Nerve-to-cancer transfer of mitochondria during cancer metastasis

Abstract

The nervous system has a pivotal role in cancer biology, and pathological investigations have linked intratumoural nerve density to metastasis¹. However, the precise impact of cancer-associated neurons and the communication channels at the nerve-cancer interface remain poorly understood. Previous cancer denervation models in rodents and humans have highlighted robust cancer dependency on nerves, but the underlying mechanisms that drive nerve-mediated cancer aggressivity remain unknown^2,3. Here we show that cancer-associated neurons enhance cancer metabolic plasticity by transferring mitochondria to cancer cells. Breast cancer denervation and nerve-cancer coculture models confirmed that neurons significantly improve tumour energetics. Neurons cocultured with cancer cells undergo metabolic reprogramming, resulting in increased mitochondrial mass and subsequent transfer of mitochondria to adjacent cancer cells. To precisely track the fate of recipient cells, we developed MitoTRACER, a reporter of cell-to-cell mitochondrial transfer that permanently labels recipient cancer cells and their progeny. Lineage tracing and fate mapping of cancer cells acquiring neuronal mitochondria in primary tumours revealed their selective enrichment at metastatic sites following dissemination. Collectively, our data highlight the enhanced metastatic capabilities of cancer cells that receive mitochondria from neurons in primary tumours, shedding new light on how the nervous system supports cancer metabolism and metastatic dissemination.

Fig. 1. Cancer metabolic dependency on nerves and intercellular transfer of functional mitochondria at the nerve–cancer interface.

a,b, Pre-denervation breast cancer model using BoNT/A injections in BALB/c mice followed by implantation of 4T1^mCherry cells. Transcriptomic analysis of 4T1^mCherry cells revealed a distinct transcriptomic signature and downregulation of metabolic processes. SSC-H, side scatter height; RNA-seq, RNA sequencing. c, Confocal micrograph of SVZ-NSCs^GFP mixed with 4T1^mCherry cells. White arrows show the establishment of neuron–cancer contacts. Scale bar, 50 μm. d, 4T1 cells FACS-isolated from coculture (left) showed increased OXPHOS capacities (Seahorse assay; mean ± s.e.m; representative profile (n = 3); right). O, oligomycin; F, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; R + A, rotenone and antimycin A; OCR, oxygen consumption rate. e,f, Time-lapse confocal microscopy (e) and flow cytometry (f) highlighted mitochondrial transfer from SVZ-NSCs^CCO-GFP to 4T1^mCherry cells (n = 6 independent cocultures). Scale bar, 10 μm. The white arrows show transferred mitochondria. g,h, 3D reconstruction shows transfer through tunnelling nanotubes between peripheral nervous system-derived 50B11-DRG^CCO-GFP cells and 4T1^mCherry cells. Red arrow shows the tunnelling nanotube structure, and white arrows show transferred mitochondria. Scale bars, 20 μm. i, Quantification of direct cell–cell contact (23.04%) versus distant (0.59%) transfers using Transwell inserts. Normalized transfer rate is calculated as the percentage of 4T1^{mCherry+/GFP+} cells among the eGFP⁺ cells in the coculture. Mean ± s.d., Student’s two-tailed unpaired t-test; ****P < 0.0001 (n = 6 independent cocultures). Ctrl, control. j, Cytochalasin B (Cyto B) reduces mitochondrial transfer. DMSO, dimethylsulfoxide. Mean ± s.d., Student’s two-tailed unpaired t-test, ***P = 0.001 (n = 3 independent cocultures). k, Mitochondrial transfer rates vary with donor cells from different origins. MEF, mouse embryonic fibroblast. Mean ± s.d. (n = 5 independent cocultures). l, 4T1^mCherry cells rendered devoid of mtDNA (ρ⁰) were cocultured with ρ⁺ SVZ-NSCs^GFP and isolated by FACS at various times to monitor the transfers. mUNG1, Y147A mutant of uracil-N-glycosylase. m, PCR of mtDNA content in FACS-sorted ρ⁰ 4T1^mCherry cells showed progressive reacquisition of mtDNA. Expression of GFP and mCherry confirmed the purity of the 4T1 cells. Nuclear DNA (nDNA) was used as the loading control. n, MitoTracker microscopy imaging of ρ⁺ 4T1, ρ⁰ 4T1 and ρ⁰ 4T1 cells FACS-sorted from coculture shows rescue in their mitochondrial morphology. o, Reacquisition of SVZ-derived mtDNA in ρ⁰ 4T1 cells restores uridine-independent growth. p,q, FACS-isolated ρ⁰ 4T1 cells rescued by mitochondrial transfer regained OXPHOS (p; Seahorse assay, mean ± s.e.m.; representative profile (n = 3)) and proliferative capacities (q; direct cell counting, mean ± s.d., n = 6 independent cultures); two-way analysis of variance (ANOVA), ****P < 0.0001; NS, not significant. a, Created in BioRender. S. Grelet (2025) https://biorender.com/0j8zovf. d, Created in BioRender. S. Grelet (2025) https://biorender.com/oxxilqq. i, Created in BioRender. S. Grelet (2025) https://biorender.com/8tdz09x. l, Created in BioRender. S. Grelet (2025) https://biorender.com/pm5yh64. Source Data

Fig. 2. Intercellular transfer of mitochondria between host neuron and cancer.

a, Histopathology of human prostate cancer with perineural invasion shows increased mitochondrial content near nerves (mitochondria are visualized by periodic acid–Schiff staining (magenta); nerves are visualized by diaminobenzidine staining (brown)). Representative profile (n = 72 patients). Scale bar, 180 μm. b, Multispectral imaging combined with machine learning-based image deconvolution, spatial correlation, and quantification indicate significantly higher mitochondrial loads in prostate cancer cells near nerves (perineural; n = 72, 40,007 cells) compared to distant cancer cells (distant; n = 58, 20,766 cells). The line shows the median, the box boundaries show the 25th and 75th percentiles, and the whiskers show the minimum and maximum values. Two-sided Welch’s t-test, ***P < 0.001. c, BoNT/A-mediated denervation reduces mitochondrial load in human prostate cancer cells (paired analysis: saline versus BoNT/A; saline: n = 10,918 cells, BoNT/A: n = 14,186 cells). Two-sided Welch’s t-test, ****P = 1.463 × 10⁻¹¹³. Clinical trial (NCT01520441). d, Mouse DRG neurons innervating the mammary gland were labelled with lentivirus (LV) to tag neuronal mitochondria before injection of 4T1^mCherry cells into mammary fat pads (MFPs). Cancer cells were isolated post-tumour growth for mitochondrial transfer analysis to detect host-derived mitochondrial transfer. e, Neuronal mitochondria were labelled using lentiviruses expressing either nuclear-localized (GFP-NLS, non-transferable) or mitochondria-localized (GFP-OMP25, transferable) eGFP under the synaptin1 (Syn1) promoter. Flow cytometry of cancer cells identified eGFP⁺ subpopulations, indicating neuronal mitochondrial transfer between mouse host neurons and cancer xenografts. f, Sanger sequencing enabled the detection of mtDNA polymorphisms between host BALB/c cell and 4T1 cancer cell mtDNA. g, Oxford Nanopore sequencing analysis of mtDNA heteroplasmy in FACS-isolated cancer cells demonstrated host-to-cancer mitochondrial transfer. BoNT/A-mediated pre-denervation at the xenograft site significantly reduced mitochondrial transfer to cancer cells (saline n = 8, BoNT/A n = 9; median values indicated). The line shows the median, the box boundaries show the 25th and 75th percentiles, and the whiskers show the minimum and maximum values. One-tailed unpaired Student’s t-test *P = 0.0316 (n = 8 saline, n = 9 BoNT/A mice). c, Created in BioRender. S. Grelet (2025) https://biorender.com/mprd95w. d, Created in BioRender. S. Grelet (2025) https://biorender.com/98cu18d. g, Created in BioRender. S. Grelet (2025) https://biorender.com/xapzg43. Source Data

Fig. 3. MitoTRACER for lineage tracing of cell–cell transfer of mitochondria.

a,b, MitoTRACER strategy. Donor neurons express mitochondria-targeted Cre recombinase (iCre) with an SV40 nuclear localization signal (NLS-iCre), linked to the OMP25 mitochondrial outer membrane domain. Recipient cells express both a loxP-DsRed-Express2-Stop-loxP-eGFP switch and the TEVp. After transfer, TEVp cleaves NLS-iCre, enabling nuclear localization and excision of DsRed-Express2, resulting in a permanent change from DsRed (red) to eGFP (green) expression. LTR, long terminal repeat. c, 4T1^{loxP-DsRed-Express2-Stop-loxP-eGFP} co-expressing both MitoTRACER and TEVp shows efficient NLS-iCre cleavage and eGFP expression activation. No unintended cleavage was detected in the absence of TEVp expression. The same sample extracts were loaded in different gels. Representative experiment (n = 3). FL, full length. d,e, Confocal microscopy (d) and flow cytometry (e) of SVZ neuron–4T1 coculture confirmed red-to-green conversion, confirming the transfers and suitability of the approach for high-throughput analysis and collection of recipient cells. WT, wild type. Scale bar, 50 μm. f, Dose-dependent increase of mitochondrial transfer with donor-to-recipient ratios (1:1 to 4:1). The centre line shows the median, the box boundaries show the 25th and 75th percentiles, and the whiskers show the minimum and maximum values. Student’s two-tailed unpaired t-test, ****P < 0.0001 (n = 5 independent cocultures). g, Time-dependent and cumulative increase of mitochondrial transfer from day 1 to day 3. The centre line shows the median, the box boundaries show the 25th and 75th percentiles, and the whiskers show the minimum and maximum values. Student’s two-tailed unpaired t-test, ****P < 0.0001 (n = 6 independent cocultures). h,i, Western blot (h) and densitometry analysis (i) of the MitoTRACER subcellular localization through the HA tag expression on the construct confirmed its mitochondrial localization. Expression of COXIV and HSP90 validated the subcellular fraction purity. The same sample extracts were loaded in different gels. Representative experiment (n = 3) (mean ± s.d.; n = 4 independent experiments). C, cytoplasmic fraction; M, mitochondrial fraction. j, Coculture using recipient cells lacking TEVp confirmed the signal’s specificity. The line shows the median, the box boundaries show the 25th–75th percentiles, and the whiskers show the minimum and maximum values. Student’s two-tailed unpaired t-test, P = 2.16463 × 10⁻¹⁰, ****P < 0.0001 (n = 5 independent cocultures). a, Created in BioRender. S. Grelet (2025) https://biorender.com/aa3gfx0. b, Created in BioRender. S. Grelet (2025) https://biorender.com/ytn18rx. Source Data

Fig. 4. Neuron-to-cancer mitochondrial transfer enhances cancer OXPHOS, stemness and resistance to metastatic stressors.

a, 4T1 recipient (green) cells sorted after MitoTRACER coculture showed spontaneous sphere formation capacities. Scale bar, 500 μm. b, Mammosphere formation assay confirmed increased stemness potential in green cells (***P = 0.0005, n = 8 independent cultures). Student’s unpaired two-tailed t-test. c,d, Recipient green cells show enhanced mitochondrial OXPHOS capacities. CC, coculture. Representative profile (n = 3); mean ± s.e.m., parental: n = 11, red: n = 13, green: n = 12 cell cultures; Student’s unpaired two-tailed t-test, *P = 0.037, ***P < 0.001, ****P < 0.0001. e, Energy map shows a more aerobic and energetic phenotype in green cells compared to the red counterpart (mean ± s.d. n = 24). ECAR, extracellular acidification rate. f, Luminescence-based assay of total cellular ATP content showed significantly higher levels in green cells (**P = 0.0044, n = 3 independent cultures). RLU, relative light units normalized per cell. Mean ± s.d., Student’s two-tailed paired t-test. g,h, Green cells exhibited increased GSH (***P = 0.0002, n = 6 independent cultures) and overall improved GSH/GSSG ratios (**P = 0.0028 (g), **P < 0.01 (h), n = 6 independent cultures). Mean ± s.d., Student’s two-tailed paired t-test. FC, fold change. i,j, Green cells exhibited higher tolerance to H₂O₂-induced oxidative stress (i; mean ± s.d., Student’s two-tailed paired t-test, NS, not significant; two-way ANOVA, P = 0.0007) and greater resistance to shear stress (j; two-way ANOVA, P < 0.001). Representative profile (n = 3). k, Modified Boyden chamber assay revealed no change in intrinsic invasion capacity between green and red cells in vitro (NS, not significant; Student’s two-tailed t-test, n = 3 independent cultures). l, In vivo, metastatic progression was enhanced in green versus red cells in mouse mammary fat pad xenografts, as observed by increased liver metastasis (mean ± s.d.; Student’s two-tailed unpaired t-test, *P = 0.01997, n = 8 mice). m, Haematoxylin and eosin liver sections showed metastatic lesions, and Ki67 immunostaining confirmed the cancerous character of the lesions. Scale bar, 4 mm. n,o, Multispectral imaging in human breast cancer and matched metastatic sites revealed increased mitochondrial content at metastatic versus primary sites. Representative image from patient breast cancer samples (n) and matching metastasis and mitochondrial score quantification curve in the samples set (o). The line shows the median, the box boundaries show the 25th and 75th percentiles, and the whiskers show the minimum and maximum values (two-sided Welch’s t-test, ***P < 0.001; n = 8 patients). k,l, Created in BioRender. S. Grelet (2025) https://biorender.com/35rzyib. Source Data

Fig. 5. Lineage tracing of the intercellular transfer of mitochondria from neuron to cancer cell during breast cancer dissemination in vivo.

a, 3D spheroids of SVZ-NSCs^MitoTRACER mixed with 4T1 recipient cancer cells confirmed mitochondria transfer, evidenced by red-to-green fluorescence conversion in 4T1 cells (white arrows). b,c, MitoTRACER spheroids were transplanted into mammary fat pads of BALB/c mice (b); after cancer progression, flow cytometry of cells isolated from primary tumours, lungs and brains revealed selective enrichment of green fluorescent cells (eGFP⁺) in lung and brain metastases compared to the primary tumour (c). Mean ± s.d.; Student’s two-tailed paired t-test, lung: *P = 0.018, brain: ***P = 1.2275 × 10⁻⁶; n = 9 mice; ANOVA, P = 0.0005. d, Schematic of the procedure for lineage tracing of mitochondrial transfer between mouse host mammary neurons and cancer cells in vivo. Lentivirus expressing Syn1-MitoTRACER construct was injected into the DRG area, innervating the lower mammary fat pads. After 10 days, the SWITCH-TEVp-expressing 4T1 recipient cells were injected. Following tumour growth, tissues (primary, lung, brain and liver) were analysed for mitochondrial transfer by flow cytometry. e, Flow cytometry of primary tumours showed eGFP⁺ cells, confirming mitochondrial transfer. Control lentivirus expressing Syn1-driven nuclear-localized Cre (LV-CRE-NLS) showed no green signal, validating the specificity of our approach. f, Ex vivo quantification of mitochondrial transfer in the primary tumour and lineage tracing of metastatic development in the lung, brain and liver showed significant enrichment of eGFP⁺ cells in metastatic sites compared to the primary tumour (ANOVA, P < 0.0001), with significant enrichment in brain and liver metastases (mean ± s.d., Student’s one-tailed paired t-test values, brain: **P = 0.0096, liver: *P = 0.0215; n = 5 mice). Top panel shows distribution of red and green cells. g, Validation using syngeneic B16-F1 melanoma cells co-injected with SVZ-NSCs^MitoTRACER as mixed-cell spheroids in C57BL/6 mice showed significant mitochondrial transfer enrichment in brain metastases (mean ± s.d.,  ANOVA, P = 0.0009; Student’s one-tailed paired t-test, brain: **P = 0.0017; n = 8 mice). Top panel shows distribution of red and green cells. b, Created in BioRender. S. Grelet (2025) https://biorender.com/culfgzj. d, Created in BioRender. S. Grelet (2025) https://biorender.com/2w7o7rr. Source Data

Extended Data Fig. 1. Related to Fig. 1 - Cancer denervation models in vivo.

(A) Ductal carcinoma in situ innervation model to evaluate nerve-cancer dependencies in breast cancer. At 4 weeks after intraductal transplant of ductal carcinoma in situ cells into mice, primary cancers were chemically denervated by BoNT/A treatment or treated with saline control (saline, n = 20 mice; BoNT/A, n = 25 mice). (B) Heatmap of differentially expressed genes between saline and BoNT/A-treated groups. Transcriptomic analysis of microdissected primary cancer cells showed 116 genes were significantly altered: 43 upregulated and 73 downregulated in the BoNT/A-treated group. (C) Gene Set Enrichment Analysis (GSEA) revealed significant downregulation of metabolic pathways, with the tricarboxylic acid (TCA) cycle being the most impacted in cancer cells derived from denervated tumors. (D) Treemap visualization of reduced Gene Ontology (GO) terms generated using the rrvgo interpretation platform confirmed the regulation of biological processes, predominantly linked to cancer cell metabolism in denervated tumors. Each square of color represents distinct clusters of GO terms grouped by semantic similarity. The size of the squares corresponds to the score of the representative GO term, with higher scores indicating greater significance in the analysis. The list of reduced GO terms obtained through rrvGO was provided in Supplementary Table S3. (E) Quantification of invasive lesions at 10 weeks through microscopy-based pathological assessment by a certified pathologist of tumors that extend beyond the originating duct, into the host stroma, after breaching the basement membrane. A significant reduction in the mean number of invasive lesions was observed in the BoNT/A-treated group compared to the saline group, indicating decreased invasiveness of cancer cells following denervation (mean ± SEM; unpaired t-test; p = 0.0014). a, Created in BioRender. S. Grelet (2025) https://biorender.com/q4jviiq. Source Data

Extended Data Fig. 2. Related to Fig. 1 - Nerve-cancer crosstalk model in vitro.

(A) Subventricular zone neuronal stem cells (SVZ-NSCs) were isolated from a fresh 6-week-old female BALB/c mouse brain. The dorsal view of the dissected mouse brain is shown (scale bar, 1 cm). (B) Lateral view of the mouse brain showing the translucent SVZ region. The diagram shows the area (orange) from which SVZ-NSCs were extracted. (C) Isolated SVZ-NSCs were cultured on laminin-coated plates. The cells differentiated into mature neurons using a differentiation medium. (D) The SVZ-NSCs were maintained as neurospheres on uncoated plates, confirming stemness (self-renewal). (E) Fluorescence microscopy of SVZ-NSCs transduced with GFP-expressing lentiviruses and cultured independently (SVZ^GFP Alone) showed moderate neurite development, evidenced by the absence of neurite extensions. SVZ-NSCs cocultured with 4T1 cells (SVZ-NSC^GFP + 4T1) showed a marked increase in SVZ-NSC differentiation, evidenced by extensive neurite outgrowth traversing the coculture. Scale: 100μm. (F) Immunohistochemistry of SVZ-NSC^GFP cocultured with 4T1 cells shows SVZ-NSCs expressing the neuronal marker Tubulin β-3 (Tubb3), confirming neuronal differentiation. Scale: 100 μm. (G) Flow cytometry analysis of nerve-cancer cocultures indicates the commitment of neuronal precursors to neurons. Nearly all neurons expressed MAP2 and Tubulin β−3 (TUBB3) but were negative for O4 and ALDH1L1, confirming that these cells were not oligodendrocytes or astrocytes. (Mean ± S.D, Student’s two-tailed unpaired t-test, n = 3). (H) Histogram plot from flow cytometry illustrating the neuronal commitment of SVZ-NSCs in the nerve-cancer coculture. EGFP-expressing SVZ-NSCs, introduced into the coculture and comprising approximately 1.6% of the total cell population in the series, were gated based on eGFP expression to assess SVZ marker expression using antibodies against O4, ALDH1L1, TUBB3, and MAP2. The omission of the primary antibodies in co-culture consisting of eGFP-expressing SVZ-NSCs mixed with 4T1 cancer cells was used as control. (I) Validation of antibodies used to quantify the neuronal commitment of SVZ-NSCs in the nerve-cancer coculture. SVZ-NSCs were treated with triiodothyronine (T3) to induce oligodendrocyte commitment and stained with the O4 antibody. To induce astrocyte differentiation, cells were treated with fetal bovine serum (FBS) and stained with the ALDH1L1 antibody. For neuronal differentiation, cells were treated with differentiation medium and stained with Tubulin β3 (TUBB3) or Microtubule-Associated Protein 2 (MAP2) antibodies. The omission of primary antibodies on SVZ-NSCs culture was used as control. (J) Calcium flux analysis in nerve-cancer coculture using Fluo-4 AM staining. (Top) Time-lapse imaging showed pulsatile calcium activity in neurons. (Bottom) Signal dynamics with 10 μM nifedipine treatment demonstrated a blockade of calcium flux, abolishing pulsatile activity. (K) Current-clamp recording of neurons in nerve-cancer coculture. Whole-cell recordings were used to record the membrane potentials of neurons. Cells clamped at −80 mV, were depolarized with incremental current ramp injections ranging from 20–200 pA, as shown in the inset. Cocultured neurons showed action potential activity. (L) Distribution of action potential threshold measurements between neurons analyzed in the nerve-cancer coculture. Action potentials were elicited in all recorded neurons (mean ± SEM n = 9 neuron cells acquisition; 100% response).

Extended Data Fig. 3. Related to Fig. 1 - Nerve-cancer crosstalk and mitochondrial metabolism.

(A) Related to Fig. 1d - Oxygen consumption rate (OCR) analysis comparing basal, maximal, and spare respiratory capacities of 4T1 cells alone versus coculture with SVZ-NSCs. Coculture led to a significant increase in all three parameters, suggesting enhanced mitochondrial function (Representative Profile (n = 3); Mean ± SEM; Student’s two-tailed unpaired t-test **** P < 0.0001). (B) MitoSOX mitochondrial indicator staining (red) of SVZ-NSCs before (SVZ-NSC) and after differentiation (NSC Diff.). There was a significant increase in mitochondrial activity observed after differentiation, evidenced by an increase in MitoSOX staining. (C) Cancer-driven neuronal differentiation markedly upregulated mitochondrial load in SVZ-NSCs. PCR of mtDNA content in SVZ-NSCs under varied concentrations of differentiation medium (0% to 80%) showed a progressive increase in mtDNA content throughout differentiation (nDNA, nuclear DNA loading control). (D) FACS analysis following mitochondrial staining (MitoTracker) shows SVZ-NSCs in three conditions: undifferentiated and cultured alone (blue, SVZ-NSCs alone), differentiated with nerve growth factor (red, SVZ-NSCs + NGF), and differentiated by coculture with 4T1 cancer cells (green, SVZ-NSCs in coculture). Representative Profile (n = 3 idenpendent cell cultures). (E) FACS and quantitative polymerase chain reaction (qPCR) of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) ratio in SVZ-NSCs alone (Alone) or in coculture with 4T1^mCherry cells (With 4T1) (mean ± S.D.). There was increased mitochondrial mass in SVZ-NSCs after exposure to 4T1 cells. (F) Fluorescence microscopy of SVZ-NSCs having eGFP-labeled mitochondria (SVZ-NSC^CCO-GFP). The mitochondria had globular morphology and were localized around the nucleus. In contrast, SVZ-NSC^CCO-GFP cocultured with 4T1^mCherry cells had mitochondria with elongated morphology and were dispersed throughout the cell cytoplasm. Scale: 10 μm. Source Data

Extended Data Fig. 4. Related to Fig. 1 - Nerve-cancer transfer of mitochondria in vitro.

(A) Quantification of mitochondrial transfer. Flow cytometry confirmed strong eGFP fluorescence in 50B11 DRG-derived cells (50B11^CCO-GFP). When cocultured with 4T1^mCherry+ cells, mitochondrial transfer was indicated by the acquisition of eGFP fluorescence in the 4T1^mCherry+ cells. The gating strategy was established using 50B11^CCO-GFP and 4T1-mCherry cells cultured independently. (B) Cell viability of cultures treated with cytochalasin B (Cyto B) for 24 h. The percentage of live cells was calculated using Hoechst/PI staining and automated counting with a fluorescence cytometer (Celigo). No significant change in cell viability was observed after treatment in both SVZ-NSC and 4T1 cancer cells. (C) Quantification of mitochondrial transfer from (Left) SVZ-NSC^CCO-GFP or (Right) 50B11 mitochondrial donor cells to cancer cell lines derived from human breast (MDA-MB-231), lung (A549), and prostate (PC3) carcinomas. The normalized transfer rate was calculated as the percentage of recipient cells within the eGFP+ population in the coculture, standardized across biological conditions. Mean ± S.D. (n = 6). (D) Related to Fig. 1m. Quantitative PCR analysis of mtDNA acquisition in ρ⁰ 4T1 cancer cells exposed to SVZ-NSCs and FACS isolated from the co-culture. Mean ± S.D. (n = 3).

Extended Data Fig. 5. Related to Fig. 2 - Nerve-cancer transfer of mitochondria in vivo.

(A) Microscopy validation of the Syn1-GFP-NLS and Syn1-GFP-OMP25 lentiviral constructs in PC12 neuronal cells and 4T1 breast cancer cells. Only neurons displayed expression of the synapsin 1-driven constructs, with eGFP localized to the nucleus (localization signal, NLS) or mitochondria (OMP25). DAPI was used as a counterstain for the nuclei. Scale: 50μm. (B) Flow cytometry validated the Syn1-GFP-OMP25 construct and confirmed neuron-specific expression by showing strong expression in PC12 neuronal cells and no activity in 4T1 cancer cells. This analysis also determined the titer of the production batch. (C) Immunohistochemical staining of mouse mammary fat pad xenograft tissue nerves using tubulin beta-3 (Tubb3) and labeling of the eGFP fluorophore confirms the expression of the transgene in the mouse host nerves. 4X magnification of the xenograft area. Zoom: Nerve fiber at 40X magnification. Magenta: tubulin beta-3, Green: eGFP, Blue: DAPI. Scale: 500 μm.

Extended Data Fig. 6. Related to Fig. 3 – MitoTRACER.

(A) Validation of the cloning constructs. We tested the plasmid constructs by co-transfection of MitoTRACER with increasing amounts of a separated TEVp-encoding plasmid into 4T1 cells stably expressing the LoxP-DsRed Express2-STOP-LoxP-eGFP recipient genetic switch but lacking the TEVp construct. Fluorescence microscopy showed activation of the Red-to-Green fluorophore switch when all components of the system were expressed in the cells by co-transfection with the TEVp. This confirmed the Red-to-Green switch and the requirement of TEVp to ensure feasibility. Scale: 100 μm. (B) Time-lapse fluorescence microscopy over 12 h of SVZ-MitoTRACER mitochondrial donor cells cocultured with 4T1 SWITCH-TEVp recipient cells, illustrating the dynamics of mitochondrial transfer. The process included the establishment of close cell-cell contacts, the development of tunneling nanotube structures, and subsequent red-to-green fluorescence transition in recipient cells. Arrowheads: blue recipient cells; yellow, tunneling nanotube structure. Captures from Video S3.