and M.D.; NRSA F31CA206416 and T32GM007752 to N.K.L.; F32CA217033 to G.L.; Flinn Foundation to E.B.; NIH CA178015, CA227807, CA222862 to E.A.C; NIH CA197699, CA186043 to T.R., NIH CA155620 to A.M.L.; NIH P50CA127297, U01CA210240, P30CA36727, 5R50CA211462 to P.M.G. readily detectable factors. Therefore, we hypothesized that this communication between PSCs and PCCs could be an Achilles heel exploitable to develop effective strategies for PDAC therapy and diagnosis. Here, starting with systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukemia inhibitory factor (LIF) is a key Gracillin paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic deletion significantly slow tumour progression and augment chemotherapy efficacy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and EMT status. Moreover, we show that, consistently in both mouse models and human PDAC, aberrant production of LIF in the pancreas is unique to pathological conditions and correlates with PDAC pathogenesis, and circulating LIF level changes correlate well with tumour response to therapy. Collectively, these findings uncover a previously unappreciated function of LIF in PDAC tumourigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. These studies underscore how a better understanding of cell-cell communications within the tumour microenvironment promotes novel strategies for cancer therapy. To comprehensively characterize the paracrine communication between PCCs and PSCs, we carried out integrated mass spectrometry (MS)-based quantitative proteomic analyses combining secretome profiling with phosphoproteomics (Fig. 1a). Phosphotyrosine proteomic analysis, performed to explore intracellular signaling events, revealed STAT3 activation as a prominent event in Rabbit Polyclonal to CDC7 PCCs in response to PSC conditioned medium (CM) stimulation, and in parallel secretomes of each cell type, MIAPaCa2 and hPSCs11 as representative lines, were individually profiled to quantitatively catalog the complete protein composition in the CM (Fig. 1b,?,cc and Extended Data Fig. 1aCd). We then conducted IP-MS assays to explore STAT3-associated proteins, especially receptor(s), and found the LIF receptor (LIFR) and its co-receptor IL6ST/GP130 as the only receptors pulled down by STAT3 in a strictly CM stimulation-dependent manner (Fig. 1d,?,ee and Extended Data Fig. 1e). Consistently, LIF was produced by hPSC in copious amounts, but not by MIAPaCa2, pinpointing LIF as the key paracrine factor for STAT3 activation in PCCs (Fig. 1c). Open in a separate window Physique 1 | Combinatorial MS analyses identified LIF as a key paracrine factor.a, Schematic workflow of the MS strategy combining secretome and phosphoproteomic analyses. Matched serum-free medium was used as control stimulation. b, Phosphotyrosine proteomic analysis of CM-stimulated intracellular signalling in PANC1 cells. c, Proteomic analysis and comparison of MIAPaCa2 and hPSC secretome presented as an MA plot. n=2 biological replicates (b,c). d,e, IP-MS assay on 3xFlag-STAT3-expressing PANC1 cells to identify CM stimulation-dependent STAT3-associated proteins. n=3 biological replicates. f,g, IB analyses of pSTAT3 in KP4 cells with LIF blockade by LIFR knockdown or anti-LIF mAb. CM harvested from hPSC. h, Lif levels in mouse pancreatic normal and tumour tissues by ELISA. NT=7; PDAC=8. i,j, RNAscope assays to examine cellular sources of mRNA expression in mouse (i) and human (j) pancreatic tissues. mRNA was co-stained to mark cancer cells. NT, mouse normal pancreatic tissues or non-tumour parts resected from the human tumour Gracillin trunks; PDAC, tumour tissues collected from mice or PDAC patients. Scale bars: black, 200 m; blue, 50 m. Representative images from at least three biological replicates per experiment were presented (f,g,i,j). LIF is usually a pleiotropic cytokine regulating cell differentiation, proliferation and survival in the embryo and the adult12, and is also involved in cancer development13C16. LIF production by PSCs was reported recently17C19, but its physiological significance in PDAC tumourigenesis is usually unclear. We firstly assessed LIF actions and found widespread response in PCCs but not in activated PSCs and normal fibroblasts, and among three reported downstream pathways12, mainly STAT3, but not ERK and Gracillin AKT, was robustly activated (Extended Data Fig..