Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma

Abstract

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification.

Fig. 1. Mitochondrial base editing results in isogenic cell lines bearing two independent truncating mutations in Mt-Nd5.

a, Schematic of the TALE-DdCBE design used. TALEs were incorporated into a backbone containing a mitochondria-targeting cassette, split-half DdCBE and uracil glycosylase inhibitor (UGI). MTS, mitochondrial targeting sequence; NES, nuclear export signal. b, Schematic of the murine mtDNA. Targeted sites within Mt-Nd5 are indicated. c, TALE-DdCBE pairs used to induce a G>A mutation at m.12,436 and m.11,944. d, Workflow used to produce Mt-Nd5 mutant isogenic cell lines. e, Heteroplasmy measurements of cells generated in d (n = 6 biological replicates). f, Immunoblot of indicative respiratory chain subunits. Representative result of three biological replicates is shown. g, Assembled complex I abundance and in-gel activity. Representative result of three biological replicates is shown. h, mtDNA copy number (n = 10, 13, 9, 15 and 8 technical replicates over n = 4, 5, 3, 5 and 3 biological replicates). i, Basal oxygen consumption rate (OCR) (n = 12, 9, 12, 9 and 12 technical replicates over n = 4, 3, 4, 3 and 4 biological replicates). j, Energy (adenylate) charge state (n = 17 technical replicates over n = 6 biological replicates). k, Proliferation rate of cell lines in permissive growth media. (n = 3 biological replicates). l, NAD⁺:NADH ratio (n = 12, 11, 12, 12 and 12 technical replicates over n = 4 biological replicates). P values were determined using a one-way ANOVA test with Sidak multiple comparisons test (e, h–i, k) or Fisher’s LSD test (j,l). Measure of centrality, mean; error bars, s.d. Number of replicates are described across conditions from left to right as presented. Source data

Fig. 2. Mutant cells undergo a metabolic shift towards glycolysis caused by cellular redox imbalance.

a, Heatmap of unlabeled steady-state abundance of select mitochondrial metabolites, arginine, argininosuccinate (AS) and terminal fumarate adducts succinylcysteine (succ.cys) and succinicGSH (succGSH) (n = 12–18 technical replicates over n = 6 biological replicates). FC, fold change. b, Labeling fate of ¹³C derived from 1-¹³C-glutamine. c, Malate m+1 abundance, derived from 1-¹³C-glutamine with indicated treatment (n = 11, 11, 11, 8, 8, 8, 6, 6 and 9 technical replicates over n = 4, 4, 4, 3, 3, 3, 3, 3 and 3 biological replicates). d, Heatmap of unlabeled steady-state metabolite abundances for select intracellular glycolytic intermediates and extracellular lactate (Ex. lactate) (n = 12–18 technical replicates over n = 6 biological replicates). e, Labeling fate of U-¹³C-glucose. f, Abundance of U-¹³C-glucose derived lactate m+3 with indicated treatment (n = 9, 9, 9, 9, 6 and 6 technical replicates over n = 3 biological replicates). g, Labeling fate of ²H derived from 4-²H₁-glucose; mitoLbNOX not shown for clarity. h, Malate m+1 abundance, derived from 4-²H₁-glucose with indicated treatment (n = 17, 17, 18, 9, 7, 8, 8, 7 and 5 technical replicates over n = 6, 6, 6, 3, 3, 3, 3, 3 and 2 biological replicates). i, IC₅₀ curves for 2-deoxyglucose (n = 4 technical replicates). Representative result of three biological replicates is shown. P values were determined using a one-way ANOVA test with Sidak multiple comparisons test (a,d) or Fisher’s LSD Test (c,f,h). Measure of centrality, mean; error bars, s.d. *P < 0.05; **P < 0.01; ***P < 0.001. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data for which asterisks are not present report non-significant changes. Source data

Fig. 3. Tumor mtDNA mutations reshape the immune microenvironment.

a, Survival of C57/BL6 mice subcutaneously injected with indicated cells (n = 14, 12, 7, 12 and 8 animals). b, Mean tumor weight at endpoint (n = 14, 12, 7, 12 and 8 individual tumors). Error bars, s.d. c, GSEA of bulk tumor RNA-seq data (n = 5–6 individual tumors per genotype). Only gene sets with P_adj < 0.1 are shown. TNFα, tumor necrosis factor alpha. d, GSEA of RNA-seq obtained from HMF database of patients with metastatic melanoma. Cancers are stratified by mtDNA status into wild type and mtDNA mutant with >50% VAF. e, UMAP of Seurat clustered whole tumor scRNA-seq from indicated samples. f, UMAP indicating cell type IDs. DC, dendritic cells; pDC, plasmacytoid dendritic cell; NK, natural killer. g, GSEA of malignant cells identified in scRNA-seq analysis. Comparison is wild-type tumors versus all mutant tumors. h–l, UMAPs colored by GSEA score for IFNα response (h), IFNγ response (i), IL2-Stat5 signaling (j), inflammatory response (k) and oxidative phosphorylation (l). m, Proportion of tumor-resident S100a9⁺ neutrophils relative to total malignant and non-malignant cells (n = 7, 6 and 3 individual tumors). Boxplots indicate mean and interquartile range; error bars, s.e.m. One-way ANOVA test with Sidak multiple comparisons test (b), two-tailed Wilcoxon signed rank test (c,d,g–l) and two-tailed Student’s t-test (m) were applied. Number of replicates are described across conditions from left to right as presented. Source data

Fig. 4. mtDNA mutation and cytoLbNOX-associated microenvironment remodeling sensitizes tumors to checkpoint blockade.

a, Schematic of the experimental plan and dosing regimen for B78-D14 tumors with anti-PD1 monoclonal antibody (mAb). b, Representative images of isolated tumors at day 21. c, Tumor weights at day 21 (n = 15, 19, 12 and 10 individual tumors). d, Schematic of experimental plan and dosing regimen for Hcmel12 tumors with anti-PD1, anti-PD-L1 or anti-CTLA4 mAbs. e, Representative images of collected tumors at day 13 for each drug regimen. f, Tumor weights at day 13 (n = 11, 11, 12 and 10 anti-PD1, n = 12 anti-PD-L1 and n = 12 anti-CTLA4 individual tumors) for each drug regimen. g, Schematic of the experimental plan and dosing regimen for 4434 tumors with anti-PD1 mAb. h, Representative images of treated tumors at day 20 and untreated tumors at endpoint. i, Tumor weights at day 21 (n = 13 individual tumors). j, Stratification of a metastatic melanoma patient cohort by mtDNA status. k, Response rate of patients to nivolumab by tumor mtDNA mutation status. P values were determined using one-way ANOVA with Sidak multiple comparisons test (c,f), one-tailed Student’s t-test (i) or chi-squared test (k). Error bars, s.d. Measure of centrality is mean. Number of replicates are described across conditions from left to right as presented. Source data

Extended Data Fig. 1. Characterising complex I truncating mutations in melanoma.

a Unique truncating variants were detected in an average of 16% of melanoma patients (n = 281) and in 19% of melanoma patients (n = 89) from the Hartwig Medical Foundation (HMF) and MSK IMPACT melanoma cohorts respectively. b Truncating and c non-truncating mutations in melanoma patients, combined from both IMPACT and HMF patient cohorts, show that truncations are enriched in complex I, compared to complexes III, IV, and V. d Immunoblot of DdCBE pair expression post-sort. αHA and αFLAG show expression of left (TALE-L) and right TALEs (TALE-R) respectively. One biological replicate performed. e Heteroplasmy measurements of cells generated (n = 6 biological replicates). f Off-target C>T activity of DdCBEs on mtDNA. Figure shows mutations detected at heteroplasmies >2% and is a measure of mutations detected relative to wild-type. These mutations likely do not impact our key observations as both models behave similarly across experiments. g Immunoblot of indicative respiratory chain subunits. Representative result of three biological replicates is shown. Volcano plot showing detected differences in protein abundance of wild-type versus h mt.12436^60% cells and i mt.11944^60% cells. Differences of p-value < 0.05 and log₂ fold change > 0.5 shown in red (n = 3 biological replicates). Heatmaps of protein abundances for j complex I, k complex II, l complex III, m complex IV and n complex V nuclear and mtDNA-encoded subunits (n = 3 biological replicates). o mtDNA copy number (n = 22 and 21 technical replicates over n = 8 and 7 biological replicates). p Expression of mitochondrial genes (n = 4 biological replicates). q Basal oxygen consumption rate (OCR) (n = 18 and 12 technical replicates over n = 6 and 4 biological replicates) r Energy (adenylate) charge state (n = 9 technical replicates over n = 3 biological replicates). s Measurements of the electrical component of the proton motive force, ΔΨ, the chemical component of the proton motive force ΔpH and total protonmotive force, ΔP (n = 4 biological replicates). t NAD+:NADH ratio (n = 9 technical replicates over n = 3 biological replicates). u GSH:GSSG ratio (n = 6, 10, 12, 8 and 10 technical replicates over n = 2, 4, 4 and 3 biological replicates). A high GSH:GSSG ratio represents a more reductive intracellular environment. v GSH:GSSG ratio (n = 9 technical replicates over n = 3 biological replicates) w Mitochondrial NADH oxidation state (n = 4 biological replicates). P-values were determined using a two-tailed Fisher’s exact test (A-C), student’s one-tailed t-test (E, O, Q, R, T, V), two-tailed Wilcoxon signed rank test (H, I), one-way ANOVA test with Sidak multiple comparisons test (J-N) or one-way ANOVA test with Fisher’s LSD test (S, U, W). Error bars indicate CI (A-C) or SD (E,O-W). Measure of centrality is mean. * P = < 0.05, ** P = <0.01, *** P = <0.001, **** P = <0.0001. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes. Source data

Extended Data Fig. 2. Independent mt-Nd5 truncations produce consistent changes in metabolic profile.

a Comparison of steady-state metabolite changes of m.12,436^60% and m.11,944^60% cells, each relative to wild-type (n= 6–9 separate wells per genotype). b Labelling fate of ¹³C derived from U-¹³C-glutamine via oxidative decarboxylation versus reductive carboxylation of glutamine. c Malate m+3 abundance, derived from U-¹³C-glutamine (n = 9 technical replicates over n = 3 biological replicates). d malate m+3:malate m+2 ratio, derived from U-¹³C-glutamine (n = 9 technical replicates over n = 3 biological replicates). e AS m+3:AS m+2 ratio, derived from U-¹³C-glutamine (n = 9 technical replicates over n = 3 biological replicates). f Labelling fate of ¹³C derived from 1-¹³C-glutamine which exclusively labels metabolites derived from the reductive carboxylation of glutamine. g Aconitate m+1 abundance, derived from 1-¹³C-glutamine (n = 9 technical replicates over n = 3 biological replicates). h Aspartate m+1 abundance, derived from 1-¹³C-glutamine (n = 9 technical replicates over n = 3 biological replicates). i AS m+1 abundance, derived from 1-¹³C-glutamine (n = 9 technical replicates over n = 3 biological replicates). j Immunoblot of siRNA mediated depletion of Mdh1. Representative result of three biological replicates is shown. k Immunoblot of cytoLbNOX expression 36hrs post-sort, detected using αFLAG. Representative result of three biological replicates is shown. l AS m+1 abundance, derived from 1-¹³C-glutamine with indicated treatment (n = 12, 11, 11, 8, 8, 6, 6, 5 and 8 technical replicates over n = 4, 4, 4, 3, 3, 2, 2, 2 and 3 biological replicates). m Labelling fate of ¹³C derived from U-¹³C-glucose. n Pyruvate m+3 abundance, derived from U-¹³C-glucose (n = 8, 8, 7, 8 and 7 technical replicates over n = 3 biological replicates). o Citrate m+2:pyruvate m+3 ratio, derived from U-¹³C-glucose (n = 7, 7, 6, 7 and 6 technical replicates over n = 3, 3, 2, 3 and 2 biological replicates). p Malate m+3:citrate m+3 ratio, derived from U-¹³C-glucose (n = 8, 8, 7, 8 and 7 technical replicates over n = 3 biological replicates). q Immunoblot of mitoLbNOX expression in sorted cells 36hrs post-transfection, detected using αFLAG. Representative result of three biological replicates is shown. r Lactate m+1 abundance, derived from 4-²H₁-glucose with indicated treatment (n = 8, 8, 9, 9, 7, 8, 8, 7 and 6 technical replicates over n = 3, 3, 3, 3, 3, 3, 3, 3 and 2 biological replicates). s NADH m+1 abundance, derived from 4-²H₁-glucose with indicated treatment (n = 8, 8, 8, 9, 7, 8, 8, 6 and 6 technical replicates over n = 3, 3, 3, 3, 3, 3, 3, 2 and 2 biological replicates). t IC₅₀ curve for metformin. IC₅₀ for wild-type = 26.31 ± 1.49mM, for mt.12436^60% = 16.60 ± 2.43mM, for mt.12436^80% = 5.89 ± 0.71mM and for mt.11944^80% = 22.93 ± 0.70mM U IC₅₀ curve for rotenone. IC₅₀ for wild-type = 0.236 ± 0.026µM, for mt.12436^60% = 0.235 ± 0.035µM, for mt.12436^80% = 0.493 ± 0.108µM and for mt.11944^60% = 0.205 ± 0.033µM and V IC₅₀ curve for oligomycin. IC₅₀ for wild-type = 13.81 ± 3.80µM, for mt.12436^60% = 13.52 ± 3.32µM, for mt.12436^80% = 7.75 ± 0.56µM and for mt.11944^80% = 13.54 ± 3.32µM (n = 4 technical replicates). Representative result of three biological replicates is shown. All P-values were determined using a one-way ANOVA test with Fisher’s LSD test. Error bars indicate SD. Measure of centrality is mean. Number of replicates are described across conditions from left to right as presented. Source data

Extended Data Fig. 3. Characterisation of mtDNA mutant tumors in mice and humans.

Representative H&E from 5 sub-sections of a wild-type, b m.12,436^40% and C m.12,436^60% tumors. d Change in detected heteroplasmy in bulk tumor samples (n = 5, 7, 12 and 11 individual tumours). e Bulk tumor mtDNA copy number (n = 14, 5, 5, 12 and 12 individual tumours). f Heatmap of steady-state abundance of metabolically terminal fumarate adducts, succinylcysteine and succinicGSH, demonstrating that metabolic changes observed in vitro are preserved in vivo (n = 9–11 individual tumours). GSEA of bulk tumor RNAseq data (n = 5-6 individual tumours) showing g mutant^40% versus wild-type and h mutant^60% versus mutant^40%. i Volcano plot depicting NES and -log₁₀(adj. p-value) derived from GSEA comparing mtDNA mutant >50% VAF to wild-type skin cancer samples from HMF. Pathways associated with the innate immune response are highlighted. j ssGSEA analysis comparing mtDNA mutant to wildtype skin cancer samples from HMF. Effect sizes are consistent with GSEA analysis, but do not reach statistical significance. Only genesets with adj. p-value <0.1 are shown unless otherwise stated. Grey, mtDNA wildtype (n = 256); red, >50% VAF (n = 57). Volcano plots showing differences in gene expression of wild-type versus k mt.12436^60% cells and L mt.11944^60% cells. Differences of p-adj < 0.05 and log₂ fold change > 1 shown in red (n = 4 biological replicates). Wilcoxon signed rank test applied. A one-way ANOVA test with Fisher’s LSD test (F), two-tailed Wilcoxon signed rank test (I, K-L) or two-tailed student’s t-test were applied. Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01. Number of replicates are described across conditions from left to right as presented. NES: normalised enrichment score. Heatmap representations of data where asterisks are not present report non-significant changes GSEA: gene-set enrichment analysis. SSGSEA: Single Sample GSEA. HMF: Hartwig Medical Foundation. Source data

Extended Data Fig. 4. scRNAseq analyses reveal distinct alterations in the tumor immune microenvironment of mtDNA mutant tumors.

UMAP indicating a Ptprc expression, b epithelial score and c aneuploidy as determined by copykat prediction. These criteria were employed as the B78 cells lack distinct transcriptional signatures to allow unambiguous identification of malignant cells. d UMAP of seurat clustered T-cell/ NK cell scRNAseq from indicated samples. e UMAP indicating cell type IDs. NK, natural killer cells. IFN, interferon. Tgd, gamma delta T-cells. Treg, regulatory T-cells. f UMAP of seurat clustered myeloid scRNAseq from indicated samples. g UMAP indicating cell type IDs. cDC, conventional dendritic cells. Malignant cells were identified for scRNAseq analysis as aneuploid cells with low or nil Ptprc (CD45) expression and high epithelial score. GSEA of non-malignant cells with significant changes for h Ifna response, i Ifng response, j IL2-STAT5 signaling, k inflammatory response and l oxidative phosphorylation. m Heatmap of tumor resident myeloid cells with significant changes (p < 0.1) in proportion relative to the total malignant and non-malignant cells (n = 7, 3, 3, and 3 individual tumours). n Heatmap of tumor resident lymphoid cells with significant changes (p < 0.1) in proportion relative to the total malignant and non-malignant cells (n = 7, 3, 3, and 3 individual tumours). o Relative PD-L1 expression within each cell cells (n = 7, 3, 3, and 3 individual tumours). 60% mutant tumors are coloured the same, presented as m.12,436G>A and m.11,944G>A from left to right. p Harvested tumor weight at day 21 (n = 15, 10, 7, 10, 9 and 5 individual tumours). Two-tailed Wilcoxon signed rank test (H-l), two-tailed student’s t-test (M,N) or one-way ANOVA test with Sidak multiple comparisons test (P) were applied. Error bars indicate SEM (O) or SD (P). Measure of centrality is mean. * P = < 0.05, ** P = <0.01. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes NES: normalised expression score. Source data

Extended Data Fig. 5. HcMel12 and 4434 mutant cells recapitulate the cellular phenotypes observed in B78-D14 cells.

a Heteroplasmy changes upon subsequent transfection of melanoma cell lines (n = 3–6 biological replicates). b Immunoblot of indicative respiratory chain subunits. Representative result of three biological replicates is shown. c mtDNA copy number (n = 9 technical replicates over n = 3 biological replicates). d Basal oxygen consumption rate (OCR) (n = 36, 21, 12 and 12 technical replicates over n = 12, 7, 4 and 4 biological replicates) e Proliferation rate of cell lines in permissive growth media (n = 12 and 6 technical replicates over n = 4 and 6 biological replicates) f Energy (adenylate) charge state (n = 24, 25, 18 and 18 technical replicates over n = 9, 9, 6 and 6 biological replicates). g NAD+:NADH ratio (n = 24, 25, 17 and 18 technical replicates over n = 9, 9, 6 and 6 biological replicates). h GSH:GSSG ratio (n = 22, 21, 15 and 16 technical replicates over n = 9, 9, 6 and 6 biological replicates). i Heatmap of unlabelled steady-state abundance of select metabolites in Hcmel12 cells. Succ. cys, succinylcysteine. (n = 24–25 technical replicates over n = 9 biological replicates). j Heatmap of unlabelled steady-state abundance of select metabolites in 4434 cells. Succ. cys, succinylcysteine. (n = 15–18 technical replicates over n = 6 biological replicates). Heatmap of k U-¹³C-glucose- and L U-¹³C-glutamine-derived select metabolites in Hcmel12 and 4434 cells (n = 6–18 separate technical replicates over n = 2–6 biological replicates). Volcano plot showing detected differences in protein abundance of m B78-D14 wild-type versus B78 mt.12436^80% cells N Hcmel12 wild-type versus Hcmel12 mt.12436^83% cells and o 4434 wild-type versus 4434 mt.12436^72% cells (n = 3 biological replicates). Heatmaps of protein abundances for p complex I, q complex II, r complex III, S complex IV and t complex V nuclear and mtDNA-encoded subunits in B78 mt.12436^80%, Hcmel12 mt.12436^83% and 4434 mt.12436^72% cells compared to their respective wild-type (n = 3 biological replicates). u Venn diagram comparing proteomic changes of mutant versus wild-type per cell line. v Significant KEGG pathway changes of mutant versus wild-type per cell. A student’s one-tailed t-test (C-H, I-L), student’s two-tailed t-test (P-T), two-tailed Wilcoxon signed rank test (M-O) or Fishers exact test (FDR = 1%) (V) were applied. Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01, *** P = <0.001, **** P = <0.0001. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes. Source data

Extended Data Fig. 6. Untreated Hcmel12 and 4334 lineage tumors recapitulate B78-D14 lineage.

a Survival of C57/BL6 mice subcutaneously injected with indicated cells (n = 8–18 animals). b Untreated tumor weight at endpoint (n = 17, 18, 11 and 8 individual tumours). c Survival of C57/BL6 and NSG mice subcutaneously injected with indicated Hcmel12 cells (n = 10 animals). d Untreated tumor weight at endpoint (n = 10 individual tumours). e Change in detected heteroplasmy in bulk tumor samples (n = 14 and 8 individual tumours). 4434 mutant tumors display a modest shift in heteroplasmy that is not seen in Hcmel12 or B78 (Extended Data Fig. 3d), likely reflecting enhanced immunogenicity of the mutant genotype. f Bulk tumor mtDNA copy number (n = 12, 9, 8 and 8 individual tumours). g Heatmap of steady-state abundance of metabolic terminal fumarate adducts, succinylcysteine and succinicGSH, demonstrating that metabolic changes observed in B78 mutant tumors are preserved in Hcmel12 in vivo (n = 9 individual tumours). h GSEA of Hcmel12 bulk tumor RNAseq data (n= 3 individual tumours) showing mutant^80% versus wild-type. Log-rank (Mantel-Cox) test (A, C), one-tailed student’s t-test applied (B, D) or two-tailed Wilcoxon signed rank test (H) were applied. Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes. Source data

Extended Data Fig. 7. Conditioned media from Hcmel12 cells does not stimulate STAT1 in BMDC and splenocyte cultures.

a Immunoblot of STAT1 and pSTAT1 isoforms from conditioned BMDCs. Representative image of three biological replicates is shown. b Densitometric ratio of normalised pSTAT1 relative to media in BMDC cultures (n = 3 biological replicates). c Heatmap of mean fluorescence intensity of specific activation markers of immune cells within BMDC cultures (n = 9 technical replicates over n = 3 biological replicates). Statistics are shown relative to negative control. d Immunoblot of STAT1 and pSTAT1 isoforms from conditioned splenocytes. Representative result of three biological replicates is shown. e Densitometric ratio of normalised pSTAT1 relative to media in splenocyte cultures (n = 3 biological replicates). f Heatmap of mean fluorescence intensity of specific activation markers of immune cells within splenocyte cultures (n = 9 technical replicates over n = 3 biological replicates). Statistics are shown relative to negative control. g Heatmap of metabolite abundance changes relative to wild-type tumors for respective tumor lineages (n = 6–38 individual tumours). Macrophages: Cd11b+ Ly6C- F4/80+. Monocytes: CD11b+ Ly6C+ F4/80-. Neutrophils: CD11b+ Ly6C+ Ly6G+. cDCs (conventional Dendritic Cells): CD11c+ MHCII+ F4/80- Ly6C-. All P-values were determined using a one-way ANOVA test with Fisher’s LSD Test (B-C,E-G). Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01, *** P = <0.001, **** P = <0.0001. Heatmap representations of data where asterisks are not present report non-significant changes. Source data

Extended Data Fig. 8. Gating strategy for flow cytometry.

a Gating strategy for Zombie+ live cells for all experiments. b Gating strategy for neutrophils, monocytes, macrophages and cDCs in BMDCs. c Gating strategy for CD4+ and CD8+ T-cells in splenocytes. d Gating strategy for neutrophils in tumors, lymph nodes and spleens. e Gating strategy for CD4+ T-cells, CD8+ T-cells, NK T-cells and macrophages in tumors, lymph nodes and spleens.

Extended Data Fig. 9. Characterisation and comparison of cytoLbNOX expressing cells in vitro and in vivo.

a Immunoblot of cytoLbNOX and catalytic mutant expression in clonal population, detected using αFLAG. Representative result of three biological replicates is shown. b Immunoblot of indicative respiratory chain subunits. Representative result of three individual runs is shown. c Basal oxygen consumption rate (OCR) (n = 36, 21, 24 and 9 technical replicates over n = 12, 7, 8 and 3 biological replicates). A significant decrease is observed in HcMel12 cytoLbNOX, akin to the decrease in basal OCR measured in m.12,436^80% cells. d mtDNA copy number (n = 9 technical replicates over n = 3 biological replicates). e Proliferation rate of cell lines in permissive growth media (n = 12, 12, 12 and 8 technical replicates over n = 4, 4, 4 and 3 biological replicates). f Energy (adenylate) charge state (n = 24, 25, 25 and 9 technical replicates over n = 9, 9, 9 and 3 biological replicates). g NAD+:NADH ratio (n = 24, 25, 25 and 9 technical replicates over n = 9, 9, 9 and 3 biological replicates). h GSH:GSSG ratio (n = 22, 21, 20 and 9 technical replicates over n = 9, 9, 9 and 3 biological replicates). i Heatmap of unlabelled steady-state abundance of select metabolites in Hcmel12 cells. Succ. cys, succinylcysteine. (n = 9–25 technical replicates over n = 3–9 biological replicates). j Survival of C57/BL6 mice subcutaneously injected with indicated cells (n = 17, 18, 12 and 9 animals). k Untreated tumor weight at endpoint (n = 17, 18, 12 and 9 individual tumours). l Survival of C57/BL6 mice subcutaneously injected with indicated cells (n = 15, 15 and 10 animals) on sustained anti-PD1 therapy. Only tumors that hit endpoint of 15mm shown for cytoLbNOX. m Tumor weight at endpoint for mice on sustained anti-PD1 therapy (n = 15, 15 and 3 individual tumours). Tumor volume changes recorded from injection date for n wild-type and m.12436^80% (n = 15 individual tumours) and o cytoLbNOX tumors (n = 10 individual tumours) on sustained anti-PD1. A one-way ANOVA test with Fisher’s LSD Test (C-I), Log-rank (Mantel-Cox) test (J,L) or one-way ANOVA with Sidak multiple comparisons test (K,M) were applied. Tumor volume calculated as 0.5*L*W² based on calliper measurements. Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01, *** P = <0.001, **** P = <0.0001. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes. Source data

Extended Data Fig. 10. Sensitisation to anti-PD1 negatively correlates to abundance of tumour-resident neutrophils.

Abundance of specific immune cells in a tumor, b tumour-draining lymph node and c spleen in untreated mice (n = 4–8 individual samples). d Schematic of the experimental plan and dosing regimen for Hcmel12 tumors with anti-PD1 monoclonal antibody (mAb) and either G-CSF or anti-Ly6G. e Tumor weight of untreated mice compared to mice treated with G-CSF or anti-Ly6G (n = 8, 8, 7, 8, 7, 8, 7, 7, 7 and 7 individual tumours). Log₂ fold change of tumor neutrophils in untreated and treated mice relative to untreated control for f G-CSF and g anti-Ly6G (n = 4–8 individual tumours). Tumor weight of mice treated with anti-PD1 or anti-PD1 and h G-CSF (n = 8, 7, 8, 8, 8, 7 and 7 individual tumours) or i anti-Ly6G (n = 8, 8, 8, 7, 8 and 8 individual tumours). Natural Killer T-cells: CD4- CD8- NK1.1+. Macrophages: Cd11b+ Ly6C- F4/80+. Neutrophils: CD11b+ Ly6C+ Ly6G+. All P-values were determined using a one-way ANOVA test with Fisher’s LSD Test (A-C,E-I). Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01, **** P = <0.0001. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes. Source data