{"paper_id":"ece9752f-6bc0-4e6e-99d3-76027d3945de","body_text":"We documented higher levels of basal glycolysis ( P  = 0.0499), lower mitochondrial respiration (ATP-linked) ( P  = 0.0400), and higher lactate secretion ( P  = 0.0004) in HPMCs from women with endometriosis compared to women without disease ( Fig. 1  A – C ). “Endo” HPMCs exhibited decreased enzymatic activity of pyruvate dehydrogenase ( P  = 0.0025) ( Fig. 1 D ).\nHPMCs from women with peritoneal endometriosis (Endo) had higher basal glycolytic extracellular acidification rates (ECARs) ( P  = 0.0499;  n  = 8) ( A ) and lower ATP-linked respiratory oxygen consumption rates (OCRs) ( P  = 0.0400;  n  = 8) ( B ) than women without endometriosis (No Endo). Consistent with Endo HPMCs having an altered metabolic phenotype, lactate secretion was higher ( P  = 0.0004;  n  = 8) ( C ) and enzymatic activity of pyruvate dehydrogenase (PDH) was lower ( P  = 0. 0.0025;  n  = 6) ( D ) than in cells from the No-Endo women. Treatment of Endo HPMCs with DCA reduced ECAR ( P  = 0.0071;  n  = 8) ( E ), increased OCR ( P  = 0.0140;  n  = 8) ( F ), suppressed lactate secretion ( P  = 0.0104;  n  = 8) ( G ), and increased PDH enzymatic activity ( P  = 0.0373;  n  = 6) ( H ) in both the presence and absence of TGF-β1. Lesion-in-a-dish coculture system. ( I ) There is no direct contact between HPMCs and stromal cells. Treatment of Endo HPMCs with DCA reduced both basal and TGF-β1–induced lactate secretion ( P  = 0.0003 and  P  = 0.0001, respectively;  n  = 3). ( J ) Treatment of Endo HPMCs with TGF-β1 stimulated proliferation of stromal cells ( P  = 0.0044;  n  = 3), but this did not occur if HPMCs were incubated with TGF-β1+DCA ( P  = 0.0002;  n  = 3). ( K ) In a mouse model, oral DCA reduced PF lactate concentrations ( P  = 0.0360) ( L ) and reduced the size of endometriosis lesions ( P  = 0.02). ( M ) Images of endometriosis lesions from mice treated with vehicle or oral 100 mg/kg DCA daily for 7 d ( n  = 10/group). ( N ) * P  < 0.05, ** P  < 0.01, *** P  < 0.001, and **** P  < 0.0001.\nTreatment of Endo HPMCs with DCA normalized their metabolic phenotype and increased PDH activity, in the presence or absence of TGF-β1 ( Fig. 1  E – H ). In a coculture system ( Fig. 1 I ), treatment of endometriosis HPMCs with DCA decreased basal and TGF-β1–stimulated HPMC lactate secretion ( P  = 0.0003) and proliferation of endometrial stromal cells ( P  = 0.0002,  Fig. 1  J  and  K ).\nIn a preclinical mouse model of endometriosis ( 4 ), treatment with oral 100 mg/kg DCA for 7 d reduced PF lactate concentrations ( P  = 0.0360) and the size of endometriosis lesions ( P  = 0.02) ( Fig. 1  L – N ).\n\nHPMCs from women with endometriosis synthesize and secrete more lactate than cells recovered from women without lesions. Lactate can stimulate cell migration and invasion, and immune escape during tumorigenesis, with the same processes implicated in the etiology of endometriosis ( 2 ). We postulate that increased lactate concentrations in pelvic PF may create an environment that promotes invasion of ectopic endometrial cells into the peritoneum so that they form lesions.\nWe have shown that Endo HPMCs exhibit a greater dependence on energy production through glycolysis under aerobic conditions than those of women who are disease-free. This abnormal cellular energy state is corrected by treatment with the small-molecule drug DCA. Notably, DCA reduced HPMC lactate release in vitro, and oral dosing of mice reduced endometriosis lesion size in vivo. DCA is a pyruvate dehydrogenase kinase (PDK) inhibitor used to treat cancer ( 5 ). Four different isoforms of PDK (PDK1–4) exist with variable expression reported: PDK1 appears to have the highest sensitivity to inhibition by DCA ( 6 ). PDK inhibition reduces phosphorylation of PDH, increasing PDH activity leading to lower release of metabolites such as lactate. DCA also inhibits proproliferative and proangiogenic transcription factors such as HIF-1α ( 5 ). In a clinical trial in patients with glioblastoma, DCA decreased tumor growth and angiogenesis ( 7 ).\nWhile we have focused on HPMCs, an overlooked source of metabolic factors in PF, DCA could also have an impact on other cell types altered in endometriosis patients. Macrophages that are present in PF or lesions of women with endometriosis exhibit a range of phenotypes ( 8 ), and treatment with DCA could drive metabolic reprogramming of macrophages toward a prorepair phenotype that could have a positive impact on the pathogenesis of the disease ( 9 ). Stromal cells in patient endometrium and lesions exhibit up-regulation of PDK1. If apoptosis of these cells is enhanced by DCA ( 10 ), this could contribute to reduced lesion growth.\nTo translate these results to women, we are using DCA in an exploratory-phase clinical trial ( ClinicalTrials.gov  identifier  NCT04046081 ). If effectiveness and acceptability are demonstrated, DCA would be a nonhormonal treatment for women with this debilitating condition.\n\nApproval was obtained from the Lothian Research Ethics Committee (LREC 11/AL/0376). Samples were collected from women attending a pain clinic who had a diagnosis of peritoneal endometriosis (Endo group) or women without macroscopic evidence of endometriosis at laparoscopy (“No Endo” group), all of whom had given informed consent. Women were not on hormones and had regular menstrual cycles. HPMCs were cultured in HOSE I media (40% Media 199, 40% IMDM, 15% heat-inactivated FBS, 0.5% penicillin/streptomycin, and 1%  l -glutamine). All cell/tissue samples were deidentified (anonymized but linked) prior to use in the study. Immortalized human endometrial stromal cells [SHT290 ( 11 )] were grown in RPMI 1640 media plus 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1%  l -glutamine, and 1% nonessential amino acids. Cells were incubated at 37 °C under 5% CO 2  in air.\nExtracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined using a glycolytic stress test kit (102340-100; Agilent) on a Seahorse XFe24 Analyzer. HPMCs were seeded in microplates at a density of 2 × 10 4  cells per well in 500 µL of HOSE I and incubated for 48 h. Cells were rinsed 3 times with 500 µL of prewarmed Seahorse Assay DMEM (no glucose or pyruvate); 525 µL of same medium was added to wells that were incubated at 37 °C for 30 min. ECAR and OCR measurements were taken simultaneously using an 8-min protocol (3-min mixing, 2-min wait, 3-min measure): 12 measurements were taken from each well; 3 times baseline prior to injection of glucose (10 mM), 3 following glucose and prior to injection of oligomycin (1 µM), 3 following oligomycin and prior to injection of 2-deoxy glucose (100 mM), and 3 following injection of 2-deoxyglucose. Basal glycolysis was calculated by subtracting the average ECARs before, and after, the injection of glucose. ATP-linked mitochondrial respiration was calculated by subtracting the average OCRs before and after the injection of oligomycin. After assay, cells were washed with PBS and protein was quantified to for normalization. Additional experiments used Endo HPMCs cultured for 16 h in HOSE 1, then switched to serum-free HOSE 1 containing vehicle (water), TGF-β1 (2 ng/mL, 240-B-010; R&D Systems), DCA (15 mM, 347795; Sigma), or TGF-β1+DCA, and incubated for 48 h before analysis of ECAR and OCR.\nLactate concentrations were determined using an enzymatic colorimetric kit (Alpha Laboratories) on a Cobas Fara centrifugal analyzer (Roche Diagnostics). PDH enzyme activity was measured using a Dipstick assay kit (ab109882; Abcam): band intensity was analyzed using ImageJ.\nEndo HPMCs were plated in 24-well plates at 1 × 10 5  cells per well (lower chamber); SHT290 were plated on Millicell-cell culture inserts (2 × 10 4  per insert; pore size, 0.4 µm; Millipore) (upper chamber) in serum-free HOSE I and RPMI 1640, respectively. After 16 h, HPMCs were incubated with vehicle (water), DCA (15 mM), TGF-β1 (2 ng/mL), or DCA+TGF-β1 for 48 h. Lactate concentrations were measured, and proliferation of SHT290 cells was determined using CellTiter96 Aqueous-one solution reagent (G3580; Promega).\nAnimal procedures were performed in accordance with UK Home Office regulations (license 70/8945). A preclinical model of endometriosis was as in ref.  4  with the minor modification that recipient mice were not ovariectomized or treated with E2. After 2 wk, mice were randomly assigned to receive vehicle (water) or DCA (100 mg/kg) daily via oral gavage for 7 d ( n  = 10/group). PF was recovered by lavage (2 mL of ice-cold PBS); endometriosis lesions were fixed in 4% neutral-buffered formalin, lactate was measured in PF, sections of lesions were stained with H&E, and the area was measured using ImageJ software.\nResults are expressed as mean ± SEM of a minimum of 3 independent experiments. Statistics were generated using GraphPad PRISM, version 6. Data were analyzed using one-way ANOVA Mann–Whitney  U  test, 1-way ANOVA Kruskal–Wallis test, and Dunn’s multiple-comparisons test, as appropriate.","source_license":"CC0","license_restricted":false}