Mitochondrial transfer from Adipose stem cells to breast cancer cells drives multi-drug resistance

Abstract

Background: Breast cancer (BC) is a complex disease, showing heterogeneity in the genetic background, molecular subtype, and treatment algorithm. Historically, treatment strategies have been directed towards cancer cells, but these are not the unique components of the tumor bulk, where a key role is played by the tumor microenvironment (TME), whose better understanding could be crucial to obtain better outcomes.

Methods: We evaluated mitochondrial transfer (MT) by co-culturing Adipose stem cells with different Breast cancer cells (BCCs), through MitoTracker assay, Mitoception, confocal and immunofluorescence analyses. MT inhibitors were used to confirm the MT by Tunneling Nano Tubes (TNTs). MT effect on multi-drug resistance (MDR) was assessed using Doxorubicin assay and ABC transporter evaluation. In addition, ATP production was measured by Oxygen Consumption rates (OCR) and Immunoblot analysis.

Results: We found that MT occurs via Tunneling Nano Tubes (TNTs) and can be blocked by actin polymerization inhibitors. Furthermore, in hybrid co-cultures between ASCs and patient-derived organoids we found a massive MT. Breast Cancer cells (BCCs) with ASCs derived mitochondria (ADM) showed a reduced HIF-1α expression in hypoxic conditions, with an increased ATP production driving ABC transporters-mediated multi-drug resistance (MDR), linked to oxidative phosphorylation metabolism rewiring.

Conclusions: We provide a proof-of-concept of the occurrence of Mitochondrial Transfer (MT) from Adipose Stem Cells (ASCs) to BC models. Blocking MT from ASCs to BCCs could be a new effective therapeutic strategy for BC treatment.

Keywords: Adipose Stem cells; Breast Cancer; Mitoception; Mitochondrial transfer; Multi-drug resistance; Tunneling nanotubes.

Fig. 1

MT occurs between pdASCs and BCCs via TNTs and Actin polymerization inhibition disrupts TNTs inhibiting MT. a-b Fluorescence microscopy of pre-labelled MCF7 or MDA-MB.231 (CellTracker-Blue) and pre-stained pdASCs (MitoTracker-Red) with F-Actin (Phalloidin-FITC). The yellow arrows point-out the pdASC mitochondria along the TNTs and into recipient BCCs (100X; Fig. 1b is the merge of two fields of view, required for the capture of the entire TNTs length). c-d Flow cytometry analysis of the cell fluorescence, for the quantization of the MT occurring from the pre-stained MitoTracker-FITC pdASCs to the pre-labelled CellTracker-Blue MCF7 or MDA-MB.231. The co-culture has been set up in presence or not of a multi-well insert that avoided the cell-to-cell contact (****P ≤ 0.0001). e–f Flow cytometry analysis of the pdASCs mitochondria fluorescence, into the recipient BCCs subset, after treatment with Antimycin A (100 nM), Cyt-B (2,5 uM), CCCP (5 uM) and MdiVi-1 (10 uM). g MTT assay for the evaluation of the viability of the cells used, in our co-culture system, after treatment with Cyt-B. For all the cell lines, the viability rate at 2,5 uM was higher than 85%. h-i Fluorescence microscopy of the co-culture with CellTracker-Blue pre-labelled MCF7 or j-k MDA-MB.231, for the detection of the F-Actin (FITC) and β-Tubulin (TRITC) in presence of Cyt-B (2,5 uM). The yellow arrows point-out the TNTs structures between the two kinds of cells (h-i) or the cytoplasmic F-actin aggregates (j-k) in both BCCs and pdASCs. (****P ≤ 0.0001)

Fig. 2

MT occurs both between pdASCs and human primary 2D and 3D cell models. a Fluorescence microscopy (magnification 100X) of pre-labelled BCAHC-1 (CellTracker-Blue) and pre-stained pdASCs (MitoTracker-Red) with F-Actin (Phalloidin-FITC). Yellow arrows point-out the pdASC mitochondria along the TNTs and into recipient BCAHC-1 cell. b Flow cytometry analysis of the cell fluorescence, for the quantization of the MT occurring from the pre-stained MitoTracker-FITC pdASCs to the pre-labelled CellTracker-Blue BCAHC-1. The co-culture has been set up in presence or not of a multi-well insert that avoided the cell-to-cell contact (****P ≤ 0.0001). c MT inhibition in co-culture between pdASCs and BCAHC-1. Flow cytometry analysis of the pdASCs mitochondria fluorescence, into the recipient BCAHC-1 subset, after treatment with Antimycin A (100 nM), Cyt-B (2,5 uM), CCCP (5 uM) and MdiVi-1 (10 uM) d MTT assay for the evaluation of the BCAHC-1 viability after treatment with Cyt-B. e–f Fluorescence microscopy of the co-culture with CellTracker-Blue pre-labelled BCAHC-1, for the detection of the F-Actin (FITC) and β-Tubulin (TRITC) in presence of Cyt-B (2,5 uM). The yellow arrows point-out the TNTs structures between the two kinds of cells (e) or the cytoplasmic F-actin aggregates (f) in both BCAHC-1 and pdASCs. (****P ≤ 0.0001). g Confocal microscopy 3D orthogonal reconstruction, z-stack technology of a 2D/3D hybrid coculture shows the pre-labelled BCC-66 (CellTracker-Blue) and pre-stained pdASCs (MitoTracker-Red) with F-Actin (Phalloidin-FITC). The white arrows in the YZ plane point-out that the pdASCs are in contact with the BCC-66 and the yellow arrow indicates the presence of exogenous mitochondria in the cytoplasm of BCC-66. h Flow cytometry analysis of the cell fluorescence, for the quantization of the MT occurring from the pre-stained MitoTracker-FITC pdASCs to the pre-labelled CellTracker-Blue BCC-66, also after treatment with Cyt-B (2 uM). The co-culture has been set up in presence or not of a multi-well insert that avoided the cell-to-cell contact

Fig. 3

Construction and validation of MitoCeption model. a MCP assay workflow, from pdASCs/hASCs hTERT mitochondria-derived isolation to the forced engulfment into the BCCs. b Confocal microscopy 3D orthogonal reconstruction, with z-stack technology, of the pre-labelled CellTracker-Blue BCCs (MCF-7) after the MCP with MitoTracker-FITC pre-labelled pdASCs derived mitochondria (c) Fluorescence microscopy and spatial co-localization statistical analysis of BCCs and MitoTracker stained pdASCs derived mitochondria at time points 0 h and 24 h (Li’s ICQ value analysis normalized on the total cells area; ****P ≤ 0.0001)

Fig. 4

MCP increases BCCs viability under chemotherapy treatment and impacts on their metabolism, modifying mitochondrial respiration. a HIF-1α expression analysis in BCCs subjected to MCP, in N-OX and H-OX conditions. The MCP significantly reverted the up-regulation of HIF-1α in the BCCs after their stimulation with the chemical hypoxia inducer cobalt chloride (100 uM). b-c Cell viability and apoptosis assay d-e of the BCCs subjected to MCP in different oxygen conditions, after treatment with the chemotherapeutic drugs DTX (50 nM) or CIS (10 uM) (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ****P ≤ 0.0001). f-g SeaHorse ATP rate assay for the analysis of the mitochondrial respiration in MCF-7 and (h-i) MDA-MB.231, 24 hours after the MCP, in different oxygen conditions. The histograms represent the ATP production rate (pmol/min), calculated during the oxygen consumption phase (****P ≤ 0.0001)

Fig. 5

ABC transporters expression is influenced by oxygen conditions and is upregulated under doxorubicin treatment. Flow cytometry (plots) and fluorescent microscopy analysis for the quantitative and qualitative evaluation of the P-gp, ABCG2 and ABCC1 expression, in MCF-7 (a-c) and MDA-MB.231 (d-f) 6 h after treatment with Doxorubicin (1 µM for MDA-MB.231; 2 µM for MCF-7), in N-OX or H-OX environment (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ****P ≤ 0.0001)

Fig. 6

MCP is protective in MDA-MB.231 treated with Doxorubicin and promotes the ABC efflux activity in BCCs. In MDA-MB.231 (a-b) treated with DOX (1 µM), the cell counts and confluence significantly increase after MCP in both N-OX (#cells/cm² = ***P ≤ 0.001, %confluence = (**P ≤ 0.01), and H-OX (#cells/cm.² = ***P ≤ 0.01, %confluence = (**P ≤ 0.05) environments. Live time-lapse microscopy (capture from movie) (c-d) of MDA-MB.231, shows the cell population growth differences after MCP in both N-OX and H-OX conditions from T0 to T24 at a single well point. The inhibition of the ABC transporter with VER (5 µM) significantly blocks the efflux capacity of P-gp and ABCG2, independently of the cell line and the oxygen levels. The MCF-7 (e–f) and MDA-MB.231 (g-h) have been cultured in N-OX or H-OX micro-environment, and subsequently treated with DOX (1 µM for MDA-MB.231; 2 µM for MCF-7) together with the metabolic regulators 2-DG (50 mM), and ROTENONE (50 µM). After 24 h, the DOX cell retention has been evaluated quantitatively and qualitatively by flow cytometry and fluorescent microscopy (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ****P ≤ 0.0001)

Fig. 7

Proposed model of ABC transporter regulatory mechanism after ADM uptake