(D-126) Cancer cells as immune vampires

Introduction:: The interaction between cancer cells with immune cells has a profound impact on metabolism related to immune evasion and tumor growth. Several paracrine and exosomal communication has been implicated in transferring proteins, metabolites, and cell organelles, in increased cell proliferation, drug resistance and immune evasion. We have recently found that cancer cell extends long extracellular tentacles known as tunneling nanotubes to hijack mitochondria from T cells and other stromal cells. Cancer cells use hijacked mitochondria to augment their metabolism and proliferation, whereas the immune cell depletes because of energy starvation. This is one of the reasons for the limited efficacy of immunotherapy, where T cells are metabolically deactivated even before the immune checkpoint inhibitor can have any effect on them. We have published this immune evasion mechanism of cancer cells in Nature Nanotechnology. Blocking of such nanotube-mediated mitochondria transfer mechanisms has a potential clinical impact in controlling the metabolic vulnerabilities of the cancer cells and reducing tumor growth. Recently we have found ways to block the nanotube-mediated mitochondria transfer by pharmacological inhibitors and RNAi-mediated gene knockdown strategies, resulting in reduced tumor growth. We introduced bioengineered RNAi therapeutics, which can selectively knockdown the exocyst proteins (such as sec3 and sec5). We have also used molecular dynamics simulation to introduce pharmacological inhibitors for the nanotube-mediated mitochondria transfer and selectively deliver it to cancer cells using a nanomedicine approach. Bioengineered therapeutics have shown a significant increase in immune response and a reduction in cancer cell metabolism resulting in reduced tumor growth.


Materials and Methods:: We have used high-resolution field-emission scanning electron microscopy and confocal microscopy to investigate the nanoscale physical communication between cancer cells and stromal cells. Mitochondria transfer has been evaluated by monitoring the fluorescently labeled mitochondria and genotyping studies. We have established a coculture of cancer and stromal cells and characterized and functional implication of the nanotube-mediated mitochondria transfer by monitoring metabolism using Seahorse. The pharmacological drugs were identified using molecular dynamics simulation and tested in vitro and in vivo for their activity. The RNAis were loaded into cationic liposomes and functionalized with DSPE-PEG for increased retention time and stability. We have tested the hypothesis in human and mouse breast (MDA-MB-231, 4T1), lung (A549 and LLC), and melanoma (B16-F10) cell lines. We have used PhAM^excised mice, which ubiquitously express a mitochondrially localized version of the Dendra2 fluorescent protein.


Results, Conclusions, and Discussions:: Results and Discussion: Trafficking of mitochondria from T cells to cancer cells via physical nanotubes is a new mechanism by which cancer cells undergo immune evasion (Fig.1). We have shown, in accordance with the literature report, that nanotubes are primarily formed by the actin cytoskeletal element (Fig.1B). Here we found exocyst-GTPase complex has a profound impact on actin cytoskeletal remodeling and nanotube formation. We have used siRNA-mediated knockdown studies to show the reduction of nanotube formation and mitochondria transport from T cells to cancer cells as a strategy to inhibit the nanotube-mediated mitochondria transfer (Fig.1C). We have used bioengineered liposomal nanoparticles to deliver the siRNA to the cancer cells, which knockdown the exocyst proteins and reduces the mitochondria transfer. On the other hand, we have used molecular dynamics simulation to introduce a novel pharmacological inhibitor that inhibits exocyst-GTPase complex and reduces nanotube-mediated mitochondria transfer. The pharmacological inhibitor can downregulate the metabolic augmentation of the cancer cells, activates the T cells metabolically, and offer reduced tumor growth. The siRNA and pharmacological inhibitors were formulated for nanotherapeutics and have significantly reduced tumor growth in aggressive breast cancer mouse tumor models. An increase in T cell population and a reduction of the metabolism of the tumor cells has been observed. A significantly increased efficacy of the immunotherapy has been observed in the 4T1 syngeneic tumor model, which is not responsive to immunotherapy alone.


Conclusions: Our study sheds insights on a novel therapeutic strategy by blocking the nanotube-mediated metabolic augmentation of cancer cells. Inhibition of such mechanism with novel pharmacological inhibitor or RNAi allows reduction of cancer cell metabolism and restoring the immune response, resulting in reduced tumor growth. The increased efficacy of the combination with PD1 inhibitors opens the possibility of developing these as novel immunotherapy agents for cancer. Taken together, the inhibition of nanotube-mediated mitochondrial hijacking of cancer cells emerges as an approach for developing next-generation anticancer therapeutics. It can be used as adjuvant therapy to reduce tumor growth and increase the objective response of traditional immunotherapy.


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References (Optional): : 1. Saha, T., Dash, C., Jayabalan, R. et al. Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells. Nat. Nanotechnol. 17, 98–106 (2022). https://doi.org/10.1038/s41565-021-01000-4

2. Dash, C.; Saha, T.; Sengupta, S.; Jang, H.L. Inhibition of Tunneling Nanotubes between Cancer Cell and the Endothelium Alters the Metastatic Phenotype. Int. J. Mol. Sci. 22, 6161 (2021). https://doi.org/10.3390/ijms22116161