Involvement of Nanoscale Cellular Communication in Obesity-Assisted Breast Cancer Severity

Introduction: Obesity is a risk factor for several cancers, including breast cancer, and contributes up to 20% of cancer-related deaths.1,2 Growing evidence from both clinical and preclinical studies indicates that increased obesity is associated with increases in cancer incidence, progression, metastasis, and therapeutic resistance.3-5 Various signaling mechanisms have been proposed related to the severity of obesity-associated breast cancer, which includes those mediated by insulin, leptin, adipokine, and aromatase signaling pathways1,3,4. Moreover, cancer-associated adipocytes impose a sustained abundance of energy available to the breast cancer cells to fulfill the high energy demand of cancer, which is known as one of the hallmarks of cancer. According to the Centers for Disease Control and Prevention (CDC), more than 70% and 40% of American adults can be classified as overweight and obese, respectively. However, treatment strategies for dealing with breast cancer in obese patients have not advanced apart from lifestyle interventions. Thus, translational research initiatives are immediately needed to provide mechanistic justifications for these striking statistics to serve this growing segment of the population better. We have shown, in a recent Nature Nanotechnology paper, that cancer cells hijack mitochondria from T cells via nanoscale physical communication and use them for their energy production.6 In the current work, we demonstrate that cancer cells acquire metabolic advantage by hijacking mitochondria from obese adipocytes through physical nanotubular communications (Figure 1). The cancer cell phenotype with increased metabolic power in obese patients results in increased proliferation and drug resistance.

Materials and

Methods: We have used human subcutaneous pre-adipocytes and MDA-MB-231 and MCF-7 cancer cell lines. We have used a primary adipocyte isolated from the mammary fat pad of a C57BL6 PhAMexcised mouse, which expresses Dendra2 fluorescent protein in mitochondria, and a human adipocyte cell line differentiated from pre-adipocytes (ATCC). Obese adipocytes were generated by incubating fully differentiated adipocytes (confirmed by CD36 staining) in basal DMEM medium supplemented with 1 mM of a 1:2:1 palmitate (C16:0), oleate (C18:1), and linoleate (C18:2). The coculture was established by mixing obese adipocytes and cancer cells in a mixture of the corresponding media. We have optimized the media composition in order to achieve the maximum viability of both cells. We have used scanning electron microscopy and optical microscopy to investigate the nanoscale communication between cancer cells and adipocytes. We have labeled the mitochondria of adipocytes by Ad-Mito-DsRed to investigate the transfer of mitochondria to cancer cells. Phalloidin green staining was used to investigate the nanotube formation using optical microscopy. We used the Nikon Ti2 Nikon Eclipse Ti camera and Agilent Novocyte flow cytometer to evaluate mitochondria transfer from adipocyte to cancer cells. Metabolic analysis was carried out using the XFe96 extracellular flux analyzer (Seahorse Bioscience). The standard Mito stress test was performed with the stepwise injection of oligomycin (1 μM), FCCP (1.0 μM), and a mixture of rotenone (0.5 μM) and antimycin A (0.5 μM) in XF base medium (Agilent) supplemented with 10mM glucose, 1mM sodium pyruvate, and 2mM l-glutamine, and adjusted to a pH of 7.4.

Results, Conclusions, and Discussions: Results and

Discussion: Here, we demonstrate the role of nanoscale physical communication between breast cancer cells and adipose tissue in promoting cancer progression. Using high-resolution scanning electron microscopy, we have found that MDA-MB-231 cells form nanoscale tubular communications with obese adipocytes (Figure 2A). The nanotubular communication can range from 10 m to approximately 300 m. The phalloidin staining of the nanotube observed in optical microscopic images has revealed that the nanotubes are primarily formed by actin cytoskeletal elements (Fig. 2B,C). The optical microscopy shows the transfer of DsRed stained mitochondria from obese adipocytes to cancer cells (Fig.2B,C). The transfer of mitochondria from adipocyte to cancer cells was preferentially unidirectional. The quantification of mitochondria transfer by flow cytometry analysis shows 60-70% of the cancer cells with DsRed+ phenotype (Figure 3A). A negligible amount of mitochondria transfer was overseeded when cancer cells and adipocytes were added to the upper and lower compartments of the Boyden chamber separated by a permeable membrane, allowing exosomal and paracrine communication and restricting the nanotube communication (Figure 3B). This observation suggests that the primary mode of mitochondria transfer is through nanotubular communication, not through exosomes. A significantly higher rate of mitochondria transfer from obese adipocytes to cancer cells has been observed than lean adipocytes, signifying the influence of obesity on cancer progression (Figure 3C). The transfer of mitochondria results in an increase in the metabolism of the cancer cells (3C). We have seen an increased metabolism of Mito-DsRed+ cancer cells than monoculture Mito- phenotype, measured in terms of oxygen consumption rate (Fig.3D). The in vivo investigation of mitochondria transfer in obese mice is in progress.

Conclusions: Here, we show mitochondria transport from obese adipocytes to cancer cells as a new mechanism for how breast cancer acquires metabolic advantage in obese patients. The increased metabolism of breast cancer cells promotes tumor progression, metastasis, and drug resistance, resulting in catastrophic outcomes for the patient. The mechanistic investigation of the nanotube-mediated mitochondria transfers and inhibition using novel pharmacological inhibitors holds tremendous potential for a therapeutic strategy for the treatment of obesity-assisted cancer progression. Studies on that line are in progress.

Acknowledgements (Optional): REFERENCE:
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2 Widschwendter, P. et al. The influence of obesity on survival in early, high-risk breast cancer: results from the randomized SUCCESS A trial. Breast Cancer Res 17, 129 (2015). https://doi.org:10.1186/s13058-015-0639-3
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5 Parekh, N., Chandran, U. & Bandera, E. V. Obesity in cancer survival. Annu Rev Nutr 32, 311-342 (2012). https://doi.org:10.1146/annurev-nutr-071811-150713
6 Saha, T. et al. Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells. Nature Nanotechnology 17, 98-106 (2022). https://doi.org:10.1038/s41565-021-01000-4