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Breast cancer cells rely on environmental pyruvate to shape the metastatic niche

Extracellular matrix (ECM) is a major component of the local environment, i.e. the niche, that can determine cell behavior1. During metastatic growth, cancer cells shape the ECM of the metastatic niche by hydroxylating collagen to promote their own metastatic growth2, 3. However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen because nutrients dictate which enzymatic reactions are active in cancer cells4, 5. Here, we discovered that breast cancer cells rely on the nutrient pyruvate to drive collagen-based ECM remodeling in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activated collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Strikingly, inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodeling and the nutrient environment in the metastatic niche.

of the enzyme collagen prolyl-4-hydroxylase (P4HA). Strikingly, inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodeling and the nutrient environment in the metastatic niche.

Keywords
Extracellular matrix metabolism; collagen remodeling; collagen modification; collagen hydroxylation; metabolic regulation; lung microenvironment; pyruvate metabolism; αketoglutarate; metastatic growth; metastatic niche; breast cancer; breast cancer-derived lung metastases The metabolic requirements of ECM production and modification can be enforced in vitro by shifting cancer cells from attached monolayer (2D) to spheroid (3D) growth. In the latter condition, soft-agar coating prevents cells from attachment and thus induces in cells the need to generate ECM for effective growth. We postulated that nutrients that drive 3D (but not 2D) growth could be a requirement for ECM metabolism. Consequently, we depleted glucose, glutamine, or pyruvate from the media and assessed growth of MCF10A H-RAS V12 and 4T1 cells in 3D compared to 2D cultures. The latter nutrient has been identified to be particularly available in the lung6, 7, which is a frequent metastatic site of breast cancers. We found that only pyruvate depletion impaired the 3D growth of breast cancer cells, while having only a minor effect on 2D growth (Figure 1a, Extended Data Figure 1a, b). This identifies pyruvate as a nutrient potentially important for ECM metabolism. In this case, we expect that ECM supplementation (Matrigel) restores 3D growth in the absence of pyruvate and decreases pyruvate uptake. Indeed, Matrigel induced the expected alterations in MCF10A H-RAS V12 cells (Figure 1b, 1c). Notably, non-tumorigenic MCF10A cells were pyruvate independent (Extended Data Figure 1c). Thus, we concluded that pyruvate supports ECM metabolism in breast cancer cells.
Next, we investigated the impact of pyruvate on collagen-based ECM production and modification by cancer cells (Extended Data Figure 1d). We used different human (MCF10A H-RAS V12 , MCF7, HCC70) and mouse (4T1, EMT6.5) breast cancer cells and assessed collagen hydroxylation (ECM modification) and collagen synthesis (ECM production). Nontumorigenic MCF10A cells were used as control. We found that pyruvate significantly increased hydroxylated collagen in all cancer cells (Figure 1d), but had no effect on nontumorigenic MCF10A cells and collagen synthesis (Figure 1d, Extended Data Figure 2a, b). We obtained similar results by targeting pyruvate uptake (by inhibiting the pyruvate transporter monocarboxylate transporter (MCT) 2) and pyruvate metabolism (by inhibiting the mitochondrial pyruvate carrier) (Figure 1e, Extended Data Figure 3a-c). These results suggest that pyruvate is required for collagen modification (i.e. hydroxylation) rather than synthesis.
As hydroxylation is essential for collagen stability, we next measured the stability of collagen produced by MCF10A H-RAS V12 and 4T1 cells using a matrix metallopeptidase (MMP) 8 assay. MMP 8 digests collagen I-III, but digestion is impaired by increased stability. If pyruvate drives collagen stability via hydroxylation, we expect that upon pyruvate depletion MMP 8 is more effective in digesting cancer cell-produced collagen. Specifically, we measured the hydroxyproline distribution between cells and supernatant because only hydroxyproline from digested collagen is released to the supernatant. We observed that pyruvate depletion significantly decreased the stability of collagen produced by MCF10A H-RAS V12 and 4T1 cells (Figure 1f). Thus, we concluded that pyruvate drives ECM remodeling by inducing collagen hydroxylation, which results in elevated collagen stability.
We then investigated the mechanism by which pyruvate drives collagen hydroxylation. We postulated that metabolites that changed in abundance upon pyruvate depletion could mechanistically link this nutrient to collagen hydroxylation. We found that upon pyruvate depletion the abundance of α-ketoglutarate, citrate and malate were reduced in MCF10A H-RAS V12 spheroids (Extended Data Figure 4a, b). Lactate, a metabolite that can be taken up by cancer cells and converted into pyruvate8, was not altered in abundance and did not influence collagen hydroxylation (Extended Data Figure 4a, c). Consequently, we measured hydroxylated collagen in the presence of α-ketoglutarate, citrate and malate and observed that only addition of α-ketoglutarate significantly increased hydroxylated collagen (  (Figure 1f). Thus, we concluded that pyruvate-induced α-ketoglutarate production drives collagen hydroxylation.
Pyruvate metabolism can be linked to α-ketoglutarate via several metabolic pathways. The most direct metabolic link is the alanine aminotransferase (ALT) reaction that converts pyruvate and glutamate to α-ketoglutarate and alanine (Extended Data Figure 1d). Measuring the 13 C carbon distribution9 in the metabolites of the ALT reaction, we observed that pyruvate contributed more carbons to alanine than to α-ketoglutarate and that glutamine (important glutamate precursor) donated about 45% of carbon to α-ketoglutarate (Extended Data Figure 5a). Moreover, alanine was secreted specifically in the presence of pyruvate (Extended Data Figure 5b) and neither alanine nor glutamate rescued hydroxylated collagen upon pyruvate depletion (Extended Data Figure 4c). These findings indicate an involvement of ALT in pyruvate-driven α-ketoglutarate production. Consequently, we measured αketoglutarate and hydroxylated collagen abundance upon ALT2 inhibition and found that both were decreased (Extended Data Figure 5c-f). Thus, we concluded that pyruvate drives α-ketoglutarate production through ALT conversion.
Next, we investigated whether α-ketoglutarate is a carbon donor for generating the P4HA substrate proline, or whether α-ketoglutarate metabolically activates P4HA via enzyme kinetics (Extended Data Figure 6a). We excluded the first mechanism by inhibiting the αketoglutarate to proline conversion via pyrroline-5-carboxylate synthase (P5CS) silencing (Extended Data Figure 6b, c). We then investigated whether α-ketoglutarate could be a metabolic activator of P4HA (Extended Data Figure 6a). Metabolic activation depends on metabolite substrate-and product-driven enzyme activity10, i.e. high substrate metabolite concentrations activate flux through a metabolic enzyme, while high product metabolite concentrations have an inhibitory effect. Thus, if α-ketoglutarate is a metabolic activator of P4HA activity, succinate as the co-product of the reaction should impair P4HA activity. Therefore, we added succinate to breast cancer spheroids and measured hydroxylated collagen in the presence of pyruvate. In accordance with the metabolic activator mechanism, we found that succinate significantly decreased hydroxylated collagen, while αketoglutarate on top of succinate rescued it (Figure 2c). These findings support the notion that P4HA is metabolically regulated by α-ketoglutarate. Interestingly, non-transformed fibroblasts (primary human skin-derived fibroblasts and immortalized human mammary myofibroblasts), which can also remodel ECM, were pyruvate, α-ketoglutarate and succinate independent, while cancer-associated myofibroblasts moderately decreased hydroxylated collagen in response to a decreased α-ketoglutarate to succinate ratio and decreased pyruvate availability (Extended Data Figure 6d-f). This suggests that metabolic regulation of P4HA is important in transformed cells and that in particular breast cancer cells (but not normal cells12) modulate this metabolic regulation via pyruvate uptake.
To date, transcriptional regulation of P4HA expression by HIF1α is considered to be the major determinant of collagen hydroxylation11. In the metastatic niche various stimuli such as hypoxia and TGFβ signaling converge in HIF1α stabilization resulting in the transcriptional up-regulation of P4HA11. To address the relationship between metabolic and transcriptional regulation of P4HA, we determined hydroxylated collagen abundance upon P4HA1 overexpression with or without pyruvate, α-ketoglutarate or succinate in normoxia or hypoxia (Figure 3a, Extended Data Figure 7a, b). While P4HA1 overexpression slightly increased hydroxylated collagen in the absence of pyruvate in normoxia, addition of αketoglutarate was necessary in normoxia and hypoxia to rescue hydroxylated collagen ( Figure 3a). Moreover, succinate addition prohibited pyruvate-driven collagen hydroxylation in P4HA1 overexpressing spheroids in normoxia and hypoxia ( Figure 3a). Accordingly, hypoxia, TGF-β stimulation and IOX2 treatment (which is a PHD2 inhibitor leading to HIF1α stabilization) did not restore hydroxylated collagen upon pyruvate depletion in different cancer cells ( Finally, we asked whether the established mechanism occurs in the metastatic niche using two different breast cancer mouse models that metastasize to the lung. We confirmed that in vivo MCT2 inhibition resulted in the same intra-and extracellular metabolite abundance changes as identified in vitro ( Figure 4a). Subsequently, we investigated in vivo ECM remodeling of the metastatic niche. We analyzed collagen hydroxylation and functional collagen I/III deposition (for which collagen stability is a prerequisite) in the 4T1 and EMT6.5 breast cancer models upon pharmacologic and genetic MCT2 inhibition. We observed that MCT2 inhibition resulted in decreased hydroxylated collagen and functional collagen I/III deposition in the lung metastatic niche (Figure 4b, c, Extended Data Figure 8a, b). Next, we performed a rescue experiment with α-ketoglutarate upon MCT2 inhibition. We found that α-ketoglutarate restored hydroxylated collagen and functional collagen I/III deposition in the metastatic niche upon MCT2 inhibition (Figure 4d, e, Extended Data Figure 8c). Moreover, we inhibited pyruvate driven α-ketoglutarate production by targeting ALT2, which phenocopied the effect of MCT2 inhibition (Figure 4f, g, Extended Data Figure 8d). These findings show that breast cancer cells in the lung metastatic niche rely on pyruvate to drive collagen-based ECM remodeling.
Subsequently, we evaluated the metastatic burden of the lung based on metastatic area, metastases number and metastatic index (metastatic area divided by primary tumor weight) in the same mouse models as described above. While primary tumor growth and weight was not significantly or only mildly affected (Extended Data Figure 9a, b), metastatic burden was substantially reduced upon pyruvate uptake and metabolism inhibition (Figure 4h, i, Extended Data Figure 9c-e). In accordance, α-ketoglutarate treatment fully restored metastatic burden upon MCT2 inhibition (Figure 4j, Extended Data Figure 9f). Thus, we concluded that inhibition of pyruvate metabolism is sufficient to impair metastatic growth in the lung.
Taken together, these results demonstrate that pyruvate serves as a pivotal nutrient in the lung metastatic niche to promote cancer cell-dependent ECM modification that supports metastatic growth ( Figure 4k). Moreover, we provide evidence that nutrients fueling metabolic regulation can be decoupled from nutrients fueling carbon biosynthetic needs. This discovery allows for the first time to target regulation by metabolite concentrations, which was so far considered to be undruggable10. In conclusion, our data suggest that targeting pyruvate uptake can normalize the aberrant collagen remodeling in the lung metastatic niche and thus impair metastatic growth.

Cell Culture
MCF10A cells expressing H-Ras V12 (MCF10A H-Ras V12 ) as well as control cells expressing an empty pLA vector (MCF10A) were generated as previously described13. MCF10A and MCF10A H-Ras V12 cells were grown in Dulbecco's modified Eagle's medium-F12 (DMEM-F12) supplemented with 5% horse serum, 1% penicillin (50 units per ml), 1% streptomycin (50 μg per ml), 0.5 μg per ml hydrocortisone, 100 ng per ml cholera toxin, 10 μg per ml insulin, and 20 ng per ml recombinant human EGF. MCF10A and MCF10A H-Ras V12 cells were cultured in 0.5 mM pyruvate condition. As MCF10A H-Ras V12 cells undergo growth inhibition upon pyruvate depletion, we used for these cells 0.02 mM pyruvate instead of complete pyruvate depletion. MCF7, HCC70, primary human skinderived fibroblasts, immortalized human mammary myofibroblasts and immortalized human mammary cancer-associated myofibroblasts14 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin (50 units per ml), 1% streptomycin (50 μg per ml) and with or without 1 mM pyruvate. 4T1 and EMT6.5 cells were cultured in Roswell Park Memorial Institute medium (RPMI) with 10% fetal bovine serum, 1% penicillin (50 units per ml), 1% streptomycin (50 μg per ml) and with or without 1 mM pyruvate. All cell lines were cultured in spheroids as described before13. Growth factor reduced Matrigel (3%) without phenol red was added into the cold DMEM-F12 media before the seeding by using tips precooled to -20°C. 3D cultures were performed as described before13, 15. All cell lines were confirmed to be mycoplasma-free by Mycoalert detection kit (Lonza). MCF10A, MCF7, HCC70 and 4T1 cell lines were purchased from ATCC. The EMT6.5 cell line was kindly provided by Professor Robin Cell permeable α-ketoglutarate (dimethyl 2-oxoglutarate) and succinate (dimethyl succinate) were purchased from Sigma-Aldrich and used at the concentration of 1.5 mM. In figure 2c and 3b α-ketoglutarate and succinate were used at the concentrations of 2 and 1.5 mM for MCF7 and HCC70, respectively. Malate and citrate were purchased from Sigma-Aldrich and used at the concentration of 5 mM. Effective transport of supplemented metabolites into the cells was confirmed by mass spectrometry analysis (Extended Data Figure 4d).
α-Cyano-4-hydroxycinnamic acid and UK5099 were purchased from Sigma-Aldrich and used at the concentration of 1.5 mM and 50 µM respectively. TGFβ1 was purchased from Peprotech and used at the concentration of 14 ng per ml. IOX2 was purchased from Sigma-Aldrich and used at the concentration of 50 µM. All the compounds were added at day 0 and analyzed at day 5. All growth experiments were performed in n≥3 biological replicates.

Collagen staining
MCF10A H-Ras V12 spheroids were transferred to fibronectin-coated plates and allowed to loosely attach to facilitate the following steps. The spheroids were fixed with 4% parafolmaldehyde in PBS for 30 min and permeabilized with 0.5% Triton X100 in PBS for 30 min. The spheroids were then incubated with either anti-Collagen I (Abcam, Ab34710; 1:500 dilution) or anti-Collagen III (Abcam, Ab7778; 1:100 dilution) antibodies overnight at 4°C, washed 3 times with PBS, incubated for 1h with the appropriate Alexa Fluor 555conjugated secondary antibody (Life Technologies, A31272) and then washed again 3 times in PBS. Nuclei were stained with a solution of 1.5 mM of 4',6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, D9542) in PBS for 15 min. Coverslips were mounted in Fluorescence Mounting Medium (Dako, S3023). The samples were visualized on a SP8X inverted confocal microscope (Leica Microsystems) equipped with a 405 nm and a white light laser. Images were acquired in form of Z-stacks using the LAS AF acquisition software (Leica Microsystems). Image analysis and fluorescence intensity quantification were performed with Imaris Image Analysis Software 8 (Bitplane).

CRISPR knockout
The MCT2 KO cell lines were generated by cloning either one or two (different exons for higher KO efficiency) CRISPR guides of MCT2 in the lentiviral CRISPR and guide expressing plasmid lentiCRISPR v2 (Plasmid #52961). From these the human MCT2 gRNA1 (GAAGAATACGGTGACAGCTT), human MCT2 gRNA2 (combination of guide: GGAAGAATACGGTGACAGCTT and guide: GCAACTATAGATTGCTTGCAA), mouse MCT2 gRNA1 (combination of guide: GCGATATCCAAGCGATCTGAC and guideGTACTAACACGACTGTTCCGC) and mouse MCT2 gRNA2 (combination of guide: GAACCACTTCCTCCCGACGGA and guide: GCGTCCTGTTTAGATCTGTTC) were selected and cloned 3' to the U6 promoter through Gibson cloning 16, 17. Lentiviral particles were produced in HEK293 cells. MCF10A H-Ras V12 and MCF7 cells were selected with puromycin 1 µg per ml. 4T1 and EMT6.5 cells were selected with puromycin 2 µg per ml. As a control, a CRISPR line lacking the guide for MCT2 was generated. CRISPR-based knockout was confirmed by western blot (Extended Data Figure 10).

Overexpression
The PHA1 expressing line was generated by cloning the 1605 base pair coding domain sequence of P4HA1 into the lentiviral pLVX-IRES vector (Clontech, 632185) through Gibson cloning. The sequence of P4HA was bought as a G block from IDT. Lentiviral particles were produced in HEK293 cells. MCF10A H-Ras V12 cells were selected with hygromycin 600 µg per ml. As a control, an empty lentiviral pLVX-IRES vector was generated. Overexpression was confirmed by qPCR (Extended Data Figure 7b) and western blot (Extended Data Figure 10).

Western blot analysis
Cells were collected and then lysed in RIPA lysis and extraction buffer (Thermo Scientific) supplemented with proteinase (Roche, 11836153001) and phosphatase (PhosSTOP™, Sigma, 4906845001). Protein amount was measured using a pierce BCA protein assay kit (Thermo Scientific). 25-40 µg of protein were loaded on a NuPAGE 4-12% denaturing Bis-Tris gel and transferred to a nitrocellulose membrane (Thermo Scientific

Mass Spectrometry analysis
The quenching and extraction procedure of 3D spheroids was performed as described before13, 15. Pseudo labeling steady state after 5 days of labeling was verified experimentally (Extended Data Figure 10f, g). Metabolite abundances and 13 C labeling patterns were either analyzed with gas or liquid chromatography mass spectrometry. Metabolites for gas chromatography mass spectrometry analysis were extracted, derivatized and measured as described before18. Metabolites for liquid chromatography mass spectrometry analysis were derivatized and measured as described before13. In brief, metabolites were resuspended in 60% acetonitrile. Metabolites were measured using a 1290 Infinity II HPLC (Agilent) coupled to a 6470 triple quadrupole mass spectrometer (Agilent). Samples were injected onto an iHILIC®-Fusion(P) column with the above-mentioned solvents.

Hydroxylated collagen and collagen stability
We assessed hydroxylated collagen based on measuring the total hydroxyproline (OHproline) content. This assumes that the majority of the total hydroxyproline originates from collagen. Hydroxyproline content was quantified by a colorimetric protocol as described by Creemers et al19. Spheroids or extracts from tissue were washed once in PBS and hydrolyzed for 3.5 hours at 135°C in 6 N HCl. Thereafter, samples were vacuum-evaporated and dissolved in demineralized water. Hydroxyproline residues were oxidized by adding chloramine-T (Sigma-Aldrich), followed by the addition of Ehrlich's aldehyde reagent (mixture of p-dimethylaminobenzaldehyde, n-propanol and perchloric acid) and incubation of the samples at 65°C for chromophore development. A standard curve was made to calculate the absolute amount of hydroxyproline per sample, which was finally normalized to the protein content of a parallel sample (spheroids) or the same sample (tissue) determined by a BCA protein assay.
To assess collagen stability via resistance to MMP-mediated degradation, breast cancer spheroids were incubated with MMP 8 (0.4 ng per µl; Bio-Techne) for 24 hours at 37°C prior to hydrolysis of cells and supernatant. Subsequently, the hydroxyproline (OH-proline) content of the cells and the supernatant was measured as described above.

Total synthesis of collagen and protein
Total collagen and protein synthesis was quantified by incubating cells with 20 uCi/ml L-2,3,4,5-3 H proline (PerkinElmer), as described before20. Briefly, after overnight labelling, cells were lysed in extraction buffer (11% acetic acid in H2O with 0.25% BSA) and collagenous proteins were precipitated by the addition of 20% trichloroacetic acid. Radioactivity was determined by liquid scintillation counting, and normalized for protein content.

Protein synthesis
Protein synthesis was analyzed using the Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit (ThermoFisher Scientific) according to the manufacturer's instructions. Briefly, tumor spheroids were incubated with 50 µM Click-iT® HPG overnight in culture medium. Breast cancer spheroids were then fixed with 3.7% formaldehyde for 15 minutes at room temperature, washed and permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. Subsequently, cells were incubated with Click-iT® reaction cocktail for 30 minutes at room temperature protected from light, washed, and fluorescence was detected by flow cytometry. This assay assumes that the majority of methionine is incorporated into non-collagen protein.

In vivo pharmacologic, genetic inhibition and metabolism
All animal experiments were approved by the local authorities in compliance with all relevant ethical regulations (including but not limited to tumor size). For injection models, mice were randomized before injection of cancer cells. All samples were analyzed blinded. Sample size was determined using power calculations with B=0.8 and P<0.05 based on preliminary data and in compliance with the 3R system: Replacement, Reduction, Refinement.
Six weeks old female BALB/c mice were inoculated with 4T1 or EMT6.5 cells either into the mammary fat pad (m.f.; 1x10 6 cells) or intravenously (i.v.; 1x10 5 cells). α-cyano-4hydroxycinnamic acid treatment was started after four days when the primary tumor nodule was established and palpable. 60 mg per kg of α-cyano-4-hydroxycinnamic acid was injected daily intraperitoneal to cancer bearing mice. α-cyano-4-hydroxycinnamic acid was dissolved in a mix containing 1.5% DMSO, 60% β-cyclodextrin, 35% polyethylene glycol (PEG) and 5% ethanol and pH neutralized with NaOH. The same mix without drug was Metabolites were harvested, extracted and analyzed as described before21. Metabolites in the blood plasma were measured in the 4T1 m.  Figure 4f) post-injection of genetically modified cells. Mice were sacrificed using nembutal, the breast tumors and lung metastases were placed in cold saline, dissected in less than 3 min, and immediately frozen using a liquid nitrogen cooled Biosqueezer (Biospec Products). The tissue was weighed (10-15 mg) and pulverized (Cryomill, Retsch) under liquid nitrogen conditions. The pulverized tissue was used to measure α-ketoglutarate and hydroxyproline as described above. 20 μL of blood plasma were extracted and analyzed as described above. Humane endpoints were determined as follows: Tumor size of 2 cm 3 . Following additional symptoms were monitored and upon detection of one of the symptoms the animal was euthanized: Loss of ability to ambulate, labored respiration, surgical infection or weight loss over 10 % of initial body weight. Housing and experimental animal procedures were approved by the Institutional Animal Care and Research Advisory Committee of the KU Leuven, Belgium.

Picro-Sirius Red and hematoxylin and eosin staining
Treatment were performed as described above, i.e. pharmacological MCT2 inhibition was initiated upon palpable primary tumor, while genetic MCT2 inhibition was present already during tumor initiation. Tissues were harvested at the same time points as described above. For the Sirius Red and hematoxylin and eosin (H&E) staining of pulmonary metastasis, dissected lung samples were gently infused via the trachea with 10% formalin and then processed as previously described. 5 µm thick sections obtained from the resulting paraffin blocks were stained with either Picro-Sirius Red or H&E. As Picro-Sirius Red stains thinner fibers in green and thicker fibers in red and collagen III are thinner than collagen I the assumption is that green is mainly collagen III while red is mainly collagen I. Picro-Sirius Red slides were analyzed with polarized microscope Imager M2 Carl Zeiss at 20X magnification. Enough images were acquired for each metastasis to cover the whole metastatic area, then GNU Image Manipulation Program (GIMP 2.10.8) was used to manually stitch all images belonging to the same metastasis together. The composite images were analyzed with ImageJ 1.45. Briefly, the polarized light images were split by RGB (red, green, blue), then the signal in the green and red channels was quantified. Only metastases with similar size were analyzed. One to four similar sized metastases per mouse were analyzed. Native collagen and highly vascularized metastases were excluded from the analysis.
Metastatic area and metastases number were quantified by ZEN blue software (2011). Metastatic index was calculated by dividing the metastatic area by the primary tumor weight. Only mice with a primary tumor of 0.8 g or greater were analyzed. All i.v. injected animals were analyzed. The animal study complies with ethical regulation and was approved by the KU Leuven ethics committee.

Statistical Analysis and software
Statistical data analysis was performed using GraphPad Prism version 7.0 (GraphPad Software Inc., CA, USA) on n≥3 biological replicates. Details on statistical tests and posttests are presented in the figure legends. In brief, two-tailed unpaired student's T-tests were performed on n≥3 biological replicates. Two-way Anova was performed in Figure 3a as described in the correspondent figure legend. Sample size for all experiments was chosen empirically. Independent experiments were pooled and analyzed together whenever possible as detailed in figure legends. Detection of outliers was performed using Grubb's test in GraphPad. All graphs show mean +/-standard deviation or standard error of the mean as indicated in the figure legends.  gallate (EGCG; 50 µM), transduced with a lentiviral vector with shRNA for either mitochondrial ALT2 (KD), GDH (KD) or scrambled control sequence in the presence of pyruvate. n=3 for EGCG and AOA treatment (c-d); n=9 for control shRNA, n=6 for GDH shRNA 1 and 2 and n=3 for ALT2 shRNA1 and 2 (MCF10A H-Ras V12 , e-f); n=3 for control shRNA and ALT2 shRNA 1 and 2 (4T1, e-f). In case ALT activity majorly contributs to αketoglutarate generation, ECGC (which inhibits the pyruvate independent conversion of glutamate to α-ketoglutarate via the enzyme glutamate dehydrogenase (GDH)), should have a minor effect on α-ketoglutarate abundance and hydroxylated collagen. Indeed, we found that this was the case. Error bars represent SD of mean from biological independent samples. Two-tailed unpaired student's T-test. Europe PMC Funders Author Manuscripts proline abundance decreases in P5CS knockdown spheroids and that they no longer respond to the α-ketoglutarate rescue upon pyruvate depletion. However, we observed that proline abundance did not significantly change in P5CS knockdown spheroids. Moreover, αketoglutarate addition still significantly increased hydroxylated collagen to a similar extent as pyruvate. n=3 (b); n=6 (control shRNA); n=3 (P5CS shRNA 1 and 2 (c)).    Error bars represent SD of mean from biological independent samples (n=3). Two-tailed unpaired student's T-test.