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Background: Neuropeptide Y (NPY) is an abundant neurohormone in human breast carcinomas that acts on a class of G-protein coupled receptors, of which NPY1R and NPY5R are the most highly expressed. This abundance is exploited for cancer imaging, but there is interest in pharmacological inhibition of the NPYRs to interrogate their functional relevance in breast cancer. We previously reported that NPY1R and NPY5R mRNA abundance is increased by hypoxia inducible factors, which sensitizes these receptors to NPY stimulation leading to enhanced migration and proliferation. Methods/Results: Here, we measured the effects of NPY1R and NPY5R antagonists in normoxia and hypoxia on migration, proliferation, invasion, and signaling in 2D and 3D models of breast cancer cell lines MDA-MB-231 and MCF7. Antagonizing NPY1R and/or NPY5R in hypoxia compared to normoxia more greatly reduced MAPK signaling, cell proliferation, cell migration and invasion, and spheroid growth and invasion. The estrogen receptor positive MCF7 cells were significantly less invasive in 3D spheres when NPY5R was specifically inhibited. There were some discrepancies in the responses of each cell line to the isoform-specific antagonists and oxygen availability, therefore further investigations are required to dissect the intricacies of NPYR signaling dynamics. In human breast tumor tissue, we show via immunofluorescence that NPY5R protein levels and colocalization with hypoxia correlate with advanced cancer, and NPY1R protein correlates with adverse outcomes. Conclusions: Antagonizing the NPYRs has been implicated as a treatment for a wide variety of diseases. Therefore, these antagonists may aid in the development of novel cancer therapeutics and patient-based treatment plans. [ABSTRACT FROM AUTHOR]

Spheroids enable the study of tumors and tumor hypoxia using a more representative model of the physiological environment compared to 2D cell culture. Spheroids can be grown in a cell suspension or when adhered to a solid scaffold. The spheroid formation method used is dependent on cell type. Here we describe the most common spheroid formation methods, including hanging drop, low adhesion plates, hydrogel, micropatterned plates, and microfluidics. After spheroids are formed, they can be used for drug treatment trials and analyzed using Western Blots, qPCR, and microscopy. Microscopy can then be used to measure the invasiveness of cells when a basement membrane is added to spheroids and for monitoring changes in the proliferation, quiescent, and necrotic zones of spheroids., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)