Journal: Cancer Discovery

Article Title: Stromal KITL/SCF Maintains Pancreas Tissue Homeostasis and Restrains Tumor Progression

doi: 10.1158/2159-8290.CD-24-1079

Figure Lengend Snippet: Mesenchymal KITL loss within PSCs accompanies pancreatic tumorigenesis. A, Uniform manifold approximation and projection (UMAP) visualization of expression of the indicated genes in normal PSCs and PSC-derived CAFs from scRNA-seq data ( n = 2 replicates pooled from n = 5 mice per arm). B, UMAP visualization of Il6 , Il33 , Col1a1 , and Col1a2 gene expression from normal PSCs and PSC-derived CAFs scRNA-seq dataset ( n = 2 replicates pooled from n = 5 mice per arm). C, UMAP visualization of Kitl transcript expression in normal PSCs and PSC-derived CAFs scRNA-seq dataset ( n = 2 replicates pooled from n = 5 mice per arm). D, Left, UMAP illustrating the cellular landscape of normal pancreatic (blue) and PDAC (red) mesenchymal cells, comprising 5,337 normal and 2,861 tumor cells. Harmony was used to integrate the datasets and correct for batch effects. Right, Monocle 3 trajectory analysis was used to depict expression of the Kitl gene along the inferred pseudotime trajectory. Cells are colored based on Kitl expression levels, with values ranging from low (black) to high (yellow), revealing the spatial and temporal expression patterns of Kitl ( n = 2 replicates pooled from n = 5 mice per arm). E, Left, qRT-PCR analysis of Kitl in quiescent (day 0) and culture-activated (day 7, on plastic) primary PSCs. Right, Quantikine ELISA KITL measurement of supernatant collected from primary PSCs in preactivated (day 2) and activated (day 10) states after 48 hours of incubation with media change on day 8 to harvest for day 10 sample. Immortalized ImPSC-1 included as the reference point. Data represents biological triplicate. F, qRT-PCR analysis of Kitl and Pdgfr α in indicated cell populations. Data show representative technical triplicates. G, Representative brightfield images of primary human PSCs and other mesenchyme. Scale bar, 50 μm. H, qRT-PCR analysis of indicated genes in primary human PSCs and other mesenchyme, shown for three independent patient samples. I, qRT-PCR analysis of Kitl in quiescent (day 0) and conditioned media–activated (day 14) primary human PSCs. Data represent biological triplicate. J, Representative images (left) and quantification (right) of RNA FISH staining for Fabp4 (green) and Kitl (red) in murine normal pancreas ( n = 3). Scale bar, 10 μm. Below, Representative RNAscope staining of GFP (green) protein and Kitl (red) mRNA in PDAC from the genetically engineered mouse model (GEMM) depicted in ( n = 3). Scale bar, 10 μm. K, Representative images and quantification of RNA FISH staining for VIM (green) and KITL (red) in human PDAC tissues between benign adjacent and PDAC regions ( n = 3). Scale bar, 20 μm. L, Representative images and quantification of RNAscope staining for Kitl (red) mRNA expression, GFP (green), and CD31 (magenta) in murine normal pancreas from Rosa26 mTmG/+ ;Fabp4-Cre mice ( n = 3). Scale bar, 20 μm. M, Representative images and quantification of RNAscope staining for GFP (green) protein and Kitl (red) mRNA in GEMM low-grade PanIN ( n = 3). Scale bar, 20 μm. N, Representative image of IHC staining for phospho-c-Kit (Y721, red) in normal murine pancreas ( n = 3). Scale bar, 10 μm. O, Representative images of IHC staining for vimentin (VIM, green) and Phospho-c-Kit (Y703, red) in benign adjacent and human PDAC tissues ( n = 3) Scale bar, 10 μm. For image quantification, at least four fields per tissue were analyzed and averaged. An unpaired t test was performed for comparisons between two groups. For comparisons more than two groups, significance was determined by the ordinary one-way ANOVA. Data are represented as mean ± SEM;*, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; Benign Adj., benign adjacent; CM, conditioned media; ns, not significant; other mes., other mesenchyme.

Article Snippet: Concentrated supernatants were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), and mouse KITL protein quantification was performed using the Mouse SCF Quantikine ELISA Kit (R&D Systems, MCK00) according to the manufacturer’s protocol.

Techniques: Expressing, Derivative Assay, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Staining, RNAscope, Immunohistochemistry