Mitochondrial Transfer of Cancer Associated Fibroblast Increases Breast Cancer Invasion

Introduction: Cancer-associated fibroblasts (CAFs) have both pro- and anti-tumorigenic roles in the tumor microenvironment. For weakly migratory breast cancer cells, CAFs increase invasion by directly leading cancer cells in collective migration. However, the role of CAFs for aggressive breast cancer cells, which are able to lead invasion independently, is unclear. Recently, CAFs have been implicated in altering the metabolic profile of breast cancer cells. Here, we aim to characterize the cooperative role of CAF-cancer cell interactions and their role in aggressive breast cancer cell migration. We observed that CAFs unidirectionally transfer mitochondria to breast cancer cells via tunneling nanotubes (TNTs), which heightens the breast cancer cells’ metabolism and increases their migration.

Materials and Methods: Spheroids were generated containing MDA-MB-231 breast cancer cells alone or in co-culture with breast CAFs expressing mCherry (2:1 ratio). Spheroids labeled with CellTracker Green were embedded in 4.5 mg/ml collagen gels with or without CAFs in collagen and invasion and migration were evaluated over time using a Zeiss LSM800 confocal microscope. Cytochalasin B and metformin were used to inhibit TNT formation. Mitochondria, actin and cell membrane were labelled with MitoTracker Red, Alexa 568-conjugated phalloidin and Alexa 633-conjugated WGA, respectively. Cancer cells were conditioned by co-culturing with CAFs expressing an inducible Caspase-9 in 2D culture and were then treated with B/B homodimerizer to induce CAF apoptosis. Mitochondria were isolated from CAFs using differential centrifugation and transferred to breast cancer cells through centrifugation at 1,500 × g for 5 min at 4'C. ATP concentration was measured using the ATPlite assay (Perkin Elmer), and ATP production rate with or without sodium pyruvate was measured using the Seahorse XF Real-Time ATP Rate Assay.

Results, Conclusions, and Discussions: While collective migration was observed in all conditions (Fig. 1A), increased outgrowth was seen in breast cancer cell-CAF co-culture spheroids when compared to spheroids containing MDA-MB-231 cells alone with or without CAFs in the surrounding collagen (Fig. 1B). Given that the presence of CAFs in the surrounding matrix did not significantly increase invasion, we hypothesized that contact-dependent communication between CAFs and cancer cells was necessary for the migratory advantage seen. Tunneling nanotube (TNT) formation (Fig. 1C) and unidirectional mitochondrial exchange from CAFs to cancer cells in 2D culture were observed. Upon blocking TNT formation with Metformin and Cytochalasin B, cancer cell migration in co-culture spheroids decreased, suggesting TNT formation is contributing to increased cancer cell migration. To test whether the transferred mitochondria alone were sufficient to promote the migration of cancer cells in 3D, breast cancer cells were pre-conditioned by either co-culture with Caspase-9 inducible CAFs in 2D or by transferring mitochondria directly before spheroid formation. Both pre-conditioning methods resulted in increased cancer cell migration and invasion. Mitochondrial transfer was also found to increase oxidative phosphorylation without reducing glycolysis, which increased the total ATP (Fig. 1D-E). To determine if increasing mitochondrial ATP production without CAF-mitochondrial transfer imparts a portion of the migratory advantage seen in co-culture or pre-conditioning, pyruvate was used to exogenously supplement the spheroid media. Interestingly, the addition of pyruvate increased the mitochondrial ATP production rate at the cost of the glycolytic ATP production rate, for an unchanged total ATP production rate and decreased spheroid invasion (Fig. 1F). Our data shows that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes that allow for the exchange of cargo between cell types. The unidirectional transfer of mitochondria from CAFs to cancer cells shifts energy metabolism pathways, increases total ATP concentration, and promotes the migration of breast cancer cells.

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