Extended Data Fig. 5: Preclinical study in KPf/fCL mouse model reveals therapeutic beneficial effects of LIF blockade by anti-LIF monoclonal antibody.
From: Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring
a–d, Anti-LIF monoclonal antibody production and characterization. a, Silver staining of control mouse IgG and purified anti-LIF monoclonal antibody used for preclinical treatment. b, c, Immunoblot analysis to evaluate the activity and specificity of anti-LIF monoclonal antibody against various forms of LIF (rhLIF, recombinant human LIF; ehLIF, eukaryotically secreted human LIF; rmLIF, recombinant mouse LIF) and IL-6 (b), revealing that the LIF monoclonal antibody has weaker neutralizing activity against mLIF compared to against hLIF (c). d, Pilot test of anti-LIF monoclonal antibody in KPf/fCL mice for dosage optimization. KPf/fCL mice at 42 days of age were administered 25 mg per kg (body weight) of LIF monoclonal antibody by three intraperitoneal injections on alternate days, and one day after the last injection tumours were collected for histology analysis. Adjacent sections were used for the indicated staining. Representative images from three independent experiments or mice are shown. e, Regimen for the preclinical therapeutic treatment. KPf/fCL mice at five weeks of age were randomly enrolled into four cohorts. In phase one, during the first 12 days, 25 mg per kg (body weight) anti-LIF monoclonal antibody or control IgG were administered by intraperitoneal injection, together with 50 mg kg−1 Gem or vehicle at standard dosage every third day for four administrations. This was followed by weekly cycles as phase two with antibodies three times and Gem two times weekly. f, Histological characterization with representative images and cell abundance quantification. g, Relative abundance of EpCAM+ cancer cells quantified by flow cytometry analysis. h, Double immunofluorescence staining to confirm the cell type-specific expression of cytosolic protein KRT19 and nuclear protein PDX1 as the PCC marker. n = 3 tumour tissues. i, Double immunofluorescence staining of nucleus-localized proteins Ki67 (used as a proliferation marker) and PDX1 (as the PCC marker), and quantification of proliferating cancer cell frequency as the fraction of proliferating cancer cells (Ki67+PDX1+DAPI+) over total cancer cells (PDX1+DAPI+). j, Cleaved Caspase 3 immunohistochemistry analysis to assess apoptosis. Scale bars: yellow, 300 µm; black, 100 µm; white, 50 µm. Statistical significance was determined by two-tailed unpaired Student’s t-test.
