Impact of platelet-derived mitochondria transfer in the metabolic profiling and progression of metastatic MDA-MB-231 human triple-negative breast cancer cells

Abstract

Introduction: An active role of platelets in the progression of triple-negative breast cancer (TNBC) cells has been described. Even the role of platelet-derived extracellular vesicles on the migration of MDA-MB-231 cells has been reported. Interestingly, upon activation, platelets release functional mitochondria into the extracellular environment. However, the impact of these platelet-derived mitochondria on the metabolic properties of MDA-MB-231 cells remains unclear. Methods: MDA-MB-231 and MDA-MB-231-Rho-0 cells were co-cultured with platelets, which were isolated from donor blood. Mitochondrial transfer was assessed through confocal microscopy and flow cytometry, while metabolic analyses were conducted using a Seahorse XF HS Mini Analyzer. The mito-chondrial DNA (mtDNA) copy number was determined via quantitative PCR (qPCR) following platelet co-culture. Finally, cell proliferation and colony formation assay were performed using crystal violet staining. Results and Discussion: We have shown that platelet-derived mitochondria are internalized by MDA-MB-231 cells in co-culture with platelets, increasing ATP production, oxygen (O₂) consumption rate (OCR), cell proliferation, and metabolic adaptability. Additionally, we observed that MDA-MB-231 cells depleted from mtDNA restore cell proliferation in uridine/pyruvate-free cell culture medium and mitochondrial O₂ consumption after co-culture with platelets, indicating a reconstitution of mtDNA facilitated by platelet-derived mitochondria. In conclusion, our study provides new insights into the role of platelet-derived mitochondria in the metabolic adaptability and progression of metastatic MDA-MB-231 TNBC cells.

Keywords: cancer; metabolic adaptability; mitochondria transfer; platelet-derived mitochondria; platelets.

FIGURE 1

MDA-MB-231 cells acquire platelet-derived mitochondria. (A) Flow cytometry histograms of CD62p expression in platelets after isolation or after 24 h co-culture with MDA-MB-231 cells. (B) Frequency of platelet activation after isolation or after 24 h co-culture with MDA-MB-231 cells. (C) Venn diagram of mitochondrial proteins from three samples: PEVs-THR, PEVs-BCC, and EVs-MDA-MB-231. (D) Amount of total and unique mitochondrial proteins for PEVs-THR and PEVs-BCC. Common mitochondrial proteins between PEVs-BCC and EVs-MDA-MB-231 were discarded to avoid possible contamination with proteins from EVs released by MDA-MB-231 cells. (E) Flow cytometry histograms of MitoTracker^™ Green (MTG) signal on MDA-MB-231 cells after 24 h co-culture. MDA-MB-231 cells were cultured with buffer or platelets previously stained with MTG. (F) MTG MFI on MDA-MB-231 cells cultured with buffer or MTG platelets. (G) Flow cytometry histograms of FSC-H for MDA-MB-231 cells cultured with buffer or platelets for 24 h. (H) FSC-H MFI for MDA-MB-231 cells cultured with buffer or platelets for 24 h. (I) Flow cytometry histograms of SSC-H for MDA-MB-231 cells cultured with buffer or platelets for 24 h. (J) SSC-H MFI for MDA-MB-231 cells cultured with buffer or platelets 24 h. (K) Confocal microscopy images of platelet-derived mitochondria internalization by MDA-MB-231 cells. These cells were previously stained with MitoTracker^™ Deep Red (MTDR) and co-cultured with MTG platelets for 24 h. Scale bar = 10 µm. A t-student statistical test was performed. *p < 0.05; **p < 0.01; ****p < 0.0001; ns = not significant.

FIGURE 2

Effect of platelet-derived mitochondria on the bioenergetic properties of MDA-MB-231 cells. (A) Gene ontology results of enriched biological process identified for mitochondrial proteins related to PEVs-BCC. GO-annotation analysis was performed using STRING V11.5 gene ontology tools. Biological processes with strength >1 and false discovery rate (FDR) < 0.05 were selected. (B) Analysis of ATP content of MDA-MB-231 cells cultured with buffer or platelets for 24 h. Results were expressed as relative light units (RLU) normalized by a total number of cells. (C) Seahorse assay results of OCR for MDA-MB-231 cells cultured with buffer or platelets. MDA-MB-231 cells were co-cultured with platelets for 24 h. After that, cells were washed, reseeded, and cultured for 48 h before performing the seahorse assay. Results were normalized to µg of a total protein of each well. (D) Basal OCR of MDA-MB-231 cells cultured with buffer or platelets. (E) ATP-linked OCR of MDA-MB-231 cells cultured with buffer or platelets. (F) Flow cytometry histograms of TMRE signal in MDA-MB-231 cells after 24 h co-culture. MDA-MB-231 cells were cultured with buffer or platelets, and mitochondrial membrane potential (ΔΨm) was analyzed using TMRE dye. (G) TMRE MFI in MDA-MB-231 cells cultured with buffer or platelets. (H) Analysis of ATP content of sorted MDA-MB-231 cells cultured with buffer or platelets for 24 h. MDA-MB-231 cells were cultured with platelets previously stained with MTG. MDA-MB-231 cells positive for MTG signal were separated according to MTG intensity, retrieving two populations. Those with low MTG intensity are named MITO-Low, and those with high MTG intensity are named MITO-High. Results are expressed as RLU normalized by the total number of cells. (I) Analysis of relative mitochondrial DNA (mtDNA) levels of sorted MDA-MB-231 cells. Relative mtDNA levels were determined using quantitative PCR (qPCR) by amplifying mtDNA and nuclear DNA (ncDNA). A t-student statistical test was performed. *p < 0.05; **p < 0.01; ns = not significant.

FIGURE 3

MDA-MB-231-Rho-0 cells restore impaired mitochondrial function by acquiring platelet-derived mitochondria. (A) Frequency of platelet activation after isolation or after 24 h co-culture with Rho-0 cells. (B) Microscopic (left panel) and macroscopic (right panel) images of colonies formed by platelet-rescued Rho-0 cells in pyruvate/uridine-free cell culture medium. Rho-0 cells were cultured with buffer or platelets for 24 h. After that, cells were washed, reseeded, and cultured for 48 h in medium with pyruvate/uridine. Next, cells were maintained in pyruvate/uridine-free medium to evidence mitochondrial function recovery and colony isolation was performed. Colonies were visualized by 0.5% crystal violet staining. (C) Analysis of relative mtDNA levels of platelet-rescued Rho-0 cells by three different donors (Rho-1, Rho-2, and Rho-3). Relative mtDNA levels were determined using quantitative PCR (qPCR) by simultaneous amplification of mtDNA and ncDNA. (D) Gel agarose electrophoresis for amplified products of mitochondrial encoded NADH dehydrogenase 1 (MT-ND1) gene and ribosomal protein lateral stalk subunit P0 (RPLP0) gene. Products of qPCR were loaded into a 2% agarose gel, and electrophoresis was performed. Gel was visualized using UV light to confirm the expected amplicon for each primer pair. (E) ATP production analysis of platelet-rescued Rho-1, Rho-2, and Rho-3 cells. ATP production was modulated with 5 µM oligomycin (Oligo) for 20 h. Results were expressed as RLU normalized to µg of total protein. (F) Seahorse assay results of OCR for platelet-rescued Rho-3 cells. Results were normalized to µg of the protein of each well. (G) Basal OCR of platelet-rescued Rho-3 cells. (H) ATP production linked to OCR of platelet-rescued Rho-3 cells. (I) Cell viability assay of MDA-MB-231 WT, Rho-0, and platelet-rescued Rho-3. Cells were treated with BAY-876 or BAY 87–2243 for 72 h as control cells were treated with vehicle (DMSO). Cell viability was determined by 0.5% crystal violet stained. Results were normalized to control. A t-student statistical test was performed. *p < 0.05; **p < 0.01; ****p < 0.0001; ns = not significant.

FIGURE 4

Platelet-derived mitochondria promote proliferation and increase clonogenic potential by augmenting the metabolic adaptability of MDA-MB-231 cells. Proliferation analysis of MDA-MB-231 cells cultured with buffer or platelets for 24 h. MDA-MB-231 cells were co-cultured with platelets. After that, cells were washed, reseeded, and proliferation was evaluated at 24 (A), 48 (B), and 72 h (C) by 0.5% crystal violet staining. Cell proliferation assay of sorted MDA-MB-231 cells cultured with buffer or platelets for 24 h. MDA-MB-231 cells were cultured with platelets previously stained with MTG. MDA-MB-231 cells positive for MTG signal were separated according to MTG intensity, retrieving two populations. Those with low MTG intensity are named MITO-Low, and those with high MTG intensity are named MITO-High. Sorted cells were seeded, and proliferation was evaluated at 24 (D), 48 (E), and 72 h (F) by 0.5% crystal violet staining. (G) Macroscopic images of colonies formed by sorted MDA-MB-231 cells in a glucose-free culture medium with 10 mM galactose and 4 mM glutamine. Sorted cells were cultured in a glucose-free cell culture medium to evidence metabolic adaptability, and colony visualization was performed by 0.5% crystal violet staining. (H) Quantification of the area covered by colonies formed in a glucose-free culture medium with 10 mM galactose and 4 mM glutamine. Results were expressed as fold change relative to PLT- (without platelets). A t-student statistical test was performed. *p < 0.05; ns = not significant.

FIGURE 5

Model of mitochondrial transfer from platelets to MDA-MB-231 cells. Activation of platelets by MDA-MB-231 human triple-negative breast cancer (TNBC) cells triggers the release of platelet-derived mitochondria. These cells then internalize these mitochondria, leading to the reconstitution of mitochondrial DNA (mtDNA), increasing ATP production, oxygen (O₂) consumption rate (OCR), and cell proliferation. Interestingly, MDA-MB-231 cells with high levels of platelet-derived mitochondria exhibit superior metabolic adaptability. This highlights the significant influence of platelet-derived mitochondria on the progression of MDA-MB-231 TNBC cells (Created with BioRender.com).