(A) Mapping of carbon atoms transition using U-13C3-lactate. Mass isotopomer distribution (MID) shows uptake of labelled lactate by MSCs cultured in α-MEM leading to the generation of α-KG (M2 αKG) via the Krebs cycle. (B) Conditioned media (CM) from mock treated and LDH inhibitor (FX11) treated Panc-1 (PDAC) cells was collected. MSCs were exposed to media alone, control conditioned media (PDAC CM), or LDH inhibitor treated conditioned media for 14 days in order to generate de novo CAFs (dn-CAF). TET enzymatic activity increases in dn-CAFs after exposure to PDAC CM and is abrogated after exposure to CM from LDH inhibitor treated Panc-1 cells (N = 2, p<0.05). (C) Quantitative analysis of 5-hMC levels by LC-MS demonstrates significant increase within dn-CAFs after treatment with Panc-1 CM, that is abrogated after exposure to CM from LDH inhibitor treated Panc-1 cells (N = 2, p<0.05). (D) CAFs were exposed to exogenous lactate and α-KG levels were analysed by ELISA. (N = 2, p<0.05) (E) 5hmC analysed in the resulting dn-CAFs by LC-MS. After a 2 week exposure to exogenous lactate, significantly increased 5hmC is observed in the dn-CAFs (N = 2, p<0.05). (F) Schematic model of lactate flux from tumor cells to MSCs during CAF differentiation, leading to aKG generation, TET activation and conversion of 5mC into 5hmC.