Fig 2: Expression of LIF is required for in vitro and in vivo malignancies of human pancreatic cancer cells. a The ability to form spheres in 3D culture, reduced by the depletion of KRAS, could be rescued by addition of LIF at 100 ng/mL. Sphere size is shown in the left panel, and sphere-forming efficiency (numbers of formed spheres/numbers of seeded cells) in the right panel (N = 6, *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001). a The ability of Panc1.0 to form spheres in 3D culture, reduced by knocking down KRAS, could be rescued by the addition of LIF, but not IL-6 at the same concentration (100 ng/mL) (N = 6, ***P < 0.001). b Representative western blots (left panel) showing the depletion of LIF protein expression by CRISPR/Cas9 and reduced phosphorylated STAT3 at Y705 in mouse pancreatic tumor cells. The ability to form spheres in 3D culture (middle panel), reduced by depletion of LIF, can be rescued by addition of mouse LIF at 100 ng/mL (N = 6, *P < 0.05, **P < 0.01). (Right panel) Overall survival rate of syngeneic mice which were orthotopically injected with mouse pancreatic cancer cells (n = 5). c Representative ELISA (left panel) detecting human LIF in culture medium of Panc2.03, which expressed inducible shRNA against human LIF. The cells were treated with doxycycline (1 µg/mL) for 14 days before ELISA assay. Reduced sphere formation efficiency (right panel) of cells where LIF had been knocked down by inducible sh-LIF in the presence of doxycycline (N = 6, **P < 0.01, ****P < 0.0001). Error bars from a to d represent the standard deviation and P-value was generated by t-test. d Representative western blots (left panel) showing LIF protein expression in Panc6.03 cell line which expressed inducible shRNA against human LIF. Induced expression of shRNA against LIF by doxycycline reduced the growth of the established Panc6.03 xenograft tumors when compared with the control (right panel). e Representative western blots (left panel) showing LIF protein expression in Panc1.0, which expressed inducible shRNA against human LIF. Tumor growth curve (right panel) of Panc1.0 xenografts where inducible LIF shRNA was expressed in response to gemcitabine at 50 mg/kg with or without doxycycline

Fig 3: LIF-neutralizing antibody acts as a therapeutic approach for treating KRAS-driven pancreatic cancers. a Representative ELISA assay showing that LIF antibodies (D25.1.4 and D62.3.2) prevent recombinant human LIF from binding to microplate pre-coated with human LIF monoclonal antibody (Invitrogen BMS242). b Representative western blots suggesting that use of a LIF-neutralizing antibody blocks the STAT3 activation/phosphorylation induced by recombinant human LIF (50 ng/mL) in human pancreatic cancer cell lines, whereas IL-6 induces STAT3 activation. c Tumor free survival rate and numbers of palpable tumors in Panc2.03 xenografts model receiving control or LIF antibody at 10 mg/kg. (d) Tumor growth in syngeneic mouse model receiving gemcitabine (50 mg/kg), LIF antibody (20 mg/kg), or gemcitabine along with LIF antibody. The cancer cells are expressed with firefly luciferase, which can be used to detect orthotopic tumor growth. Twice a week of treatments started on the fourteenth day post-tumor cell injections (N = 5, ****P < 0.0001). e Overall survival rate of syngeneic mouse model receiving gemcitabine, LIF antibody, or gemcitabine along with LIF antibody. f Tumor growth curve of PDX (patient-derived xenograft)_PC105 receiving gemcitabine (100 mg/kg) or gemcitabine along with LIF antibody (10 mg/kg). The treatment of gemcitabine was conducted once a week and LIF antibody was authorized twice a week (N = 6, *P < 0.05). g Tumor growth curve of PDX_PC187 receiving control IgG, gemcitabine (100 mg/kg), LIF antibody (10 mg/kg), or gemcitabine along with LIF antibody (10 mg/kg). The treatment of gemcitabine was conducted once a week and LIF antibody was authorized twice a week (N = 7, *P < 0.05; **P < 0.01). d, f, g Error bars: mean ± SEM and P-value was generated by Two-way ANOVA

Fig 4: Inhibition of MAPK-signaling pathway represses expression of LIF. a Representative western blots showing the amounts of LIF, phosphorylated ERK and phosphorylated AKT in Panc1.0 cells. Inhibition of the MAPK-signaling pathway by small molecules selumetinib, GSK-1120212, and PD0325901 suppressed the expression of LIF, whereas inhibiting the activity of AKT by MK-2206 did not. The inhibitory efficiency of the small molecules was confirmed by the phosphorylation levels of AKT at S-473 and the phosphorylation of ERK. The cells were treated with compounds for 48 h before harvested for western blot analysis. b Growth of Panc1.0 in 2D and 3D culture suppressed by MEK inhibitors, GSK-1120212, or PD0325901 at 1 µM. The suppression of growth can be rescued by adding LIF in culture medium. Crystal violet staining was used to visualize cell growth in 2D culture. Sphere formation assay was used to determine the growth in 3D (N = 6; **P < 0.01; ***P < 0.001). Error bar represents the standard deviation and P-value was generated by t-test

Fig 5: Oncogenic KRAS regulates the expression of LIF, but not other IL-6 family cytokines. a Representative western blots showing the amounts of LIF, KRAS, and phosphorylated STAT3 at Y-705 in multiple human and mouse pancreatic cancer cell lines where wild-type or mutant KRAS have been stably knocked down by shRNAs. b Representative western blots showing increased expression of LIF, KRAS, and phosphorylated STAT3 at Y-705 in mouse pancreatic adenocarcinoma cell line (iKRAS*). Representative qPCR showing increased mRNA expression of LIF and KRAS in the same cell line (N = 3; **P < 0.01; ****P < 0.0001). Error bar represents the standard deviation and P-value was generated by t-test. Its expression of KRAS was reactivated by adding 1 µg/mL of doxycycline in growth medium. iKRAS* line was harvested and established from pancreatic tumor developed in a p48Cre; TetO-KrasG12D; Rosa26rtTa/+; p53R172H/+mouse. c Western bot suggesting increased expression of LIF in KRAS mutant human pancreatic cancer cell lines, Panc1.0 and Panc2.03, which had higher RasGTP level when compared to KRAS wild-type cell line, BxPC3. RasRBD pull-down assay was used to determine RasGTP level. The cells were serum-starved for 24 h before harvested for RasRBD pull-down. d Oncomine analysis suggesting upregulation of human LIF, but not IL-6, in the human cancer cell lines where KRAS is mutant when compared with cells expressing wild-type KRAS.The analysis in the pan-cancer cell lines (KRAS WT vs mutant) (Nature volume 483, pages 570–575 (29 March 2012)) was performed by using Oncomine Platform Software (Invitrogen; https://www.oncomine.org/resource/login.html)