Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis

Abstract

Mitochondrial DNA (mtDNA) mutations are frequently observed in cancer, but their contribution to tumor progression is controversial. To evaluate the impact of mtDNA variants on tumor growth and metastasis, we created human melanoma cytoplasmic hybrid (cybrid) cell lines transplanted with wildtype mtDNA or pathogenic mtDNA encoding variants that partially or completely inhibit oxidative phosphorylation. Homoplasmic pathogenic mtDNA cybrids reliably established tumors despite dysfunctional oxidative phosphorylation. However, pathogenic mtDNA variants disrupted spontaneous metastasis of subcutaneous tumors and decreased the abundance of circulating melanoma cells in the blood. Pathogenic mtDNA did not induce anoikis or inhibit organ colonization of melanoma cells following intravenous injections. Instead, migration and invasion were reduced, indicating that limited circulation entry functions as a metastatic bottleneck amidst mtDNA dysfunction. Furthermore, analysis of selective pressure exerted on the mitochondrial genomes of heteroplasmic cybrid lines revealed a suppression of pathogenic mtDNA allelic frequency during melanoma growth. Collectively, these findings demonstrate that functional mtDNA is favored during melanoma growth and enables metastatic entry into the blood.

Extended Data Figure 1.. A375 mtDNA depletion and establishment of ρ0 clones.

a, Mitochondrial genome (mtDNA) to nuclear genome (nDNA) ratios in A375 parental line and serial diluted clones following two week treatment with 5 μM or 10 μM ddC (dideoxycytidine). n.d., mtDNA not detected. b,c, Mitochondrial oxygen consumption rate (mito-OCR) (b) and representative oxygen consumption rates (c) for 20,000 cells (per well) of A375 parental line and ddC treated clones. Mitochondrial inhibitors (oligomycin, CCCP, antimycin A) were injected at the indicated timepoints. The number of samples analyzed per treatment is indicated. Data are mean ± s.e.m.

Extended Data Figure 2.. Generation of cybrid cell lines.

a, Illustration of the cellular fractionation and staining utilized during generation of A375 cybrids. Donor mtDNA lines were stained with MitoTracker Green and Hoechst prior to cytoplast generation. Cytoplasts were generated, enrich via flow cytometry, and then fused with mtDNA depleted A375 ρ0 cells that were pre-stained with SYTO59. Successful cybrid cells were further enriched based on the presence of SYTO59 signal, MitoTracker Green signal, and the absence of Hoechst signal. b, Representative epifluorescence images of stained mtDNA donor 143B_nuclear Wildtype_mtDNA cells following treatment with DMSO vehicle (left) and cytochalasin B (right) and centrifugation to generate cytoplasts. Scale bars, 25 μm. c, Enrichment of cytoplasts following differential centrifugation with treatment of DMSO (top) or cytochalasin B (bottom). Cell gating populations are displayed with black dashed lines and sorted cytoplast population is shown with red dashed lines. d, Flow cytometric enrichment of PBS mixed (top) and PEG fused cybrid cells (bottom). Cell gating populations are displayed with black dashed lines. Fusion positive populations were sorted for an enrichment of MitoTracker Green in the PEG fused cybrid cells relative to the PBS mixed cells.

Extended Data Figure 3.. Genetic validation of homoplasmic A375 cybrid lines.

a,b,c, Representative Sanger sequencing of mtDNA regions surrounding mt.3460 (a), mt.8993 (b), and mt.6692 (c) for wildtype cybrid line (top) and indicated variant cybrid lines (bottom). Pathogenic variant allelic locations are highlighted in red. d,e, Standard curve for ddPCR probes specific to mt.8993 T>G (d) and mt.3460 G>A (e) constructed from purified plasmids for each variant. The coefficient of determination (R²) is provided for each curve. f,g, Representative ddPCR 2-dimensional plot with probes specific to the ATP6 T8993G in homoplasmic ATP6 cybrids (f) and wildtype cybrids (g). h,i, Representative ddPCR 2-dimensional plot with probes specific to the ND1 G3460A in homoplasmic ND1 cybrids (h) and wildtype cybrids (i).

Extended Data Figure 4.. Retention of mtDNA variants after subcutaneous tumor development.

a,b,c, Representative Sanger sequencing of mtDNA regions surrounding mt.3460 (ND1) (a), mt.8993 (ATP6) (b), and mt.6692 (CO1) (c) from DNA of xenograft subcutaneous tumors for each respective cybrid lines. Pathogenic variant allelic locations are indicated in red.

Extended Data Figure 5.. Representative Ki67⁺ staining of subcutaneous cybrid tumors.

a-d, Representative Ki67 staining images of xenograft subcutaneous tumors of the indicated mtDNA haplotype. Highlighted are proliferative regions with high abundance of Ki67⁺ nuclei (top yellow box) and non-proliferative regions with low abundance of Ki67⁺ nuclei (bottom blue box). Scale bar for full section, 5000 μm. Scale bar for zoomed region, 200 μm.

Extended Data Figure 6.. Histological analysis of subcutaneous tumors.

a, Quantitation of tumor regions from representative H&E sections (Fig 2e) with QuPath Analysis Pixel Classification algorithm. b,c, Representative regions (b) and cross-sectional quantitation (c) of discohesive regions in H&E sections of indicated xenograft subcutaneous cybrid tumors. Scale bar, 500 µM. P values indicate comparison with WT group. The number of tumors analyzed per treatment is indicated. Data are mean ± s.e.m. (c). Statistical significance was assessed using one-way ANOVA with Dunn’s multiple comparison adjustment (c).

Extended Data Figure 7.. Isolation of circulating human melanoma cells from mouse blood.

a,b, Flow cytometric analysis and gating strategy to identify human melanoma cells in negative control mouse blood (a) or blood of xenograft subcutaneous tumor bearing mouse (b).

Extended Data Figure 8.. Reduced migration and invasion of dysfunctional cybrid lines at low glucose availability.

a,b, Continuous (a) and final measurement (b) of oxygen consumption by a monolayer culture of indicated cybrid lines at 1 mM and 25 mM glucose. P values indicate comparison of 1 mM and 25 mM oxygen consumption. c-e, Viability for indicated cybrid lines at 25 mM (a), 5 mM (b), and 1 mM (c) glucose concentrations after 24 hours of culture as a confluent monolayer. f,g, Representative wound healing migration images after 24 hours of culture in 25 mM glucose and 1 mM glucose media. Image is composed of individually stitched images with contrast enhanced for viewing purposes. h, Representative Boyden transwell migration of cybrid cells after 24 hours of culture in 1 mM glucose media. Image contrast was enhanced for viewing purposes. The number of cells analyzed per treatment is indicated. Data are mean ± standard error of the mean (a,b) and median ± interquartile range (c-e). Statistical significance was assessed using two-way ANOVA with Šídák’s multiple comparisons test (b).

Extended Data Figure 9.. Heteroplasmic ND1 and CO1 alleles are lost after passage in culture.

a,b, Single cell ddPCR (sc-ddPRCR) analysis of heteroplasmy at ATP6 mt.T8993G (a) and ND1 mt.G3460A (b) directly following cybrid fusion of respective mutant cytoplasts with WT cybrid clones. c, Sanger sequencing at CO1 mt.6692del directly following cybrid fusion of CO1 cytoplasts with WT cybrid clones. Heteroplasmic allele is indicated with black arrow and red font. d, Bulk ddPCR analysis of heteroplasmic frequency at mt.G3460A for ND1/WT cybrids after clonal line establishment. e, Sanger sequencing at CO1 mt.6692del for three representative CO1/WT cybrids after clonal line establishment. Data are median ± interquartile range (a,b) and mean ± SEM (d).

Extended Data Figure 10.. Heteroplasmy assessment of ATP6/WT subcutaneous tumors at increased initial subcutaneous injection cell count.

a, Subcutaneous tumor volume over time after xenograft of 10,000 cells from heteroplasmic ATP6/WT clones. b-e, Single cell ddPCR analysis of heteroplasmy at mt.T8993G for ATP6/WT heteroplasmic clones of subcutaneous xenograft of 10,000 cells following tumor growth of indicated clones. P values reflect comparisons with the initial passage. The number of cells analyzed per treatment is indicated. Data are median ± interquartile range (b-e). Statistical significance was assessed using non-parametric Kruskal-Wallis test with Dunn’s multiple comparison adjustment (b-e).

Figure 1.. mtDNA pathogenic variants in A375 homoplasmic cybrid lines impact mitochondria ETC activity.

a, Schematic of electron transport chain with complexes labeled to display subunits with mtDNA variants investigated in this study. b,c, ddPCR quantitation of allelic fraction for (b) ATP6 allele mt.T8993G and (c) ND1 allele mt.G3460G in homoplasmic cybrid lines. d, Western blot analysis for the indicated mtDNA encoded proteins in homoplasmic cybrid cell lines. β-Actin levels are shown as loading control. e, Mitochondrial genome (mtDNA) to nuclear genome (nDNA) ratios in the indicated homoplasmic cybrid lines. n.d., not detected. P values reflect comparisons with the A375 parental line. f, Mitochondrial mass as assessed by flow cytometric analysis of MitoTracker Green staining in the indicated homoplasmic cybrid lines. P values reflect comparisons with the A375 parental line. MFI, mean fluorescence intensity. g, Mitochondrial oxygen consumption rates (Mito-OCR) in indicated homoplasmic cybrid lines. P values reflect comparisons with the A375 parental line. The number of samples analyzed per treatment is indicated. Data are mean (b,c), mean ± s.e.m. (e,f), and median ± interquartile range (g). Statistical significance was assessed using one-way ANOVA with Dunn’s multiple comparison adjustment (d) and nested one-way ANOVA with Dunn’s multiple comparison adjustment (f,g).

Figure 2.. Functional mtDNA is dispensable for primary melanoma growth.

a, Illustration of subcutaneous injection to assess primary tumors of homoplasmic cybrid lines. b, Frequency of tumor formation for indicated injection counts across homoplasmic cybrid lines. c, Tumor growth rate of indicated cybrid lines following 10,000 cell subcutaneous injection. Homoplasmic growth data (black circle) is repeated as a reference in all panels. P values reflect comparison with wildtype (WT) group. d, Quantitation of the percentage of Ki67⁺ nuclei across subcutaneous tumors. P values reflect comparison with wildtype (WT) group. e, Representative H&E images of subcutaneous tumors. Scale bar, 5000 μm. f, Quantitation of necrotic region as a percentage of tumor cross-sectional area. P values reflect comparison with wildtype (WT) group. g, Representative immunohistochemistry images for pimonidazole from subcutaneous tumors of the indicated mtDNA haplotype. Scale bar, 500 μm. h, Quantitation of pimonidazole positive (hypoxic) regions as a percentage of tumor cross-sectional area. P values reflect comparison with wildtype (WT) group. i, Western blot analysis of mitochondrial outer membrane protein TOMM20 and matrix protein HSP60. β-Actin expression is shown as loading control. P values reflect comparison with wildtype (WT) group. j, Mitochondrial genome content (mtDNA/nDNA) for subcutaneous tumors of the indicated mtDNA haplotype. The number of samples analyzed per group is indicated. Data are mean ± s.e.m. (d,f,h,i,j). Statistical significance was assessed using exponential growth least squares fitting on the mean values of replicates followed by extra sum-of-squares F test with Holm-Sidak’s adjustment (b) and one-way ANOVA with Dunn’s multiple comparison adjustment (d,f,h,i,j).

Figure 3.. Spontaneous metastasis is reduced by dysfunctional mtDNA.

a, Illustration of subcutaneous injection to assess spontaneous metastasis. b,c, Representative images (b) and quantitation (c) of metastatic bioluminescence signal of organs from mice with late-stage subcutaneous xenografts of indicated cybrid lines. P values indicate comparisons with the A375 (parental) group. d, Frequency of circulating melanoma cells from mice (as a percentage of DAPI negative events) with late-stage subcutaneous xenografts of indicated cybrid lines. P values indicate comparisons with WT group. e, Illustration of acute pharmacological evaluation of IACS-010759 to assess metastasis in late-stage patient derived xenograft model UT10. f, Tumor diameter of xenografted UT10 mice treated with IACS-010759 or vehicle. g,h, Representative images (g) and quantitation (h) of organ bioluminescence signal of UT10 xenograft following treatment with IACS-010759 or vehicle. i, Circulating melanoma cells normalized to total blood volume of xenografted UT10 mice following treatment with IACS-010759 or vehicle. The number of tumors or mice analyzed per treatment is indicated. Data are mean ± s.e.m. (c,d,h,i). Statistical significance was assessed using non-parametric Kruskal-Wallis test with Dunn’s multiple comparison adjustment (c), nested one-way ANOVA with Dunn’s multiple comparison adjustment (d), two-tailed unpaired t-test with Welch’s correction (h,i).

Figure 4.. Dysfunctional mtDNA disrupts migratory potential in human melanoma.

a-d, Reactive oxygen species (a), glucose consumption (b), lactate excretion (c), and cell count (d) for indicated cybrid cells after 24 hour adherent and detached culture. P values indicate comparison with WT group for each condition. MFI, mean fluorescence intensity e, Detached viability of indicated cybrid lines following 24 hour of culture. P values indicate comparison with WT group. f, Percentage of apoptotic cells following 24 hour culture in adherent or detached conditions. P values indicate comparison with WT group. g, Illustration of intravenous injection of cybrid lines to assess organ colonization. h,i, Representative bioluminescence imaging of live mice (h) and quantitation of metastatic bioluminescence signal of organs (i) following intravenous injection of indicated cybrid lines. P values indicate comparisons with WT group. j, 24 hour wound healing assay quantification for indicated cybrid lines at 25 mM, 5 mM, and 1 mM glucose concentrations. Gap distance was quantified from the difference of the 0 hour and 24 hour wound gap. P values indicate comparisons with WT + vehicle group for each glucose concentration. k, Relative transwell invaded cells at 1 mM glucose concentration. P values indicate comparisons with WT + vehicle group for each glucose concentration. The number of samples, tumors, or mice analyzed per treatment is indicated. Data are mean ± s.e.m. (a), median ± interquartile range (i,j,k). Statistical significance was assessed using one-way ANOVA with Dunn’s multiple comparison adjustment (e), non-parametric Kruskal-Wallis test with Dunn’s multiple comparison adjustment (i), two-way ANOVA with Dunn’s multiple comparison adjustment (a,b,c,d,f), and one-way ANOVA with Dunn’s multiple comparison adjustment (j,k).

Figure 5.. Purifying intracellular mtDNA selection is a feature of melanoma growth.

a, Overview of ATP6/WT heteroplasmic cybrid generation. b, Bulk ddPCR analysis of heteroplasmic frequency at mt.T8993G for ATP6/WT cybrids after clonal line establishment. Clones selected for experiments are indicated with red arrows. c, Single cell ddPCR (sc-ddPCR) analysis of heteroplasmy at mt.T8993G for selected ATP6/WT cybrid clones. d, Mitochondrial oxygen consumption rate of ATP6/WT heteroplasmic clones. e, Illustration of heteroplasmic selection experiment. Initial cells were passaged for extended in vitro culture or xenografted for subcutaneous tumors in NSG mice. f, Single cell ddPCR analysis of heteroplasmy at mt.T8993G for ATP6/WT heteroplasmic clones at passage 5 (p5) and passage 10 (p10) of in vitro culture. Three replicates were independently passaged and analyzed for each clone. g, Subcutaneous tumor diameter over time after xenograft of 100 cells from heteroplasmic ATP6/WT clones. h, Single cell ddPCR analysis of heteroplasmy at mt.T8993G for ATP6/WT heteroplasmic clones of subcutaneous xenograft of 100 cells following tumor growth. i, Illustration of heteroplasmic selection assay following tail-vein IV injection of ATP6/WT heteroplasmic clones. P values reflect comparisons with the initial passage. j, Bioluminescence imaging of live mice following intravenous injection of 1,000 cybrid cells. k, Quantification of organ bioluminescence total flux following intravenous injections. l,m, Single cell ddPCR analysis of heteroplasmy at mt.T8993G for ATP6/WT heteroplasmic clones tumor nodules following intravenous injection. The number of samples analyzed per treatment is indicated. Data are mean ± s.e.m. (b,g), median ± interquartile range (c,d,f,h,k,l). Statistical significance was assessed one-way ANOVA with Tukey’s multiple comparison adjustment (d), nested one-way ANOVA with Dunn’s multiple comparison adjustment (f,h), one-way ANOVA with Dunn’s multiple comparison adjustment (l), and non-parametric Kruskal-Wallis test with Dunn’s multiple comparison adjustment (m).