowed CD8+ T cells to overcome this TSP1 interaction to sustain these functions. TSP1 exposed CD8+ T cells have a decreased rate of glycolysis; however, targeting CD47 restored glycolysis when CD8+ T cells were exposed to TSP1, suggesting CD47 mediated metabolic reprogramming of T cells. Additionally, non-responding patients to anti-PD-1 therapy had increased T cells expressing CD47 and circulating levels of TSP1 compared with responding patients. Since CD47/TSP1 signaling axis negatively impacts CD8+ T cells and non-responding patients to anti-PD-1 therapy have increased CD47/TSP1 expression, we targeted CD47 in combination with anti-PD-1 in a melanoma model. Targeting CD47 in combination with anti-PD-1 treatment further decreased tumor burden compared with monotherapy and control.ConclusionCD47/TSP1 expression could serve as a marker to predict patient response to immune checkpoint blockade treatment, and targeting this pathway may preserve T cell activation, proliferation, effector function, and bioenergetics to reduce tumor burden as a monotherapy or in combination with anti-PD-1.

Background Immune checkpoint blockade therapy targeting PD-L1 has recently been approved for metastatic triple negative breast cancer (TNBC) patients. However, a 7% response rate calls for better models and strategies to stimulate TN-tumor immunogenicity to increase patient response. Overexpression of the receptor CD47 impairs innate and adaptive tumor immunosurveillance when engaged to its counter receptor SIRPα or ligand thrombospondin-1. Co-expression of CD47 and PD-L1 is implicated with disease progression in TNBC patients. We examined through murine models and tumor organoid platforms whether targeting CD47 sensitizes TNBC tumors to PD-L1 therapy, focusing on the modulation of cellular bioenergetics as a potential mechanism and potentially predict response. Methods The effects of targeting CD47 and PD-L1 were examined through orthotopic syngenic 4T1 and EMT-6 TNBC murine models. Due to predicting patient therapeutic response challenges, tumor organoid platforms investigated mechanisms of tumor sensitization to anti-PD-L1 by targeting CD47. Organoids were constructed by embedding murine TNBC tumor tissue and AH1 CD8+ T cells in a specialized ECM mimicking hydrogel. Immunohistochemistry was performed on organoid, human and murine TNBC tumor tissue. Cellular bioenergetics was analyzed through Seahorse® bioanalyzer. Results Staining of human TNBC biopsies found elevated CD47 expression, signifying a potential therapeutic target. Targeting CD47 or in combination with anti-PD-L1 resulted in decreased tumor volume and weight in a TNBC murine model. The decrease in tumor burden was correlated with increased intratumoral granzyme B secreting CD8+ T cells. Additionally, targeting CD47 within organoids increased IFNγ and granzyme B released, indicating enhanced CD8+ T cell cytolytic capacity. Differential cellular bioenergetics was observed between cancer and T cells suggesting a shift in metabolism in the tumor microenvironment. CD47 targeted T cells had an increased glycolytic rate compared to WT T cells. Conversely CD47 targeted TNBC cells had a decreased glycolytic rate, which may be correlated with decreased PD-L1 expression. Conclusions Targeting CD47 enhanced granzyme B and IFNγ expression suggesting potential mechanisms to increase tumor immunogenicity. CD47 targeted monotherapy or combination with anti-PD-L1 preserves T cell bioenergetics and antitumor function, resulting in decreased TNBC tumor burden. Alternatively, CD47 targeted TNBC had a decreased glycolytic rate and decreased PD-L1 expression, which is reported to regulate glycolysis through Akt/mTOR signaling. Targeting CD47 on T cells enhances their bioenergetics and antitumor function while decreasing TNBC cell bioenergetics, making them more susceptible to immune cell killing. Our data indicates that CD47 targeted monotherapy or combination with anti-PD-L1 may enhance TNBC patient response and improve overall survival. Acknowledgements DSP and SS are supported by the Wake Forest Comprehensive Cancer Center Breast Cancer Center of Excellence Pilot Award. DSP is also supported by the ASTRO-BCRF Career Development Award (637969) while ERS is supported by NIGMS T32 (GM127261). Ethics Approval Animal studies were approved by the Institutional Care and Use Committee, Wake Forest Health Sciences.

Obesity and poor diet often go hand-in-hand, altering metabolic signaling and thereby impacting breast cancer risk and outcomes. We have recently demonstrated that dietary patterns modulate mammary microbiota populations. An important and largely open question is whether the microbiome of the gut and mammary gland mediates the dietary effects on breast cancer. To address this, we performed fecal transplants between mice on control or high-fat diets (HFD) and recorded mammary tumor outcomes in a chemical carcinogenesis model. HFD induced protumorigenic effects, which could be mimicked in animals fed a control diet by transplanting HFD-derived microbiota. Fecal transplants altered both the gut and mammary tumor microbiota populations, suggesting a link between the gut and breast microbiomes. HFD increased serum levels of bacterial lipopolysaccharide (LPS), and control diet–derived fecal transplant reduced LPS bioavailability in HFD-fed animals. In vitro models of the normal breast epithelium showed that LPS disrupts tight junctions (TJ) and compromises epithelial permeability. In mice, HFD or fecal transplant from animals on HFD reduced expression of TJ-associated genes in the gut and mammary gland. Furthermore, infecting breast cancer cells with an HFD-derived microbiome increased proliferation, implicating tumor-associated bacteria in cancer signaling. In a double-blind placebo-controlled clinical trial of patients with breast cancer administered fish oil supplements before primary tumor resection, dietary intervention modulated the microbiota in tumors and normal breast tissue. This study demonstrates a link between the gut and breast that mediates the effect of diet on cancer. Significance: This study demonstrates that diet shifts the microbiome in the gut and the breast tumor microenvironment to affect tumorigenesis, and oral dietary interventions can modulate the tumor microbiota in patients with breast cancer.

Summary: Recent identification of a mammary gland-specific microbiome led to studies investigating bacteria populations in breast cancer. Malignant breast tumors have lower Lactobacillus abundance compared with benign lesions, implicating Lactobacillus as a negative regulator of breast cancer. Diet is a main determinant of gut microbial diversity. Whether diet affects breast microbiome populations is unknown. In a non-human primate model, we found that consumption of a Western or Mediterranean diet modulated mammary gland microbiota and metabolite profiles. Mediterranean diet consumption led to increased mammary gland Lactobacillus abundance compared with Western diet-fed monkeys. Moreover, mammary glands from Mediterranean diet-fed monkeys had higher levels of bile acid metabolites and increased bacterial-processed bioactive compounds. These data suggest that diet directly influences microbiome populations outside the intestinal tract in distal sites such as the mammary gland. Our study demonstrates that diet affects the mammary gland microbiome, establishing an alternative mechanistic pathway for breast cancer prevention. : Using a non-human primate model of women’s health, Shively et al. demonstrate that diet plays a critical role in determining microbiota populations in tissues outside the gut, such as the mammary gland. These microbial populations modulate localized bile acid and bacterial-modified metabolites to potentially influence anticancer signaling pathways. Keywords: breast, mammary gland, diet, microbiome, bile acid, hippurate, oxidative stress

BACKGROUND: A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. However, whether CD47 protects cancer cells from cytotoxic chemotherapy is unknown. METHODS: We tested the effect of CD47 blockade on cancer cell survival using a 2-dimensional high-throughput cell proliferation assay in 4T1 breast cancer cell lines. To evaluate blockade of CD47 in combination with chemotherapy in vivo we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. RESULTS: Our high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. CONCLUSIONS: Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, absence of CD47 in did not elicit a protective effect but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by the lack of druggable targets and an incidence of brain metastasis from the primary site of approximately 35%. There is no standard treatment for managing brain metastasis associated with TNBC; therefore, new strategies are urgently needed to overcome disease mortality. The CD47/SIRPα signaling pathway is implicated in tumor progression due to bypassing innate and adaptive immune surveillance. Most strategies targeting this pathway focus on targeting the receptor CD47; however, targeting SIRPα as a potential strategy to mitigate tumor burden remains understudied. Analysis of gene expression database shows that SIRPα expression is significantly elevated in invasive breast cancer when compared to primary. Furthermore, single-cell data indicates that SIRPα is expressed in basal epithelial cells in TNBC tumors aside from the myeloid compartment. Our immune staining against SIRPα in patient biopsies shows a five-fold increase in SIRPα expression in metastatic brain tumors compared to the primary lesions. Therefore, targeting SIRPα may be a new immunotherapeutic strategy to treat breast cancer brain metastases. Anti-SIRPα treatment of mice bearing brain metastatic 4T1br3 orthotopic tumors showed reduced tumor volume and tumor weight by over 50% compared to isotype control-treated mice. Furthermore, in a model of intracardial brain metastasis, treatment with SIRPα antibody was associated with a 60% increase in survival compared to isotype control-treated mice. RNA sequencing of tumors indicated that SIRPα blockade is associated with a reduction in genes linked to mitochondrial respiratory chain and increases in negative regulation of the cell cycle. Furthermore, in vitro SIRPα targeting enhanced the cell-mediated cytotoxicity of microglia against 4T1Br3 breast cancer cells. This suggests that SIRPα blockade may influence both tumor and innate immune cells to limit brain metastatic breast cancer growth and enhance survival.

// David R. Soto-Pantoja 1, 3 , Adam S. Wilson 1 , Kenysha YJ. Clear 1 , Brian Westwood 1 , Pierre L. Triozzi 2, 3 and Katherine L. Cook 1, 3 1 Department of Surgery, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2 Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 3 Comprehensive Cancer Center of Wake Forest Baptist Medical Center, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA Correspondence to: Katherine L. Cook, email: klcook@wakehealth.edu Keywords: breast cancer, melanoma, immune therapy, ipilimumab, CTLA4 Received: March 22, 2017 Accepted: July 23, 2017 Published: August 03, 2017 ABSTRACT The unfolded protein response (UPR) is a stress pathway controlled by GRP78 to mediate IRE1, PERK, and ATF6 signaling. We show that targeting GRP78, IRE1, and PERK differentially regulates macrophage polarization. Specifically, PERK targeting enhanced macrophage proliferation and macrophage-mediated killing but not GRP78 or IRE1. Targeting UPR in cancer cells also differentially affected macrophage cytolytic capacity. Tumoral IRE1 or GRP78 inhibition enhanced macrophage-mediated cancer cell clearance. Conditioned media from GRP78-silenced cancer cells caused reciprocal regulation of CD80 and CD206, suggesting control of plasticity by secreted factors. GRP78 targeting in mice resulted in a cytokine shift and increased tumoral CD80+/CD68+ cells, suggesting an M1-like profile. Targeting UPR in both macrophage and cancer cells indicates that PERK or GRP78 reduction enhances macrophage clearance of cancer cells. Recent evidence suggests that macrophage polarization influences immune checkpoint therapy resistance. To determine whether UPR effects immunotherapy resistance, analysis of matched melanoma patient PBMC before/after developing ipilimumab resistance demonstrated increased UPR signaling and an M2-like macrophage population, supporting a novel role of UPR signaling and innate immune regulation in anti-CTLA-4 therapy resistance. These data suggest that targeting GRP78 or PERK promotes an anti-tumor immune response by either directly promoting macrophage cytolytic activity or indirectly by shifting tumoral cytokine secretion.

The unfolded protein response (UPR) is an endoplasmic reticulum stress pathway controlled by the protein chaperone, glucose-regulated protein 78 (GRP78), to mediate inositol-requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6) signaling. UPR signaling has been shown to be upregulated in many different types of cancers, including breast cancer and melanoma, and is associated with the development of therapeutic resistance. These data suggest the importance of targeting UPR signaling as a possible cancer therapy. We have previously shown GRP78 to be upregulated in human breast tumor samples and leads to endocrine targeted therapy resistance. We recently showed inhibiting GRP78 in human orthotopic xenografts potentiates tamoxifen therapy effectiveness in sensitive tumors and restores endocrine therapy responsiveness in resistant tumors. In these GRP78-inhibited tumors there was a significant increase of CD68 positive macrophage population, suggesting that targeting UPR signaling has critical effects on the tumor microenvironment. Therefore, consideration of each UPR signaling component and how it effects the different cellular compartments of the tumor microenvironment need to be investigated to optimally induce both an antitumor immune effect and inhibit tumor epithelial cell growth. We now show deletion of GRP78, IRE1, and PERK through RNAi differentially regulates macrophage polarization. Specifically, PERK inhibition enhances macrophage proliferation and macrophage-mediated phagocytosis of 4T1 breast cancer cells, but not GRP78 or IRE1 inhibition. Targeting UPR signaling in the breast cancer cells also differentially affected macrophage cytolytic capacity; Specific breast cancer cell inhibition of IRE1 or GRP78 enhanced macrophage-mediated phagocytosis. Conditioned media from control or GRP78 silenced ZR-75-1 breast cancer cells indicated reciprocal regulation of CD206 and CD80; suggesting regulation of macrophage plasticity by GRP78-controlled secreted factors. GRP78 targeting in mice resulting in a cytokine shift and increased tumoral CD80+/CD68+ cells, suggesting that GRP78 inhibition favors a M1-like macrophage profile. Inhibition of UPR components in both macrophage and breast cancer cells, similar to what would be observed in systemic cancer therapies, indicated that either PERK or GRP78 inhibition enhances macrophage cytolytic clearance of breast cancer cells. Taken together, these data suggest that targeting GRP78 or PERK promotes an anti-tumor immune response by either directly promoting macrophage cytolytic activity (PERK targeting) or indirectly by shifting tumoral cytokine secretion (GRP78 targeting). Citation Format: Cook KL, Wilson A, Westwood B, Soto-Pantoja DR. Targeting of the unfolded protein response signaling arms differentially regulates macrophage proliferation, plasticity, and breast cancer cell clearance [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-06.

OBJECTIVES: Western diet consumption (WD) is associated with increased risk and poor prognosis for breast cancer (BC) due to altered inflammation, metabolism, and microbial colonization. This study will determine whether intervention with probiotics (prbx) and muscadine grape extract (MGE) reduces these effects. METHODS: Female C57Bl/6 mice were placed on either control diet (CD) or WD (45% fat and 25% sugar). Mice were randomized into six groups per diet: diet alone, antibiotics (abx), prbx, MGE, MGE + abx, and MGE + prbx (n = 8). Prbx groups received 1 × 10(5) CFU of a 10-strain probiotic 3x weekly. MGE (0.1 phenolics/mL) and abx (5 mg/mL streptomycin, 1 mg/mL ampicillin, 1 mg/mL colistin) were administered in drinking water. Body weights were measured, and feces was collected for 16S sequencing. EchoMRI was performed on mice (total body adiposity) and livers at the end of the study (13 weeks). Immunohistochemistry (IHC) was used to compare mammary gland (MG) and visceral adipose (VA) inflammation (CD68 and MCP-1). To determine whether dietary interventions effected breast cancer growth, female C57Bl6 mice consuming either CD or WD, were injected with 2.5 × 10(5) E0771-luc triple negative breast cancer cells into the R4/5 MG. Tumors progressed to 100 mm3 prior to treatment. Size was monitored with calipers and IVIS for 21 days. RESULTS: MGE + prbx administration in WD-fed mice resulted in reduced body weight. All intervention groups displayed reduced VA and MG weight compared to WD-fed mice. Significant intervention-mediated gut microbial alterations included changes in proportional abundance of Bacteroidetes, Lactococcus, Lactobacillus, and Bifidobacterium taxa. Interventions modulated VA and MG inflammatory markers. Dietary intervention with MGE, prbx, and MGE + prbx reduced E0771 tumor growth rate in CD-fed mice, but not in WD-fed mice. CONCLUSIONS: Our data suggests that MGE + prbx modulates diet-induced metabolic, inflammatory, and microbial factors. Treatment with MGE + prbx reduced tumor growth rate in CD-fed mice, but not WD. WD-fed mice developed aggressive tumors that treatments were unable to attenuate. Further analyzing tumor tissue will determine whether MGE + prbx altered the tumor microenvironment to improve CD-associated BC prognosis. FUNDING SOURCES: Chronic Disease Research Fund.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype with a 5-year survival rate significantly worse when compared to other breast cancer types. There are no targeted therapy options, limiting these patients to cytotoxic chemotherapy regimens. Our group demonstrated increased tumoral Pseudomonas abundance in breast cancer patients receiving neoadjuvant chemotherapy, suggesting that systemic anticancer therapy administration may modulate microbiota populations. The purpose of our study was to determine whether gut microbiota populations correlate with chemotherapeutic responsiveness and may be a predictive biomarker outcome. We wanted to determine the impact of shifting the microbiome on chemotherapy efficacy. To do so, 8-week old female BALB/c mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were either untreated (control group), treated with 1x weekly 2.5 mg/kg doxorubicin (DOX) for 4 weeks, or treated with doxorubicin +antibiotics (mixture of streptomycin, ampicillin, and colistin in the drinking water to ablate the microbiome). Tumor size was monitored. Tumors and lungs were collected after the study. Fecal samples were collected at T0 (before treatment) and T4 (after treatment). Mice receiving DOX were stratified into DOX-responders or DOX-nonresponders based upon tumor size. Mice from DOX-responders and DOX +antibiotics groups displayed reduced tumor weight and decreased lung metastatic burden. 16S-bacterial sequencing indicates elevated fecal Ruminococcus correlates with DOX-nonresponsiveness and increased abundance of Oscillospira and Bacteroidales is associated with better therapeutic outcome. Protein analysis of tumor tissue indicates a significant increase in apoptosis in DOX-responders and DOX +antibiotic groups. To determine whether modulating the gut microbiome influences drug responsiveness, BALB/c mice were stratified into a control group or group receiving lard diet-derived fecal transplant (LDFT) by oral gavage. Mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were treated with 1x weekly 2.5 mg/kg DOX for 4 weeks. Tumors and lungs were collected at the end of the study. Fecal samples were collected at T0 (before gavage), T3 (after 3 weeks of LDFT and before DOX-treatment), and T7 (at end of study). All tumors from LDFT-group were larger and displayed reduced chemotherapy responsiveness when compared with control animals, suggesting gut microbiome populations can modulate chemotherapy resistance. Taken together, our data demonstrate that chemotherapy efficacy is modulated by gut microbiome and suggest that modulation of the gut microbiome through dietary or probiotic interventions may affect therapeutic outcomes. Moreover, fecal microbiota populations could be used as a predictive biomarker of chemotherapeutic responsiveness. Citation Format: Kenysha Clear, Adam Wilson, Akiko Chiba, Katherine L. Cook, Alaa Bawaneh. Gut microbiome populations modulate neoadjuvant chemotherapy responsiveness in preclinical triple-negative breast cancer murine model [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B15.

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancers and predominately affects young and minority women. TNBC is characterized by a high recurrence rate, with about 34% of TNBC patients relapsing around 2.6 years. This type of breast cancer lacks targeted therapy options and is limited to chemotherapy treatment options. The purpose of our study was to determine whether gut microbiota populations correlate with chemotherapeutic responsiveness and may be a predictive biomarker of outcome. Moreover, we want to determine the impact of targeting the microbiome on chemotherapy efficacy. In this study, 8-week old female BALB/c mice were injected with TNBC 4T1-luciferase cells into their L4/5 mammary fat pad. Once tumors developed, mice were either untreated (control group), treated with 1 x weekly 2.5 mg/kg IV doxorubicin (DOX), or treated with doxorubicin + antibiotics (mixture of streptomycin, ampicillin, and colistin in the drinking water to ablate host bacterial populations). Tumors were collected at the end of the study. Fecal samples were collected at time point 0 weeks (T0; before therapy when tumors were approximately 100 mm3) and time point 4 weeks (T4; after treatment). We subdivided the doxorubicin treated group into DOX-responders (tumors stopped growing or shrank) or DOX-nonresponders (tumors continued to grow on treatment). Tumors from the DOX-responders and DOX + antibiotics groups were significantly smaller and mice from these groups displayed reduced lung weight, suggesting decreased lung metastatic burden. 16S-bacterial sequencing analysis was performed on breast tumor tissue and feces. We demonstrate that at T0, elevated fecal Ruminococcus correlates with DOX-nonresponsiveness. Furthermore, at T4, we show increased fecal abundance of Oscillospira and Bacteroidales are associated with better therapeutic outcome. Protein analysis of primary 4T1 breast tumor tissue indicate a significant elevation of apoptosis (cleaved caspase-3 protein levels) in DOX-responders and DOX + antibiotic treated mice. We also stained breast tumor tissue and lung tissue for bacteria to confirm presence of microbiota by immunohistochemistry. Taken together our data demonstrates that chemotherapy efficacy is modulated by the microbiome. Moreover, fecal microbiota populations could be used as a predictive biomarker of chemotherapeutic responsiveness and suggests that modulation of the gut microbiome through dietary or probiotic interventions may affect therapeutic outcomes. Citation Format: Alaa Bawaneh, Adam S. Wilson, Kenysha YJ Clear, Akiko Chiba, Katherine L. Cook. Gut microbiota population may be used to predict chemotherapeutic responsiveness in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 650.

The development of immunotherapy is a major recent advance in clinical oncology. However the majority of patients do not respond and those who do, experience resistance and relapse. Therefore, understanding the molecular mechanisms of immune checkpoint inhibitor resistance is critical to develop combinatorial drug strategies to potentiate therapeutic responsiveness to reduce mortality. Highly secretory cells, such as T-cells, have a larger endoplasmic reticulum (ER) to handle the increased protein translation capacity required by the cell. Therefore, T-cells may be highly sensitive to ER stress. The high nutrient needs of the tumor deplete resources in the microenvironment subjugating infiltrating T-cells to reduced nutrient availability resulting in stress. Our data indicate that co-culturing melanoma cells with T-cells increase T-cell specific unfolded protein response (UPR) signaling. Stimulating ER stress decreased cytolytic T-cell function in TALL-104 T-cells, enabling Mun2b melanoma survival. Knockdown of PERK restored T-cell killing capacity in ER stress induced cells. In an ex-vivo model of antigen-specific mediated T-cell death, Pmel-1 T-cells stimulated with gp100 displayed increased B16 melanoma killing when treated with GSK2606414 (a small molecule PERK inhibitor). PERK inhibition also elevated glycolysis proteins and mitochondrial bioenergetics in T-cells, suggesting PERK inhibition increases T-cell metabolism. As proof-of-concept, we determined the effect of PERK inhibition in a syngeneic B16 melanoma model. Male C57/Bl6 mice were inoculated with B16 melanoma cells. At 5 days post injection, mice were treated with a control IgG, antisense morpholino targeting PERK, PD1 antibody, or a combination of PERK morpholino and PD1 antibody. PERK morpholino treatment alone was sufficient to reduce tumor volume by 46.5%. PD1 antibody therapy alone reduced tumor volume by 47.4%. The combination of PD1 antibody and PERK targeting therapy significantly reduced B16 melanoma tumor volume by 64.5% demonstrating that targeting PERK in vivo enhanced immunocheckpoint therapy efficacy. Treated B16 tumors were homogenized and infiltrating T-cells isolated by flow cytometry. Gated CD3+ infiltrating leukocytes were then counted for CD8 and PD1 expression. Tumor infiltrating CD8+ T-cells doubled with PERK inhibition alone, giving further proof indicating that PERK may be a novel immune checkpoint target. Small molecule PERK inhibitors were previously shown to cause β-islet damage and insulin resistance; therefore we tested the effect of our PERK morpholino on glucose tolerance in the B16 melanoma model. PERK morpholino treatment had no overall impact on glucose tolerance and therefore may be a novel therapeutic to induce T-cell metabolism and potentiate immune checkpoint therapy efficacy with reduced off-target toxicities. Citation Format: David R. Soto-Pantoja, Yismeilin R. Feliz-Mosquea, Kenysha YJ Clear, Adam S. Wilson, Katherine L. Cook. Targeting PKR-like endoplasmic reticulum kinase modulates metabolism to promote T-cell effector function and PD1 immunotherapy responsiveness [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 449.

Dysfunction of infiltrating CD8+ effector T cells can be induced by hypoxia and aberrant tumor metabolite uptake in the microenvironment causing an “exhausted” T cell phenotype that limits anti-tumor immunosurveilance. Hence- therapeutic strategies aimed at improving T cell bioenergetics have the potential to reinvigorate T cell responses to reduce tumor burden. CD47 is a widely expressed receptor that controls phagocytic activity by engaging its counter receptor, SIRPα, in macrophages. Also autonomously or by binding to its ligand Thrombospondin-1 CD47 activation can control cell fate under stress. Our prior work shows that targeting CD47 on CD8+ T cells enhanced cytotoxicity against cancer cells. Moreover, depletion of CD8+ T cells in murine models reversed the anti-tumor effect of anti-CD47 therapy. Our new data shows that targeting CD47 reduced the growth of B16 melanoma tumors by approximately 50% (1607 ± 213.7, saline vs. 815.1 ± 67.8, CD47 (-) *p Citation Format: Yismelin R. Feliz-Mosquea, Elizabeth Stirling, Katherine L. Cook, Adam Wilson, Manish Bharadwaj, Anthony J. Molina, Liliya Yamaleyeva, Pierre L. Triozzi, David R. Soto-Pantoja. Anti-CD47 immunotherapy regulates T cell metabolism and hypoxia in the tumor microenvironment [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 3528.

Macrophage mediated elimination of cancer cells is critical for control of tumor growth. Cancer cells evade immune recognition by expressing receptors that send inhibitory signals to prevent macrophage mediated cytotoxicity. Clinical studies show that overexpression of the cell surface receptor CD47 is a poor prognostic factor in cancer, allowing tumors to bypass immunosurveillance and activation pro-growth pathways. Prior studies have described that CD47 cell surface expression on cancer cells inhibits phagocytic activity through engagement of its counter receptor SIRPα on macrophages. However, the role of CD47 expression on macrophage function is not known. Here, we show that CD47 is expressed on primary mouse macrophages, RAW 264.7 and the U937 cell lines. Moreover, knockdown of CD47 on macrophages enhanced the tumoricidal capacity against melanoma and breast cancer cells as measured by cell impedance and LDH release. Blockade of CD47 enhanced the macrophage-mediated cytotoxicity against B16 melanoma and 4T1 cells when compared to untreated effector/target pairs. Blockade of CD47 on differentiated U937 human monocytes also enhanced tumoricidal activity against the triple-negative breast cancer cell line MDA-MB-231 by over 50% when compared to untreated co-cultures. This indicates that expression of CD47 on macrophages may play a role in polarization and macrophage tumoricidal capacity against cancer cells beyond the known interactions with SIRPα. Our previous studies show that blockade of CD47 preserves T cell viability under stress by increasing glycolytic flux. Activation of M1 cytotoxic macrophages requires a shift in metabolism from oxidative phosphorylation to glycolysis. Our data shows that blockade of CD47 on RAW 264.7 macrophages increases glycolytic flux when compared to untreated controls as measured by extracellular acidification rate (ECAR) using a Seahorse© XF analyzer. The increase in glycolysis was associated with a significant elevation of glucose uptake by macrophages treated with anti-sense CD47 morpholino, indicating that targeting this receptor regulates glucose metabolism to improve macrophage tumoricidal activity. Interestingly, blockade of CD47 in the 4T1 breast cancer cell line reduced ECAR and reduced glucose uptake indicating that targeting CD47 reverses Warburg metabolism in cancer cells. Overall, these results show a new role of CD47 expression that extends beyond suppressing macrophage mediated killing through engagement of SIRPα. Therefore, anti-CD47 immunotherapy may regulate immunometabolism to improve clinical outcomes of cancer patients. Citation Format: Ashley A. Smith, Adam S. Wilson, David R. Soto-Pantoja. Targeting CD47 expression on macrophages regulates immunometabolism enhancing tumoricidal activity against cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5661. doi:10.1158/1538-7445.AM2017-5661

CD47 is a widely expressed cell surface receptor that serves as a counter-receptor for SIRPα in recognition of self by the innate immune system. On the other hand CD47 also serves as a signaling receptor for the matricellular protein thrombospondin-1 (TSP-1), regulating cell growth and survival. Clinical studies consistently show that increased expression of CD47 is an independent poor prognosis factor in several types of cancer, including breast cancer. Moreover elevated expression of CD47 is associated with development of resistance to chemotherapy by inhibiting cell death pathways. Therefore, agents targeting CD47 are attractive to overcome therapeutic resistance in breast cancer. Using a high-throughput assay in a 1536-well microplate format we screened a comprehensive set of approved and late stage development oncology drugs in combination with an anti-CD47 morpholino to find combinatorial strategies that are more cytotoxic against breast cancer cells. Potency and efficacy measurements during the primary screen identified compounds that synergized with anti-CD47 to augment their cytotoxicity effect. One of the compound groups that demonstrated increased synergism with CD47 were the anthracycline family of drugs. We further validated these results in a syngeneic model of breast cancer and demonstrated that blockade of CD47 in combination with doxorubicin improves chemotherapeutic response when compared to mice administered doxorubicin alone. Furthermore the anti-tumor response with anti-CD47 combination is associated with a reduction in glycolytic flux and a selective up regulation of mitophagy. Overall this indicates that blockade of CD47 in the clinic may synergize with chemotherapeutic strategies to improve patient curative responses. Citation Format: David D. Roberts, Ashley Smith, John M. Sipes, Adam Wilson, Lesley Mathews-Griner, Rajarshi Guha, Craig J. Thomas, Marc Ferrer, David R. Soto-Pantoja. High-throughput matrix screening reveals synergistic chemotherapeutic combinations with blockade of CD47 to enhance cytotoxicity in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1352.