Fig 1: EV-KRAS complex facilitates inflammation through the IL-17A/FGF21 axis. (a) HE staining of lung tissue from mice (blocked with IL-17R/Fc or anti-FGF21 antibody) intratracheally administered extracellular vesicle (EV) KRAS complex (liposome-EV-KRAS). (b) ELISA for TNFa, IL-17A and IL-6 from lung tissue from mice (blocked with IL-17R/Fc or anti-FGF21 antibody) intratracheal administered liposome-EV-KRAS. (c) Adenosine triphosphate (ATP) cell proliferation assay showing proliferation of Jurkat cells in response to lung tumour lysate with and without IL-17A depletion using IL-17A immunoprecipitation. (d) Luciferase assay for FGF21 promotor assessment, TC1 cells treated with respective lung tumour lysate with and without IL-17A depletion using IL-17A immunoprecipitation. (e) Western blot for PI3K, SIRT1, NF?B- TC1 cells treated with respective lung tumour lysate with and without FGF21 depletion using FGF21 immunoprecipitation. (f) qRT-PCR for genes involved in EMT pathway- TC1 cells treated with respective lung tumour lysate with and without FGF21 depletion using FGF21 immunoprecipitation. For in vivo treatment mice were administered with 50 µl of 10 × 1010 liposome particles/ml and for in vitro treatment 2 × 108 liposome particle/ml for respective group were used. Data are mean ± SEM from three replicates using one-way ANOVA, **p < 0.01.
Fig 2: Proteins interacting with extracellular vesicular (EV) G12D-mutant KRAS are different from those that interact with intracellular G12D-mutant KRAS. (a) A representative image shows silver stained proteins from EV and cell lysates pulled down with streptavidin after running PAGE. (b) Heat map of proteins highly abundant or absent in EV-KRAS complex (EV-KRAS) group compared to the cell lysate-KRAS complex (Ly-KRAS) group, scale represents fold change in absolute abundance. (c) Pie-chart showing the number of proteins in the EV-KRAS subset analyzed by mass spectrometry (MS). (d) To confirm mass spectrometry data, western blots for Fn1, tubulin-ß and KRAS were performed showing the differences in streptavidin pull down proteins from EV and cell lysates. Data are from three replicates.
Fig 3: Extracellular vesicular (EV) KRAS complex is more potent in promoting lung tumour growth than KRAS complex from EVs donor cells: TC1 cells were treated for three consecutive days with liposome containing KRAS complex from EV or EV-donor cells (Cell lysate; Ly) and then administered intratracheally to mice. (a) Uptake of liposome-EV-KRAS in TC1 cells. (b) Timeline of in vivo experiment for TC1 cell treatment for lung tumour development showing development of TC1 tumour following intratracheal administration of TC1 cells (PBS, liposome alone, liposome-EV-KRAS, or lysate KRAS complex (liposome-Ly-KRAS). (c) Pictorial representation of TC1 tumour development in mouse lung. (d) HE staining of TC1 tumours. (e) Tumour volume and number of nodules in lung tumours. (f) Immunostaining of CD11b and Gr-1 positive cells in lung tissue (scale: 20 µm). (g) Flow cytometry analysis of CD8 positive IFN? and Granzyme B cells. (h) ELISA for IL-17A, IL-6 and FGF21. (i) Heat map showing expression of genes associated with in epithelial–mesenchymal transition. For TC1 lung tumour development, the respective liposome-KRAS treated TC1 cells (2 × 105 cells in 50 µl) were administered using the IMIT instillation procedure. Data are mean ± SEM from five biological replicates, *p < 0.05, **p < 0.01 using one-way ANOVA.
Fig 4: Fn1 plays a central role in extracellular vesicle (EVs) KRAS mediated inflammation in vivo: (a) Enrichment analysis based on MS analysis using the STRING database. (b) Enrichment analysis by the STRING database using proteins that were abundant in the EV KRAS complex (EV-KRAS) based on MS analysis and the cytokines upregulated following EV-KRAS intratracheal administration. (c) Pathway analysis showing KRAS mediated inflammation. (d) HE staining of lung tissue from the mice, intratracheal administered with PBS, liposome-EV-KRAS and Fn1 depleted EV-KRAS complex (liposome-EV-KRAS-Fn1-D). (e) ELISA for lung tissue TNFa, IL-17A and IL-6 from mice after intratracheally being administered liposome-EV-KRAS-Fn1-D. (f) HE staining of C57BL/6J wild and IL-17A KO mice lung tissue from the mice intratracheally administered PBS and liposome-EV-KRAS. (g) ELISA for IL-6 and FGF21 from C57BL/6J wild and IL-17A receptor KO mice lung tissue after intratracheal administration of PBS and liposome-EV-KRAS. (h) ELISA for IL-17A, TNFa, and IL-6 after treatment with PBS, liposome-EV-KRAS, liposome-EV-KRAS-Fn1-D, or liposome-EV-KRAS-Fn1-Dexogenously supplemented with Fn1. Mice were administered with 50 µl of 10 × 1010 liposome particles/ml. Data are mean ± SEM from five replicates, *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA.
Fig 5: Effect of KRAS proximal proteins on tumour cell proliferation, lung inflammation, cytokine production and immune cell activity in B6 mice: (a) Effect of mutant KRAS extracellular vesicle (EV) complex on the proliferation of H1299, LLC1 and TC1 cells. (b) Schematic diagram showing intratracheal administration of KRAS complex. (c) Haematoxylin and eosin (HE) staining of lung tissue treated with KRAS complex from EVs and cell lysate (Ly) loaded in liposome (liposome‐EV‐KRAS/ liposome‐Ly‐KRAS). (d) Comparison of cytokine expression in lung tissue harvested from mice treated with PBS, liposome, liposome‐EV‐KRAS and liposome‐Ly‐KRAS, fold change represent pixel density. (e) ELISA for IL‐6, IL‐17A, TNFα, and FGF21 from lung tissue lysate showing T cell response to KRAS complex loaded in liposome following intratracheal administration. (f) Confocal microscopy for IL‐17A and TNFα positive cells in lung tissue (scale: 10 μm). Mice were administered with 50 μl of 10 × 1010 liposome particles/ml. Data are mean ± SEM from five replicates, **p < 0.01 using one‐way ANOVA.
Supplier Page from Sino Biological, Inc. for Human KRAS / K-Ras (Q61H) Protein (His Tag)