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. 2023 Mar 6;12:e85494. doi: 10.7554/eLife.85494

Figure 1. Cell-contact-mediated transfer of macrophage mitochondria leads to increased cancer cell proliferation.

(a) CD14+ monocytes harvested from human blood are transduced and differentiated for 6 days. Mito-mEm +macrophages (green) are co-cultured with MDA-MB-231 cells (231 cells) expressing mito-RFP (magenta; right image). (b) Confocal image showing transferred mitochondria (green, arrowhead) in a 231 cell (magenta, cell outline in white). (c) Representative flow cytometry plots depicting mitochondrial transfer (black box) within a population of co-cultured mito-RFP+ 231 cells (right) compared to monoculture control (left) with background level of mEmerald (mEm) fluorescence set at 0.2%. (d) Aggregate data of mitochondrial transfer rates across macrophage donors. Each data point represents one replicate (N=14 donors). (e) Analysis of proliferative capacity by quantifying Ki-67 levels and DNA content in co-cultured 231 cells after 24 hr. Percentage of cancer cells within a specific cell cycle phase with or without transfer is shown. A significantly different percent of recipient cells occupies G2/M (black) phases of the cell cycle compared to non-recipient cells (N=4 donors; statistics for G2/M only). (f) Co-cultured recipient 231 cells have a significantly higher specific growth rate compared to non-recipients (N=60 cells (control), 115 (recipient) over 4 donors indicated as shades of gray). (g) Schematic of mitochondrial isolation and bath application on MDA-MB-231 cells. Mitochondria are isolated from mito-mEmerald expressing THP-1 monocytes and bath applied at 20–30 µg/mL for 24 hr. Cancer cells which had taken up mEm+ mitochondria are then FACS-isolated and plated for 48 hr for further analyses. (h) Representative confocal image showing mito-RFP-expressing 231 cell (magenta) that had taken up macrophage mitochondria (green, grey arrow). (i) 48 hr after FACS-isolating 231 cells with macrophage mitochondria, flow cytometry was used to determine percent of daughter cells which still contain mEm+ mitochondria. N=3 biological replicates. (j), Cell cycle analysis of daughter cells 48 hr after FACS-isolation of 231 cells that had taken up macrophage mitochondria. N=3 biological replicates. For all panels, standard error of the mean (SEM) is displayed and scale bars are 10 µm. Mann-Whitney (d), two-way ANOVA (e, j), Welch’s t-test (f, i), *p<0.05; **p<0.01; ****p<0.0001.

Figure 1.

Figure 1—figure supplement 1. Macrophages transfer mitochondria to cancer cells.

Figure 1—figure supplement 1.

(a) Representative flow cytometry plots of mito-RFP MDA-MB-231 monocultured cells used as a control (top) and mito-RFP 231/mito-mEm macrophage co-cultures after 24 hr (bottom). (b) Panel of cancer cell lines – MDA-MB-231 (‘231’), MDA-MB-468 (‘468’) and A375 – co-cultured with mEm-positive (transduced, ‘+’) or mEm-negative (untransduced, ‘-’) macrophages. Mitochondrial transfer rates determined by flow cytometry, as described in (a). Different donors (N=3) indicated as shades of gray. (c) Left: Bar graph quantifying mitochondrial transfer to MCF10A cells after 24 hr. (N=3 donors indicated by shades of gray). Right: Confocal image showing macrophage mitochondria (green, arrowhead) in a MCF10A cell (magenta). (d) Stills from a time-lapse showing a 231 cell (mCherry-TOMM20, magenta) containing transferred macrophage mitochondria (mEmerald-TOMM20, green, arrowhead). Timepoints indicated in upper left. (e) Single Z-plane of a 231 cell labeled with mitochondrial dye TMRM (magenta) with macrophage mitochondria (green, arrowhead) containing DNA (gray). 83% of transferred mitochondria contain DNA (N=16 cells). (f) Schematic depicting trans-well experiments. 231 cells (magenta) were plated alone (left), separated from macrophages (green; middle), or plated together (right). (g) Percent of cancer cells with transfer in conditions depicted in f, (N=3 donors). (h), Conditioned media (CM) experiments showing percent of cancer cells with transfer when co-cultured in media type listed on the x-axis (N=2 experiments). Each dot represents one replicate for all panels. Error bars represent standard error of the mean (SEM) and scale bars are 10 µm. Two-way ANOVA (b, g), Mann-Whitney test (c), ****p<0.0001.
Figure 1—figure supplement 2. Cancer cells will macrophage mitochondria exhibit increased proliferation.

Figure 1—figure supplement 2.

(a) Cancer cells and macrophages were cocultured for 24 hours and scRNA-seq was performed. Ingenuity Pathway Analysis of scRNA-seq data reveals significant changes in canonical cell proliferation pathways in co-cultured cancer cells that received macrophage mitochondria compared to co-cultured cancer cells that did not. (b) Representative flow cytometry plots of mito-RFP 231/mito-mEm macrophage co-cultures stained for Ki67 and DNA content. (c) 24 hr data from Figure 1e but separated as percent of cancer cells in each phase of the cell cycle with (triangle) or without transfer (circle). (d) 48 hr of co-culture as described in (c) with aggregate data in stacked bar graphs to the right (stats displayed for G2/M only). Each pair in (c) and (d) represents cells within one technical replicate and each donor is represented by color (N = 4 donors for both data sets). (e) Time-lapse imaging was used to quantify the amount of co-cultured 231 cells that did (black) or did not complete (gray) cytokinesis over a 48-hr period (N = 416 cells, 4 donors). Error bars represent SEM. Two-way ANOVA (c–e), ***p<0.001; ****p<0.0001.
Figure 1—figure supplement 3. Mitochondrial transfer leads to sustained increased growth rate in daughter cancer cells.

Figure 1—figure supplement 3.

(a) QPI data of mass over time measurements for a single triad consisting of one parent cell (black) and two daughters that inherited (gray) or did not inherit (light gray) the parent’s macrophage mitochondria. Specific growth rate (slope of best fit line normalized by average mass) is listed next to line trace of corresponding cell. (b) QPI data of specific growth rate of 5 individual triads (indicated by color). Parent and daughter cells are indicated with shape (legend on graph). (c) QPI data of normalized mass in picograms (pg) over time in hours (hrs) for daughter cells that did (black) or did not (gray) inherit the parent’s macrophage mitochondria normalized to daughter cell initial mass. Error bars represent SEM. (d) Cell cycle analyses MDA-MB-231/macrophage co-cultures after 24 hr 1 µM Palbociclib treatment. Two-way ANOVA comparing G1/G0 fractions in each condition, ****p<0.0001. When comparing G2/M fractions across conditions **p=0.0017. n=1 in technical triplicate. (e) MDA-MB-231/macrophage cocultures were treated with 10 µM Palbociclib for 24 hr. Mitochondrial transfer rates were determined via flow cytometry as previously described. Unpaired t-test p=0.4675, n=1 in technical triplicate.