Macrophage subtypes inhibit breast cancer proliferation in culture.

Macrophages are a highly plastic cell type that adopt distinct subtypes and functional states depending on environmental cues. These functional states can vary wildly, with distinct macrophages capable of displaying opposing functions. We sought to understand how macrophage subtypes that exist on two ends of a spectrum influence the function of other cells. We used a co-culture system with primary human macrophages to probe the effects of macrophage subtypes on breast cancer cell proliferation. Our studies revealed a surprising phenotype in which both macrophage subtypes inhibited cancer cell proliferation compared to cancer cells alone. Of particular interest, using two different proliferation assays with two different breast cancer cell lines, we showed that differentiating macrophages into a "pro-tumor" subtype inhibited breast cancer cell proliferation. These findings are inconsistent with the prevailing interpretation that "pro-tumor" macrophages promote cancer cell proliferation and suggest a re-evaluation of how these interpretations are made.

CSPG4-targeting CAR-macrophages inhibit melanoma growth.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophage's ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma. STATEMENT OF SIGNIFICANCE: We engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth in vivo . Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.

Transferred mitochondria accumulate reactive oxygen species, promoting proliferation.

Recent studies reveal that lateral mitochondrial transfer, the movement of mitochondria from one cell to another, can affect cellular and tissue homeostasis. Most of what we know about mitochondrial transfer stems from bulk cell studies and have led to the paradigm that functional transferred mitochondria restore bioenergetics and revitalize cellular functions to recipient cells with damaged or non-functional mitochondrial networks. However, we show that mitochondrial transfer also occurs between cells with functioning endogenous mitochondrial networks, but the mechanisms underlying how transferred mitochondria can promote such sustained behavioral reprogramming remain unclear. We report that unexpectedly, transferred macrophage mitochondria are dysfunctional and accumulate reactive oxygen species in recipient cancer cells. We further discovered that reactive oxygen species accumulation activates ERK signaling, promoting cancer cell proliferation. Pro-tumorigenic macrophages exhibit fragmented mitochondrial networks, leading to higher rates of mitochondrial transfer to cancer cells. Finally, we observe that macrophage mitochondrial transfer promotes tumor cell proliferation in vivo. Collectively these results indicate that transferred macrophage mitochondria activate downstream signaling pathways in a ROS-dependent manner in cancer cells, and provide a model of how sustained behavioral reprogramming can be mediated by a relatively small amount of transferred mitochondria in vitro and in vivo.

Genetic Modification of Primary Human Myeloid Cells to Study Cell Migration, Activation, and Organelle Dynamics.

Myeloid dendritic cells (DCs) and macrophages are mononuclear phagocytes with key roles in the immune system. As antigen-presenting cells, they link innate detection of microbes with programming adaptive immune responses. Myeloid DCs and macrophages also play critical roles in development, promote tissue homeostasis, and direct repair in response to injury and inflammation. As cellular migration and organelle dynamics are intimately connected with these processes, it is necessary to develop tools to track myeloid cell behavior and function. Here, we build on previously established protocols to isolate primary human myeloid cells from peripheral blood and report an optimized method for their genetic modification with lentiviral vectors to study processes related to cell migration, activation, and organelle dynamics. Specifically, we provide a protocol for delivering genetically encoded fluorescent markers into primary monocyte-derived DCs (MDDCs) and monocyte-derived macrophages (MDMs) to label mitochondria, peroxisomes, and whole cells. We describe the isolation of primary CD14+ monocytes from peripheral blood using positive selection with magnetic beads and, alternatively, isolation based on plastic adherence. Isolated CD14+ cells can be transduced with lentiviral vectors and subsequently cultured in the presence of cytokines to derive MDDCs or MDMs. This protocol is highly adaptable for cotransduction with vectors to knock down or overexpress genes of interest. These tools enable mechanistic studies of genetically modified myeloid cells through flow cytometry, fluorescence microscopy, and other downstream assays. © 2022 Wiley Periodicals LLC. Basic Protocol: Transduction of MDDCs and MDMs with lentiviral vectors encoding fluorescent markers Alternate Protocol 1: Isolation of monocytes by plastic adhesion Alternate Protocol 2: Transduction of MDDCs and MDMs with lentiviral vectors to knock down or overexpress genes of interest Support Protocol 1: Production and purification of lentiviral vectors for transduction into primary human myeloid cells Support Protocol 2: Flow cytometry of MDDCs and MDMs Support Protocol 3: Fixed and live-cell imaging of fluorescent markers in MDMs and MDDCs.

Novel mechanism for OSM-promoted extracellular matrix remodeling in breast cancer: LOXL2 upregulation and subsequent ECM alignment.

BACKGROUND: Invasive ductal carcinoma (IDC) is a serious problem for patients as it metastasizes, decreasing 5-year patient survival from > 95 to ~ 27%. The breast tumor microenvironment (TME) is often saturated with proinflammatory cytokines, such as oncostatin M (OSM), which promote epithelial-to-mesenchymal transitions (EMT) in IDC and increased metastasis. The extracellular matrix (ECM) also plays an important role in promoting invasive and metastatic potential of IDC. Specifically, the reorganization and alignment of collagen fibers in stromal ECM leads to directed tumor cell motility, which promotes metastasis. Lysyl oxidase like-2 (LOXL2) catalyzes ECM remodeling by crosslinking of collagen I in the ECM. We propose a novel mechanism whereby OSM induces LOXL2 expression, mediating stromal ECM remodeling of the breast TME. METHODS: Bioinformatics was utilized to determine survival and gene correlation in patients. IDC cell lines were treated with OSM (also IL-6, LIF, and IL-1ß) and analyzed for LOXL2 expression by qRT-PCR and immunolabelling techniques. Collagen I contraction assays, 3D invasion assays, and confocal microscopy were performed with and without LOXL2 inhibition to determine the impact of OSM-induced LOXL2 on the ECM. RESULTS: Our studies demonstrate that IDC patients with high LOXL2 and OSM co-expression had worse rates of metastasis-free survival than those with high levels of either, individually, and LOXL2 expression is positively correlated to OSM/OSM receptor (OSMR) expression in IDC patients. Furthermore, human IDC cells treated with OSM resulted in a significant increase in LOXL2 mRNA, which led to upregulated protein expression of secreted, glycosylated, and enzymatically active LOXL2. The expression of LOXL2 in IDC cells did not affect OSM-promoted EMT, and LOXL2 was localized to the cytoplasm and/or secreted. OSM-induced LOXL2 promoted an increase in ECM collagen I fiber crosslinking, which led to significant fiber alignment between cells and increased IDC cell invasion. CONCLUSIONS: Aligned collagen fibers in the ECM provide pathways for tumor cells to migrate more easily through the stroma to nearby vasculature and tissue. These results provide a new paradigm through which proinflammatory cytokine OSM promotes tumor progression. Understanding the nuances in IDC metastasis will lead to better potential therapeutics to combat against the possibility.

Co-Expression of VEGF and IL-6 Family Cytokines is Associated with Decreased Survival in HER2 Negative Breast Cancer Patients: Subtype-Specific IL-6 Family Cytokine-Mediated VEGF Secretion.

Breast cancer cell-response to inflammatory cytokines such as interleukin-6 (IL-6) and oncostatin M (OSM) may affect the course of clinical disease in a cancer subtype-dependent manner. Furthermore, vascular endothelial growth factor A (VEGF) secretion induced by IL-6 and OSM may also be subtype-dependent. Utilizing datasets from Oncomine, we show that poor survival of invasive ductal carcinoma (IDC) breast cancer patients is correlated with both high VEGF expression and high cytokine or cytokine receptor expression in tumors. Importantly, epidermal growth factor receptor-negative (HER2-), but not HER2-positive (HER2+), patient survival is significantly lower with high tumor co-expression of VEGF and OSM, OSMRß, IL-6, or IL-6Rα compared to low co-expression. Furthermore, assessment of HER2- breast cancer cells in vitro identified unique signaling differences regulating cytokine-induced VEGF secretion. The levels of VEGF secretion were analyzed by ELISA with siRNAs for hypoxia inducible factor 1 α (HIF1α) and signal transducer and activator of transcription 3 (STAT3). Specifically, we found that estrogen receptor-negative (ER-) MDA-MB-231 cells respond only to OSM through STAT3 signaling, while ER+ T47D cells respond to both OSM and IL-6, though to IL-6 to a lesser extent. Additionally, in the ER+ T47D cells, OSM signals through both STAT3 and HIF1α. These results highlight that the survival of breast cancer patients with high co-expression of VEGF and IL-6 family cytokines is dependent on breast cancer subtype. Thus, the heterogeneity of human breast cancer in relation to IL-6 family cytokines and VEGF may have important implications in clinical treatment options, disease progression, and ultimately patient prognosis.