174P - Human adipose stem cells transfer their mitochondria to breast cancer patient derived organoids increasing drug resistance

Background

Mitochondrial transfer (MT) from mesenchymal stem cells showed to restore damaged cell function in inflammatory disease models. We showed that breast cancer (BC) cells acquire mitochondria from human adipose stem cells (hASCs) via tunneling nanotubes (TNTs), promoting multidrug resistance. Here we aimed at evaluating if MT occurs in patient-derived organoids (PDO) co-cultured with hASC.

Methods

We generated PDOs and matched hASCs from consenting BC patients. Fresh tissues were mechanically and enzymatically digested and cultured with proper medium. Organoids were characterized by immunocytochemistry and hASCs by flow cytometry (FC). A 2D-3D co-culture was set up, plating hASCs with PDOs with/without insert: hASCs mitochondria were stained with MitoTracker Red CMXRos® and PDOs cytoplasm with Cell Tracker Blue®. MT was analyzed by immunofluorescence microscopy and FC. Moreover, a TNT inhibitor, Cytochalasin B, was added to the co-culture to evaluate if TNTs are involved in MT. PDOs were subjected to Mitoception (MCP) and treated with cisplatin. Cell viability was assessed with CCK8® assay.

Results

We successfully generated and characterized PDOs from luminal BC patients, and showed that they maintained the same hormone receptor profile and showed cell heterogeneity. Furthermore, we generated primary hASCs from the same patients which showed a FC CD45-CD324-CD34-CD29+CD44+CD73+CD90+CD105+ pattern. We set up a hybrid co-culture model with 3D PDOs and 2D hASCs, showing that MT occurs massively in direct co-culture, but also (at lower level) with insert. Indeed, when treating with Cytochalasin B MT was not blocked, indicating that it occurs with additional mechanisms than TNTs. To validate the effect of MT on drug resistance, we forced hASCs-mitochondria internalization into PDOs via MCP, and treated them with cisplatin, observing an increase in PDOs viability with respect to those not subjected to MCP.

Conclusions

We confirmed that MT occurs in a more physiological model such as PDOs and matched hASCs, in which it reduces drug response. It appears as a key process that could drive tumor aggressiveness, whose better understanding could help to design more effective treatment strategies to overcome drug resistance.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

The authors.

Funding

MIUR - PRIN 2022.

Disclosure

F. Papaccio: Financial Interests, Institutional, Funding, Liberal Contribution: Merck. All other authors have declared no conflicts of interest.