Anti-CD47 immunotherapy as a therapeutic strategy for the treatment of breast cancer brain metastasis.

The presence of cell surface protein CD47 allows cancer cells to evade innate and adaptive immune surveillance resulting in metastatic spread. CD47 binds to and activates SIRPα on the surface of myeloid cells, inhibiting their phagocytic activity. On the other hand, CD47 binds the matricellular protein Thrombospondin-1, limiting T-cell activation. Thus, blocking CD47 is a potential therapeutic strategy for preventing brain metastasis. To test this hypothesis, breast cancer patient biopsies were stained with antibodies against CD47 to determine differences in protein expression. An anti-CD47 antibody was used in a syngeneic orthotopic triple-negative breast cancer model, and CD47 null mice were used in a breast cancer brain metastasis model by intracardiac injection of the E0771-Br-Luc cell line. Immunohistochemical staining of patient biopsies revealed an 89% increase in CD47 expression in metastatic brain tumors compared to normal adjacent tissue (p ≤ 0.05). Anti-CD47 treatment in mice bearing brain metastatic 4T1br3 orthotopic tumors reduced tumor volume and tumor weight by over 50% compared to control mice (p ≤ 0.05) and increased IBA1 macrophage/microglia marker 5-fold in tumors compared to control (p ≤ 0.05). Additionally, CD47 blockade increased the M1/M2 macrophage ratio in tumors 2.5-fold (p ≤ 0.05). CD47 null mice had an 89% decrease in metastatic brain burden (p ≤ 0.05) compared to control mice in a brain metastasis model. Additionally, RNA sequencing revealed several uniquely expressed genes and significantly enriched genes related to tissue development, cell death, and cell migration tumors treated with anti-CD47 antibodies. Thus, demonstrating that CD47 blockade affects cancer cell and tumor microenvironment signaling to limit metastatic spread and may be an effective therapeutic for triple-negative breast cancer brain metastasis.

Targeting the CD47/thrombospondin-1 signaling axis regulates immune cell bioenergetics in the tumor microenvironment to potentiate antitumor immune response.

BACKGROUND: CD47 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. METHODS: A 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. RESULTS: Human 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. CONCLUSION: CD47/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.

Immuno-reactive cancer organoid model to assess effects of the microbiome on cancer immunotherapy.

Immune checkpoint blockade (ICB) therapy has demonstrated good efficacy in many cancer types. In cancers such as non-resectable advanced or metastatic triple-negative breast cancer (TNBC), it has recently been approved as a promising treatment. However, clinical data shows overall response rates (ORRs) from ~ 3-40% in breast cancer patients, depending on subtype, previous treatments, and mutation status. Composition of the host-microbiome has a significant role in cancer development and therapeutic responsiveness. Some bacterial families are conducive to oncogenesis and progression, while others aid innate and therapeutically induced anti-tumor immunity. Modeling microbiome effects on anti-tumor immunity in ex vivo systems is challenging, forcing the use of in vivo models, making it difficult to dissect direct effects on immune cells from combined effects on tumor and immune cells. We developed a novel immune-enhanced tumor organoid (iTO) system to study factors affecting ICB response. Using the 4T1 TNBC murine cell line and matched splenocytes, we demonstrated ICB-induced response. Further administration of bacterial-derived metabolites from species found in the immunomodulatory host-microbiome significantly increased ICB-induced apoptosis of tumor cells and altered immune cell receptor expression. These outcomes represent a method to isolate individual factors that alter ICB response and streamline the study of microbiome effects on ICB efficacy.

Circulating Immune Bioenergetic, Metabolic, and Genetic Signatures Predict Melanoma Patients' Response to Anti-PD-1 Immune Checkpoint Blockade.

PURPOSE: Immunotherapy with checkpoint inhibitors is improving the outcomes of several cancers. However, only a subset of patients respond. Therefore, predictive biomarkers are critically needed to guide treatment decisions and develop approaches to the treatment of therapeutic resistance. EXPERIMENTAL DESIGN: We compared bioenergetics of circulating immune cells and metabolomic profiles of plasma obtained at baseline from patients with melanoma treated with anti-PD-1 therapy. We also performed single-cell RNA sequencing (scRNAseq) to correlate transcriptional changes associated with metabolic changes observed in peripheral blood mononuclear cells (PBMC) and patient plasma. RESULTS: Pretreatment PBMC from responders had a higher reserve respiratory capacity and higher basal glycolytic activity compared with nonresponders. Metabolomic analysis revealed that responder and nonresponder patient samples cluster differently, suggesting differences in metabolic signatures at baseline. Differential levels of specific lipid, amino acid, and glycolytic pathway metabolites were observed by response. Further, scRNAseq analysis revealed upregulation of T-cell genes regulating glycolysis. Our analysis showed that SLC2A14 (Glut-14; a glucose transporter) was the most significant gene upregulated in responder patients' T-cell population. Flow cytometry analysis confirmed significantly elevated cell surface expression of the Glut-14 in CD3+, CD8+, and CD4+ circulating populations in responder patients. Moreover, LDHC was also upregulated in the responder population. CONCLUSIONS: Our results suggest a glycolytic signature characterizes checkpoint inhibitor responders; consistently, both ECAR and lactate-to-pyruvate ratio were significantly associated with overall survival. Together, these findings support the use of blood bioenergetics and metabolomics as predictive biomarkers of patient response to immune checkpoint inhibitor therapy.

In Vitro Cell Impedance Assay to Examine Antigen-Specific T-Cell-Mediated Melanoma Cell Killing to Support Cancer Immunotherapy Drug Discovery.

The design of cancer immunotherapy drugs is essential for the continued investigation of novel drug regimens to improve responses and increase the survival of cancer patients. Methods to examine the interaction of effector immune cells with target cancer cells are limited by labor-intensive labeling that can be examined at specific time points. In this report, we examine an antigen-dependent model of effector cytotoxic (CD8+) T-cell-mediated cytotoxicity of target murine melanoma cells using a real-time cell impedance assay. The real-time monitoring allows measurement of viability and kinetics, allowing for a better understanding of effector/target cell interactions to support drug discovery.

Metabolic Implications of Immune Checkpoint Proteins in Cancer.

Expression of immune checkpoint proteins restrict immunosurveillance in the tumor microenvironment; thus, FDA-approved checkpoint inhibitor drugs, specifically PD-1/PD-L1 and CTLA-4 inhibitors, promote a cytotoxic antitumor immune response. Aside from inflammatory signaling, immune checkpoint proteins invoke metabolic reprogramming that affects immune cell function, autonomous cancer cell bioenergetics, and patient response. Therefore, this review will focus on the metabolic alterations in immune and cancer cells regulated by currently approved immune checkpoint target proteins and the effect of costimulatory receptor signaling on immunometabolism. Additionally, we explore how diet and the microbiome impact immune checkpoint blockade therapy response. The metabolic reprogramming caused by targeting these proteins is essential in understanding immune-related adverse events and therapeutic resistance. This can provide valuable information for potential biomarkers or combination therapy strategies targeting metabolic pathways with immune checkpoint blockade to enhance patient response.

Metabolomic Analysis Reveals Unique Biochemical Signatures Associated with Protection from Radiation Induced Lung Injury by Lack of cd47 Receptor Gene Expression.

The goal of this study was to interrogate biochemical profiles manifested in mouse lung tissue originating from wild type (WT) and cd47 null mice with the aim of revealing the in vivo role of CD47 in the metabolic response to ionizing radiation, especially changes related to the known association of CD47 deficiency with increased tissue viability and survival. For this objective, we performed global metabolomic analysis in mouse lung tissue collected from (C57Bl/6 background) WT and cd47 null mice with and without exposure to 7.6 Gy whole body radiation. Principal component analysis and hierarchical clustering revealed a consistent separation between genotypes following radiation exposure. Random forest analysis also revealed a unique biochemical signature in WT and cd47 null mice following treatment. Our data show that cd47 null irradiated lung tissue activates a unique set of metabolic pathways that facilitate the handling of reactive oxygen species, lipid metabolism, nucleotide metabolism and nutrient metabolites which may be regulated by microbial processing. Given that cd47 has pleiotropic effects on responses to ionizing radiation, we not only propose this receptor as a therapeutic target but postulate that the biomarkers regulated in this study associated with radioprotection are potential mitigators of radiation-associated pathologies, including the onset of pulmonary disease.

Thrombospondin-1 interactions regulate eicosanoid metabolism and signaling in cancer-related inflammation.

The metabolism of arachidonic acid and other polyunsaturated fatty acids produces eicosanoids, a family of biologically active lipids that are implicated in homeostasis and in several pathologies that involve inflammation. Inflammatory processes mediated by eicosanoids promote carcinogenesis by exerting direct effects on cancer cells and by affecting the tumor microenvironment. Therefore, understanding how eicosanoids mediate cancer progression may lead to better approaches and chemopreventive strategies for the treatment of cancer. The matricellular protein thrombospondin-1 is involved in processes that profoundly regulate inflammatory pathways that contribute to carcinogenesis and metastatic spread. This review focuses on interactions of thrombospondin-1 and eicosanoids in the microenvironment that promote carcinogenesis and how the microenvironment can be targeted for cancer prevention to increase curative responses of cancer patients.