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Figure 1. Multiplex immunofluorescence (multiplex IF) staining for markers of suppressive T regulatory (CD4+FoxP3+) and exhausted T cells (CD8+PD-1+TIM3+Tox/Tox2+). This mouse liver metastasis harbors abundant expression of both T cell populations.
Test the impact of suppressive T cells on progression and treatment resistance of breast cancer liver metastasis
Recent ground breaking work demonstrated that anti-tumor cytotoxic CD8 T cells are selectively depleted in liver metastases. However, our profiling of human breast cancer liver metastases showed that some tumors harbor abundant levels of both CD8 and CD4 T cells. These metastases also had elevated expression of T cell exhaustion markers, suggesting that some breast cancer liver metastases may maintain suppressive T cells populations. This idea was confirmed in a mouse model of breast cancer liver metastasis where abundant regulatory and exhausted T cells were detected in metastasis (Figure 1). The goal of this project is to assess the effects of tumor cell signaling, starting with tumor cell-heme metabolism, on these pro-tumor T cell populations and immunotherapy resistance in breast cancer liver metastases. This work will include targeted inhibition and activation of heme metabolism in breast cancer liver metastasis models followed by analysis of T cell populations using flow cytometry and multispectral imaging approaches.
Future work will continue to test the impact of tumor cell signaling on recruited immune cells. This will be achieved by comparing pathways differentially expressed in breast cancer liver metastases that have abundant immune cell infiltration with those that are immune ignored or have few immune infiltrates.
Assess the effects of tumor cell secreted metabolites on tissue resident immune cell function
The liver is a unique immune tolerant organ composed of many specialized cells; however, the role of tissue resident immune cells in breast cancer liver metastasis remains underexplored. My postdoctoral work showed that secreted byproducts of tumor cell heme metabolism suppress normal macrophage function including efferocytosis or dead cell engulfment. Conversely, these metabolites enhance pro-tumor macrophages function such as T cell suppression. The goal of this project is to test the impact of these metabolites on liver resident macrophages, or Kupffer cell, function. This work will include utilization and development of assays to assess Kupffer cell efferocytosis (as seen in Figure 2) and antigen presentation in tissue culture and animal models.
Current work is also determining the molecular mechanism used by heme metabolites to affect macrophages. In the future we hope to assess the impact of these metabolites on additional liver resident cell populations such as hepatic stellate cells and hepatocytes.
Figure 2. Kupffer cells (liver resident macrophages) were co-cultured with dead tumor cells dyed with a fluorescent marker that emits a bright red signal in the high pH of the macrophage lysosome. In this experiment, the IncuCyte imaging system was used to track red signal or dead cell engulfment known as efferocytosis.
Figure 3. Summary figure of metabolites and metabolic enzymes altered in 66Cl-4 mammary tumors upon treatment with the heme oxygenase-1 (HO-1) inhibitor tin mesoporphyrin (SnMP). In green are metabolites and orange are enzymes that were altered with SnMP treatment.
Test the effect of tumor cell-heme metabolism on metabolic rewiring in breast cancer liver metastasis
Previous studies demonstrate that tumor cells acquire a unique metabolic program to survive in the liver, a metabolically active and hypoxic organ. Our preliminary data suggests that the heme degrading enzyme heme oxygenase-1 (HO-1) regulates additional metabolic pathways. For example, inhibition of HO-1 in mammary tumors shifted metabolism from glycolysis to fatty acid oxidation (Figure 3). The goal of this project is to understand if tumor cell heme metabolism contributes to the metabolic shift that supports liver metastasis.
Future work will assess the impact of heme metabolism on a immune suppressive metabolite milieu in liver metastasis models and identify the mechanism by which HO-1 regulates additional metabolic pathways.
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