band intensities were quantified with imagej. cells were collected with a cell scraper, transferred into eppendorf tubes, and centrifuged at 6, 500 rpm for 5 min. the pellet was resuspended in 200 µl of lysis buffer supplemented with 10 µl of proteinase k ( 10 mg ml; applichem ). samples were incubated overnight at 55 °c and then for 10 min at 95 °c to inactivate proteinase k and centrifuged at 17, 000g for 10 min at room temperature. the supernatant, containing crude dna, was retained for qpcr analysis. dna was isolated from approximately 3 × 10 immortalized fibroblasts from pdgfrα – egfp transgenic mice and from a431 cells using a qiaamp dna mini kit ( 51306, qiagen ). both cell types were cocultured for 24 h, followed by facs isolation of mouse fibroblasts. dna was isolated and analyzed by pcr using primers that amplify the gene encoding human mitochondrial 16s rna. pcr products were visualized on a 2 % agarose gel. the pcr product was purified using a qiaquick pcr purification kit ( 28104, qiagen ) and sequenced. species - specific and cell - type - specific snps were determined by comparing individual chromatograms using snapgene software ( gsl biotech ). scrna - seq and spatial transcriptomics 10x visium analysis was performed on the basis of published scc datasets. for scrna - seq, prefiltered data were used according to the quality control procedures of each paper. the logged count per 10k ( cp10k ) was used for normalization. the distribution of expression of miro2 was compared using a wilcoxon rank - sum test. for spatial transcriptomics, the spots were normalized using the logged cp10k expression. to analyze the colocalization of miro2 mrna positive spots with caf subtypes, we first assigned spots to fibroblasts or cafs ( positive for pdgfra ), to an icaf subtype ( positive for pdgfra and mmp11 ), to a myof ibroblast caf subtype ( positive for pdgfra and acta2 ), to an adicaf subtype ( positive for pdgfra and cfd ) or to inhba + fibroblasts ( expression of inhba detected ). to assess statistical significance of miro2 and caf subtype spots ( inhba ), we computed the six nearest neighbors of each spot and compared, using fisher ’ s exact test, the enrichment of neighboring spots expressing both miro2 and the caf subtype marker or inhba versus noncolocalized spots. for spatial transcriptomics data, the leading edge annotations were obtained from the authors. the effect of miro2 knockdown in genome-wide knockdown screens was analyzed using depmap ( depmap. org ). tcga clinical, survival and rna - seq data from primary tumors of 8, 911 participants across 30 solid cancer types were downloaded from the ucsc xena data hub ( https: / / xena. ucsc. edu ) using the ucscxenatools r package ( version 1. 4. 8 ). gene expression values were downloaded as log2 - transformed rsem normalized counts. the continuous miro2 gene expression was used for survival analysis censored at 5 years of follow - up. hazard ratios were computed using the cox proportional hazard model implemented in the ‘ coxph ’ function from the r package survival ( version 3. 5 - 7 ). for visualization, gene expression was divided into terciles and kaplan – meier survival curves were computed using the r package ggsurvfit ( version 0. 3. 1 ). the leading edge expression signature was compose d by 91 genes upregulated at the leading edge compared to the tumor core. for each tcga sample, a signature enrichment score was computed using the ‘ gsva ’ method from the gene set variation analysis r bioconductor package ( version 1. 46. 0 ). the correlation between the leading edge signature score and miro2 expression was assessed using pearson ’ s correlation. random - effects meta - analysis across all cancer types was conducted with the ‘ metagen ’ function from the r package meta ( version 7. 0 - 0 ). total rna was isolated as described above. rna - seq was performed after poly ( a ) enrichment and true - seq library preparation on a novaseq 6000 sequencer ( illumina ). fastq files were trimmed using trimmomatic ( version 0. 36 ), and processed using salmon ( version 1. 10. 2 ) using default parameters. the count matrix was processed using tximport to obtain gene - level counts and transcripts per million ( tpm ) estimates. pca was computed on the standar diz ed tpm expression. differential expression among control, mitotracker - high and mitotracker - low fibroblasts was computed using pydeseq2 ( https: / / github. com / owkin / pydeseq2 ), a python variant of deseq2 ( ref. ). significance levels were cut at 10. to compare the markers of mitotracker - high fibroblasts with known caf subtypes, we defined a gene signature for mitotracker - high fibroblasts using genes that were significantly differentially expressed compared to both mitotracker - low fibroblasts ( adjusted p < 0. 01 ) and control populations ( adjusted p < 0. 01 ) and overexpressed in the mitotracker - high population ( fold change ( fc ) > 1. 25 ). we computed the enrichment of this signature in known caf subtype signatures with fisher ’ s exact test, using genes quantified in the rna - seq experiment as background. single - cell data from human sccs were used as a reference to deconvolve visium 10x spots. a negative binomial model was trained on the discovery cohort with default parameters to estimate cell - type - specific average gene expression profiles. next, the cell2location model was applied, setting the prior to n = 15 average cells per spot and α = 20 for relaxed regularization, which produced estimated counts of each cell type per spot. we next sampled from the model ’ s posterior distribution to determine cell - type - specific gene expression per spot. fibroblast - specific expression estimates per spot were used to score caf subtypes, leveraging marker genes from human sccs. additionally, we assessed miro2 expression using the estimated raw counts for keratinocytes. samples were prepared using sp3 technology. briefly, cell pellets were lysed in radioimmunoprecipitation assay buffer ( 50 mm tris, 150 mm nacl, 1 % trit on x - 100, 0. 5 % sodium deoxycholate and 0. 1 % sds ) and lysates were sonicate d in an ultrasonic bath. protein amount was quantified with a bicinchoninic acid assay ( pier23225, pierce biotechnology, thermo fisher scientific ). then, 10 µg of protein was used for downstream analyses. after reduction of proteins with 5 mm dithioth reitol ( dtt ), they were alkylated with 5. 5 mm iodoacetamide and quenched with 5 mm dtt. sp3 beads ( sera - mag speedbeads, ge healthcare, 45152105050250 and 65152105050250 ) were added to protein lysates in a 10: 1 ratio. binding to the beads was induced by the addition of 100 % ethanol. after rinsing of beads with 80 % ethanol, samples were digested overnight with trypsin ( 1: 25 ) in 100 mm ammonium bicarbonate, ph 8. peptides were desalted using stage tips and adjusted to a concentration of 100 ng µl in 0. 1 % formic acid. peptides were analyzed by lc – ms / ms on a vanquish neo ultrahigh - performance lc instrument coupled to an orbitrap exploris 480 ( both from thermo fisher scientific ) as previously described. briefly, samples were applied to fused silica c18 column tips ( inner diameter: 75 μm; new objective ), produced in house with reprosil - pur 120 c18 aq ( 1. 9 μm, length: 20 cm; dr. maisch ) using a mixture of water ( solvent a ) and 80 % acetonitrile in water ( solvent b ), both acidified with of 0. 1 % formic acid. samples were separated at a flow rate of 250 nl min within 85 min ( 5 – 30 % solvent b ). data were acquired by data - independent acquisition ( dia; full ms, 350 – 1, 200 m / z; 120, 000 resolution; maximum injection time, 60 ms; 28 ms / ms scans with a width of 30. 4 m / z; 1 - da overlap ). a normalized automatic gain control target value of 300 %, resolution of 30, 000 and normalized stepped collision energy of 25. 5 %, 27 % and 30 % were used.