Journal: CNS Neuroscience & Therapeutics

Article Title: Interleukin‐3 Modulates Macrophage Phagocytic Activity and Promotes Spinal Cord Injury Repair

doi: 10.1111/cns.70181

Figure Lengend Snippet: IL‐3 is specifically expressed in astrocytes at 14 dpi. (A) Immunofluorescent staining of CD31 (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (B) Immunofluorescent staining of PDGFRβ (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (C) Immunofluorescent staining of CD68 (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (D) Immunofluorescent staining of NG2 (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (E) Immunofluorescent staining of GFAP (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. The region of interest (ROI) represents the boxed region on the left. Asterisks indicate the injured core. Scale bars: 200 μm (left panel) and 20 μm (right panel). n = 3 animals per group. (F) Quantification of the proportion of IL‐3 + CD31 + cells in CD31 + cells, IL‐3 + PDGFRβ + cells in PDGFRβ + cells, IL‐3 + CD68 + cells in CD68 + cells, IL‐3 + NG2 + cells in NG2 + cells, or IL‐3 + GFAP + cells in GFAP + cells at 14 dpi. ND, no determined, ** p < 0.01; **** p < 0.001 by one‐way ANOVA followed by Tukey's post hoc test.

Article Snippet: The primary antibodies used in this study were as follows: rat anti‐IL‐3 (1:10, 503902, Biolegend), rabbit anti‐IL‐3Rα (1:100, 141039, US Biological), goat anti‐CD31 (1:200, AF3625, R&D Systems), rat anti‐F4/80 (1:100, 14‐4801‐82, Invitrogen), goat anti‐PDGFRβ (1:100, AF1042‐SP, R&D Systems), rat anti‐GFAP (1:200, 13‐0300, Invitrogen), rabbit anti‐GFAP (1:100, 16825‐1‐AP, Proteintech), goat‐Iba1 (1:200, NB100‐1028, Novus), rat anti‐CD68 (1:300, MCA1957, AbD Serotec), goat anti‐5‐HT (1:5000, #20080, Immunostar), and rabbit anti‐NeuN (1:500, ab177487, Abcam).

Techniques: Staining

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: IL33 + stromal cells are abundant in human and mouse PDA. A, Human IHC staining of IL33 in matched adjacent normal (“Adj. Normal”) and PDA regions. S, stromal area; T, tumor area. B, UMAP visualization of human scRNA-seq dataset split into adjacent normal and PDA groups. n = number of patients in each dataset. C, Feature plot of IL33 transcription levels in human scRNA-seq. D, UMAP visualization of murine scRNA-seq dataset split into healthy, PanIN, and PDA groups. E, Dot plot representation of Il33 transcription levels across cell types in the murine scRNA-seq dataset. F, Co-IF staining of murine tissues [healthy (wildtype) aka WT, PanIN aka KC ( Ptf1a-Cre; LSL-Kras G12D ), and PDA aka KPC ( Ptf1a-Cre; Trp53 R172H/+ ;LSL-Kras G12D )]. IL33 (green), PDGFRα/β (red), E-Cadherin (white), DAPI (blue). IL33 CTCF was quantified per individual ROI; each ROI encompasses one PDGFRα/β + cell. N = 3 mice were quantified per group. N in the figure represents the number of ROIs measured per group. P values represent one-way ANOVA testing between groups. Line = mean CTCF.

Article Snippet: Primary antibodies were used at the following concentrations: mouse IL33 (1:100, #AF3625, R&D Systems), PDGFRα (1:500, #3164S, Cell Signaling), pSTAT3 (Y705; 1:1,000, #9145S, Cell Signaling), STAT3 (1:1,000, #9139S, Cell Signaling), p-SMAD2 (S465/467)/SMAD3 (S423/425; 1:500, #8828S, Cell Signaling), SMAD2/3 (1:1,000, #8685S, Cell Signaling), α-tubulin (1:2,000, #3873S, Cell Signaling), vinculin (1:2,000, #13901S, Cell Signaling).

Techniques: Immunohistochemistry, Staining

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Stromal IL33 promotes PDA growth. A, Genetic scheme of Pdgfra-CreER T2/+ ;Il33 f/f murine model. Tamoxifen induces activation of the Cre-ERT2 fusion protein, allowing recombination to occur. B, Experimental design for the Pdgfra-CreER T2/+ ;Il33 f/f orthotopic tumor model. OT, orthotopic, CreER = Pdgfra-CreER T2/+ , CreER;Il33 f/f = Pdgfra-CreER T2/+ ;Il33 f/f . C, Western blot of PDGFRα + cells sorted from CreER and CreER;Il33 f/f orthotopic tumors. Two tumors/mice were pooled in each lane. D, Relative and absolute tumor sizes from CreER and CreER;Il33 f/f orthotopic tumors. (E + F) Immunostainings of CreER and CreER;Il33 f/f tumors: E, = Co-IF staining of Ki67 (green), PDGFRα/β (red), E-Cadherin (white), and DAPI (blue), F, = IHC staining of Cleaved Caspase-3 (CC3). In quantification, each dot represents one animal. G, Treatment schedule for the Pdgfra-CreER T2/+ ;Il33 f/f orthotopic tumor model adapted for scRNA-seq. H, UMAP visualization of orthotopic scRNA-seq dataset split into CreER and CreER;Il33 f/f groups. I, Waterfall plot depicting differential pathway enrichment in tumor cells based on the Hallmark collection of annotations. Positive normalized enrichment scores are enriched in the control group. Pathways of interest are bolded. padj = Bonferroni-corrected P value. J, Violin plot depicting expression of Il1rl1 (ST2) in select leukocytes from scRNA-seq. K, Experimental design for Il1rl1 +/+ and Il1rl1 −/− orthotopic tumor experiment. L, Relative and absolute tumor sizes from Il1rl1 +/+ and Il1rl1 −/− orthotopic tumors. Tumor weight/body weight ratios are relative to the control group. Histogram data are mean ± standard deviation. Experiments with two conditions were compared using a two-tailed Student t test.

Article Snippet: Primary antibodies were used at the following concentrations: mouse IL33 (1:100, #AF3625, R&D Systems), PDGFRα (1:500, #3164S, Cell Signaling), pSTAT3 (Y705; 1:1,000, #9145S, Cell Signaling), STAT3 (1:1,000, #9139S, Cell Signaling), p-SMAD2 (S465/467)/SMAD3 (S423/425; 1:500, #8828S, Cell Signaling), SMAD2/3 (1:1,000, #8685S, Cell Signaling), α-tubulin (1:2,000, #3873S, Cell Signaling), vinculin (1:2,000, #13901S, Cell Signaling).

Techniques: Activation Assay, Western Blot, Staining, Immunohistochemistry, Control, Expressing, Standard Deviation, Two Tailed Test

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Loss of stromal IL33 alters the ST2 + immune cell secretome, resulting in a shift in CAF differentiation. A, Gene expression of activation markers split by CreER and CreER;Il33 f/f from scRNA-seq. B, Waterfall plot depicting differential pathway enrichment in fibroblasts based on the Hallmark collection of annotations. Negative normalized enrichment scores are enriched in the experimental group. Pathways of interest are bolded. No genesets were enriched in the control group with Bonferroni-corrected P value (padj) of < 0.05. C, Chord diagram visualizing differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) predicted to interact with fibroblasts. Edge widths are proportional to predicted interaction strength. D, UMAP visualization of fibroblasts from the CreER and CreER;Il33 f/f scRNA-seq datasets. E, Gene expression of markers representing CAF subtypes. F, Il33 expression in each CAF population split by experimental group. G, Histogram depicting the frequency of each CAF population across the CreER and CreER;Il33 f/f scRNA-seq datasets. H, Chord diagram visualizing differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) fibroblast-derived ligands and their predicted interaction partners. Edge widths are proportional to predicted interaction strength.

Article Snippet: Primary antibodies were used at the following concentrations: mouse IL33 (1:100, #AF3625, R&D Systems), PDGFRα (1:500, #3164S, Cell Signaling), pSTAT3 (Y705; 1:1,000, #9145S, Cell Signaling), STAT3 (1:1,000, #9139S, Cell Signaling), p-SMAD2 (S465/467)/SMAD3 (S423/425; 1:500, #8828S, Cell Signaling), SMAD2/3 (1:1,000, #8685S, Cell Signaling), α-tubulin (1:2,000, #3873S, Cell Signaling), vinculin (1:2,000, #13901S, Cell Signaling).

Techniques: Expressing, Activation Assay, Control, Derivative Assay

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Inactivation of stromal IL33 enables cytotoxic T-cell activity. A, IHC staining of F4/80 in CreER and CreER ; Il33 f/f tumors. B, scRNA-seq gene expression of curated proinflammatory and immunosuppressive markers, grouped by cell type and split by experimental group. C, Chord diagram visualizing ligands differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) in CreER and CreER;Il33 f/f tumors that interact with CD8 + T cells. Edge widths are proportional to predicted interaction strength. Chemokines are bolded. D and E, Co-IF staining of CreER and CreER;Il33 f/f tumors: ( D ) = CD8 (green), Granzyme-B (red), E-Cadherin (white) and DAPI (blue), ( E ) = CD4 (yellow), Foxp3 (magenta), and DAPI (cyan). For staining quantification, each dot represents one animal, and values were compared using a two-tailed Student t test. Histogram data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: mouse IL33 (1:100, #AF3625, R&D Systems), PDGFRα (1:500, #3164S, Cell Signaling), pSTAT3 (Y705; 1:1,000, #9145S, Cell Signaling), STAT3 (1:1,000, #9139S, Cell Signaling), p-SMAD2 (S465/467)/SMAD3 (S423/425; 1:500, #8828S, Cell Signaling), SMAD2/3 (1:1,000, #8685S, Cell Signaling), α-tubulin (1:2,000, #3873S, Cell Signaling), vinculin (1:2,000, #13901S, Cell Signaling).

Techniques: Activity Assay, Immunohistochemistry, Expressing, Staining, Two Tailed Test, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Expression of fibroblast IL33 is extrinsically induced by epithelial KRAS G12D and requires JAK1/2-STAT3 activation throughout tumorigenesis. A, Genetic scheme of the iKRAS G12D mouse. Doxycycline induces reversible expression of KRAS G12D in pancreatic epithelial cells. B, Diagram representing the various iKRAS G12D treatment models and collection points across tumorigenesis. Cae, caerulein; OT, orthotopic. C, UMAP visualization of iKRAS G12D scRNA-seq dataset. Projection on the left is colored by cell type (all datasets merged). Projections on the right are split by iKRAS G12D “ON” and “OFF” status and are colored by timepoint. D, Feature plot representation of Il33 expression levels split by iKRAS G12D “ON” and “OFF” status (all timepoints merged). E, Violin plots depicting fibroblast Il33 expression level per timepoint and split by iKRAS G12D “ON” and “OFF” status. Wilcoxon rank sum tests were performed between iKRAS G12D “ON” and “OFF” pairings per each timepoint, and Bonferroni adjusted P values are displayed above violins. F, GSEA enrichment plots of the Hallmark “IL6_JAK_STAT3_SIGNALING” pathway based on fibroblast iKRAS G12D “ON” and “OFF” differential gene expression analysis within each timepoint. G, Treatment scheme for iKRAS G12D “ON” model + JAK1/2 inhibitor. H, Co-IF staining of IL33 (green), PDGFRα/β (red), E-Cadherin (white), DAPI (blue). IL33 CTCF was quantified per individual ROI; each ROI encompasses one PDGFRα/β + cell. N = 3 mice were quantified per group. N in the figure represents the number of ROIs measured per group. P values represent a two-tailed Student t test. Line = Mean CTCF. I, Genetic scheme of Pdgfra-CreER T2/+ ; Stat3 f/f ( CreER;Stat3 f/f ) murine model. Tamoxifen induces activation of the Cre-ERT2 fusion protein, allowing recombination to occur. J, Diagram representing the treatment schedule for the CreER;Stat3 f/f orthotopic tumor model. K, Expression levels of Il33 in CAFs from J as measured by RT-qPCR. Values are normalized to Ppia (Cyclophilin A) and relative to the CreER group. Two-tailed Student t test was performed to compare groups; data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: mouse IL33 (1:100, #AF3625, R&D Systems), PDGFRα (1:500, #3164S, Cell Signaling), pSTAT3 (Y705; 1:1,000, #9145S, Cell Signaling), STAT3 (1:1,000, #9139S, Cell Signaling), p-SMAD2 (S465/467)/SMAD3 (S423/425; 1:500, #8828S, Cell Signaling), SMAD2/3 (1:1,000, #8685S, Cell Signaling), α-tubulin (1:2,000, #3873S, Cell Signaling), vinculin (1:2,000, #13901S, Cell Signaling).

Techniques: Expressing, Activation Assay, Staining, Two Tailed Test, Quantitative RT-PCR, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Tumor cell-initiated autocrine signaling drives IL33 upregulation in pancreatic fibroblasts. A, Ex vivo culture scheme for iKRAS G12D ; Trp53 R172H/+ (cell line 9805) CM generation and healthy pancreatic fibroblasts (cell line CD1WT). B, Western blot of CD1WT whole cell lysates after 24 hours of treatment with DMEM, iKRAS G12D “OFF” CM, iKRAS G12D “ON” CM, or concurrent iKRAS G12D “ON” CM and JAK1/2i. C, RT-qPCR of CD1WT after treatment with DMEM, JAK1/2i (4 hours, 0.3 μmol/L), iKRAS G12D “ON” CM (24 hours), or pretreatment of iKRAS G12D “ON” CM for 20 hours followed by spike-in of JAK1/2i (0.3 μmol/L) for an additional 4 hours (24 hours total iKRAS G12D “ON” CM treatment). Groups were compared with ordinary one-way ANOVA. D, Western blot of CD1WT whole cell lysates after 24 hours of treatment with DMEM, iKRAS G12D “ON” CM, rIL6 (left) or rLIF (right). E, Representative western blot of CD1WT whole cell lysates after treatment with DMEM, iKRAS G12D “OFF” CM, or iKRAS G12D “ON” CM for increasing intervals of time. Densitometry quantification for IL33 normalized to loading control (α-tubulin) and pSTAT3 normalized to total STAT3 are shown. Quantification is relative to the 0-hour timepoint. Ordinary one-way ANOVA was performed to compare each timepoint to the control. F, RT-qPCR of CD1WT after treatment with DMEM, iKRAS G12D “OFF” CM, or iKRAS G12D “ON” CM for increasing intervals of time. Values are log 10 transformed to better visualize large changes in gene expression level. Ordinary one-way ANOVA was performed to compare each timepoint to the 0-hour timepoint. Only comparisons with P value < 0.05 are shown. G, Experimental scheme to block autocrine signaling in CD1WT. CD1WT were treated with DMEM, iKRAS G12D “ON” CM, or rLIF for 18 hours, and then, the resulting CM was set aside. Cells were washed with PBS and then given back their original 18-hour CM or given GolgiStop (1.3 μL/2 mL) + fresh DMEM, iKRAS G12D “ON” CM, or rLIF media. Cells were incubated for an additional 6 hours before harvesting CD1WT RNA and protein. H, RT-qPCR of CD1WT after autocrine blocking experiment. Two-tailed Student t test was performed to compare groups of interest (all tested comparisons shown). I, Western blot of CD1WT whole cell lysates after autocrine blocking experiment. In all experiments with iKRAS G12D CM, doxycycline is used as a vehicle control. In all experiments with JAK1/2i (ruxolitinib), DMSO was used as a vehicle control. All replicates represent complete, independent experiments. RT-qPCR values are normalized to Ppia (Cyclophilin A) and relative to the untreated DMEM group. Histogram data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: mouse IL33 (1:100, #AF3625, R&D Systems), PDGFRα (1:500, #3164S, Cell Signaling), pSTAT3 (Y705; 1:1,000, #9145S, Cell Signaling), STAT3 (1:1,000, #9139S, Cell Signaling), p-SMAD2 (S465/467)/SMAD3 (S423/425; 1:500, #8828S, Cell Signaling), SMAD2/3 (1:1,000, #8685S, Cell Signaling), α-tubulin (1:2,000, #3873S, Cell Signaling), vinculin (1:2,000, #13901S, Cell Signaling).

Techniques: Ex Vivo, Western Blot, Quantitative RT-PCR, Control, Transformation Assay, Expressing, Blocking Assay, Incubation, Two Tailed Test, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Tumor cell KRAS G12D initiates upregulation of fibroblast IL33, promoting immunosuppression in PDA. Working model. During PanIN and PDA, KRAS G12D -dependent tumor cell-derived signaling factors initiate fibroblast autocrine signaling, including the JAK1/2-STAT3 pathway. This fibroblast reprogramming results in the upregulation of IL33. Furthermore, at least one additional autocrine loop is required for IL33 upregulation (possibly also dependent on pSTAT3). CAF IL33 is secreted in response to oxidative stress, in which it signals to ST2 + immune cells ILC2s and Tregs, promoting an immunosuppressive TME and tumor growth. When stromal IL33 is removed, ILC2s and Tregs exhibit an altered secretory gene signature, and a shift in CAF and myeloid cell polarization is seen. This ultimately results in the recruitment and activation of CD8 + T cells, and the suppression of tumor growth.

Article Snippet: Primary antibodies were used at the following concentrations: mouse IL33 (1:100, #AF3625, R&D Systems), PDGFRα (1:500, #3164S, Cell Signaling), pSTAT3 (Y705; 1:1,000, #9145S, Cell Signaling), STAT3 (1:1,000, #9139S, Cell Signaling), p-SMAD2 (S465/467)/SMAD3 (S423/425; 1:500, #8828S, Cell Signaling), SMAD2/3 (1:1,000, #8685S, Cell Signaling), α-tubulin (1:2,000, #3873S, Cell Signaling), vinculin (1:2,000, #13901S, Cell Signaling).

Techniques: Derivative Assay, Activation Assay

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: IL33 + stromal cells are abundant in human and mouse PDA. A, Human IHC staining of IL33 in matched adjacent normal (“Adj. Normal”) and PDA regions. S, stromal area; T, tumor area. B, UMAP visualization of human scRNA-seq dataset split into adjacent normal and PDA groups. n = number of patients in each dataset. C, Feature plot of IL33 transcription levels in human scRNA-seq. D, UMAP visualization of murine scRNA-seq dataset split into healthy, PanIN, and PDA groups. E, Dot plot representation of Il33 transcription levels across cell types in the murine scRNA-seq dataset. F, Co-IF staining of murine tissues [healthy (wildtype) aka WT, PanIN aka KC ( Ptf1a-Cre; LSL-Kras G12D ), and PDA aka KPC ( Ptf1a-Cre; Trp53 R172H/+ ;LSL-Kras G12D )]. IL33 (green), PDGFRα/β (red), E-Cadherin (white), DAPI (blue). IL33 CTCF was quantified per individual ROI; each ROI encompasses one PDGFRα/β + cell. N = 3 mice were quantified per group. N in the figure represents the number of ROIs measured per group. P values represent one-way ANOVA testing between groups. Line = mean CTCF.

Article Snippet: Primary antibodies were used at the following concentrations: human IL33 (1:50, #AF3625, R&D Systems), Cleaved Caspase 3 (1:100, #9664L, Cell Signaling), F4/80 (1:200, #70076, Cell Signaling), and myeloperoxidase (1:200, #AF3667, R&D Systems).

Techniques: Immunohistochemistry, Staining

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Stromal IL33 promotes PDA growth. A, Genetic scheme of Pdgfra-CreER T2/+ ;Il33 f/f murine model. Tamoxifen induces activation of the Cre-ERT2 fusion protein, allowing recombination to occur. B, Experimental design for the Pdgfra-CreER T2/+ ;Il33 f/f orthotopic tumor model. OT, orthotopic, CreER = Pdgfra-CreER T2/+ , CreER;Il33 f/f = Pdgfra-CreER T2/+ ;Il33 f/f . C, Western blot of PDGFRα + cells sorted from CreER and CreER;Il33 f/f orthotopic tumors. Two tumors/mice were pooled in each lane. D, Relative and absolute tumor sizes from CreER and CreER;Il33 f/f orthotopic tumors. (E + F) Immunostainings of CreER and CreER;Il33 f/f tumors: E, = Co-IF staining of Ki67 (green), PDGFRα/β (red), E-Cadherin (white), and DAPI (blue), F, = IHC staining of Cleaved Caspase-3 (CC3). In quantification, each dot represents one animal. G, Treatment schedule for the Pdgfra-CreER T2/+ ;Il33 f/f orthotopic tumor model adapted for scRNA-seq. H, UMAP visualization of orthotopic scRNA-seq dataset split into CreER and CreER;Il33 f/f groups. I, Waterfall plot depicting differential pathway enrichment in tumor cells based on the Hallmark collection of annotations. Positive normalized enrichment scores are enriched in the control group. Pathways of interest are bolded. padj = Bonferroni-corrected P value. J, Violin plot depicting expression of Il1rl1 (ST2) in select leukocytes from scRNA-seq. K, Experimental design for Il1rl1 +/+ and Il1rl1 −/− orthotopic tumor experiment. L, Relative and absolute tumor sizes from Il1rl1 +/+ and Il1rl1 −/− orthotopic tumors. Tumor weight/body weight ratios are relative to the control group. Histogram data are mean ± standard deviation. Experiments with two conditions were compared using a two-tailed Student t test.

Article Snippet: Primary antibodies were used at the following concentrations: human IL33 (1:50, #AF3625, R&D Systems), Cleaved Caspase 3 (1:100, #9664L, Cell Signaling), F4/80 (1:200, #70076, Cell Signaling), and myeloperoxidase (1:200, #AF3667, R&D Systems).

Techniques: Activation Assay, Western Blot, Staining, Immunohistochemistry, Control, Expressing, Standard Deviation, Two Tailed Test

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Loss of stromal IL33 alters the ST2 + immune cell secretome, resulting in a shift in CAF differentiation. A, Gene expression of activation markers split by CreER and CreER;Il33 f/f from scRNA-seq. B, Waterfall plot depicting differential pathway enrichment in fibroblasts based on the Hallmark collection of annotations. Negative normalized enrichment scores are enriched in the experimental group. Pathways of interest are bolded. No genesets were enriched in the control group with Bonferroni-corrected P value (padj) of < 0.05. C, Chord diagram visualizing differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) predicted to interact with fibroblasts. Edge widths are proportional to predicted interaction strength. D, UMAP visualization of fibroblasts from the CreER and CreER;Il33 f/f scRNA-seq datasets. E, Gene expression of markers representing CAF subtypes. F, Il33 expression in each CAF population split by experimental group. G, Histogram depicting the frequency of each CAF population across the CreER and CreER;Il33 f/f scRNA-seq datasets. H, Chord diagram visualizing differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) fibroblast-derived ligands and their predicted interaction partners. Edge widths are proportional to predicted interaction strength.

Article Snippet: Primary antibodies were used at the following concentrations: human IL33 (1:50, #AF3625, R&D Systems), Cleaved Caspase 3 (1:100, #9664L, Cell Signaling), F4/80 (1:200, #70076, Cell Signaling), and myeloperoxidase (1:200, #AF3667, R&D Systems).

Techniques: Expressing, Activation Assay, Control, Derivative Assay

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Inactivation of stromal IL33 enables cytotoxic T-cell activity. A, IHC staining of F4/80 in CreER and CreER ; Il33 f/f tumors. B, scRNA-seq gene expression of curated proinflammatory and immunosuppressive markers, grouped by cell type and split by experimental group. C, Chord diagram visualizing ligands differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) in CreER and CreER;Il33 f/f tumors that interact with CD8 + T cells. Edge widths are proportional to predicted interaction strength. Chemokines are bolded. D and E, Co-IF staining of CreER and CreER;Il33 f/f tumors: ( D ) = CD8 (green), Granzyme-B (red), E-Cadherin (white) and DAPI (blue), ( E ) = CD4 (yellow), Foxp3 (magenta), and DAPI (cyan). For staining quantification, each dot represents one animal, and values were compared using a two-tailed Student t test. Histogram data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: human IL33 (1:50, #AF3625, R&D Systems), Cleaved Caspase 3 (1:100, #9664L, Cell Signaling), F4/80 (1:200, #70076, Cell Signaling), and myeloperoxidase (1:200, #AF3667, R&D Systems).

Techniques: Activity Assay, Immunohistochemistry, Expressing, Staining, Two Tailed Test, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Expression of fibroblast IL33 is extrinsically induced by epithelial KRAS G12D and requires JAK1/2-STAT3 activation throughout tumorigenesis. A, Genetic scheme of the iKRAS G12D mouse. Doxycycline induces reversible expression of KRAS G12D in pancreatic epithelial cells. B, Diagram representing the various iKRAS G12D treatment models and collection points across tumorigenesis. Cae, caerulein; OT, orthotopic. C, UMAP visualization of iKRAS G12D scRNA-seq dataset. Projection on the left is colored by cell type (all datasets merged). Projections on the right are split by iKRAS G12D “ON” and “OFF” status and are colored by timepoint. D, Feature plot representation of Il33 expression levels split by iKRAS G12D “ON” and “OFF” status (all timepoints merged). E, Violin plots depicting fibroblast Il33 expression level per timepoint and split by iKRAS G12D “ON” and “OFF” status. Wilcoxon rank sum tests were performed between iKRAS G12D “ON” and “OFF” pairings per each timepoint, and Bonferroni adjusted P values are displayed above violins. F, GSEA enrichment plots of the Hallmark “IL6_JAK_STAT3_SIGNALING” pathway based on fibroblast iKRAS G12D “ON” and “OFF” differential gene expression analysis within each timepoint. G, Treatment scheme for iKRAS G12D “ON” model + JAK1/2 inhibitor. H, Co-IF staining of IL33 (green), PDGFRα/β (red), E-Cadherin (white), DAPI (blue). IL33 CTCF was quantified per individual ROI; each ROI encompasses one PDGFRα/β + cell. N = 3 mice were quantified per group. N in the figure represents the number of ROIs measured per group. P values represent a two-tailed Student t test. Line = Mean CTCF. I, Genetic scheme of Pdgfra-CreER T2/+ ; Stat3 f/f ( CreER;Stat3 f/f ) murine model. Tamoxifen induces activation of the Cre-ERT2 fusion protein, allowing recombination to occur. J, Diagram representing the treatment schedule for the CreER;Stat3 f/f orthotopic tumor model. K, Expression levels of Il33 in CAFs from J as measured by RT-qPCR. Values are normalized to Ppia (Cyclophilin A) and relative to the CreER group. Two-tailed Student t test was performed to compare groups; data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: human IL33 (1:50, #AF3625, R&D Systems), Cleaved Caspase 3 (1:100, #9664L, Cell Signaling), F4/80 (1:200, #70076, Cell Signaling), and myeloperoxidase (1:200, #AF3667, R&D Systems).

Techniques: Expressing, Activation Assay, Staining, Two Tailed Test, Quantitative RT-PCR, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Tumor cell-initiated autocrine signaling drives IL33 upregulation in pancreatic fibroblasts. A, Ex vivo culture scheme for iKRAS G12D ; Trp53 R172H/+ (cell line 9805) CM generation and healthy pancreatic fibroblasts (cell line CD1WT). B, Western blot of CD1WT whole cell lysates after 24 hours of treatment with DMEM, iKRAS G12D “OFF” CM, iKRAS G12D “ON” CM, or concurrent iKRAS G12D “ON” CM and JAK1/2i. C, RT-qPCR of CD1WT after treatment with DMEM, JAK1/2i (4 hours, 0.3 μmol/L), iKRAS G12D “ON” CM (24 hours), or pretreatment of iKRAS G12D “ON” CM for 20 hours followed by spike-in of JAK1/2i (0.3 μmol/L) for an additional 4 hours (24 hours total iKRAS G12D “ON” CM treatment). Groups were compared with ordinary one-way ANOVA. D, Western blot of CD1WT whole cell lysates after 24 hours of treatment with DMEM, iKRAS G12D “ON” CM, rIL6 (left) or rLIF (right). E, Representative western blot of CD1WT whole cell lysates after treatment with DMEM, iKRAS G12D “OFF” CM, or iKRAS G12D “ON” CM for increasing intervals of time. Densitometry quantification for IL33 normalized to loading control (α-tubulin) and pSTAT3 normalized to total STAT3 are shown. Quantification is relative to the 0-hour timepoint. Ordinary one-way ANOVA was performed to compare each timepoint to the control. F, RT-qPCR of CD1WT after treatment with DMEM, iKRAS G12D “OFF” CM, or iKRAS G12D “ON” CM for increasing intervals of time. Values are log 10 transformed to better visualize large changes in gene expression level. Ordinary one-way ANOVA was performed to compare each timepoint to the 0-hour timepoint. Only comparisons with P value < 0.05 are shown. G, Experimental scheme to block autocrine signaling in CD1WT. CD1WT were treated with DMEM, iKRAS G12D “ON” CM, or rLIF for 18 hours, and then, the resulting CM was set aside. Cells were washed with PBS and then given back their original 18-hour CM or given GolgiStop (1.3 μL/2 mL) + fresh DMEM, iKRAS G12D “ON” CM, or rLIF media. Cells were incubated for an additional 6 hours before harvesting CD1WT RNA and protein. H, RT-qPCR of CD1WT after autocrine blocking experiment. Two-tailed Student t test was performed to compare groups of interest (all tested comparisons shown). I, Western blot of CD1WT whole cell lysates after autocrine blocking experiment. In all experiments with iKRAS G12D CM, doxycycline is used as a vehicle control. In all experiments with JAK1/2i (ruxolitinib), DMSO was used as a vehicle control. All replicates represent complete, independent experiments. RT-qPCR values are normalized to Ppia (Cyclophilin A) and relative to the untreated DMEM group. Histogram data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: human IL33 (1:50, #AF3625, R&D Systems), Cleaved Caspase 3 (1:100, #9664L, Cell Signaling), F4/80 (1:200, #70076, Cell Signaling), and myeloperoxidase (1:200, #AF3667, R&D Systems).

Techniques: Ex Vivo, Western Blot, Quantitative RT-PCR, Control, Transformation Assay, Expressing, Blocking Assay, Incubation, Two Tailed Test, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Tumor cell KRAS G12D initiates upregulation of fibroblast IL33, promoting immunosuppression in PDA. Working model. During PanIN and PDA, KRAS G12D -dependent tumor cell-derived signaling factors initiate fibroblast autocrine signaling, including the JAK1/2-STAT3 pathway. This fibroblast reprogramming results in the upregulation of IL33. Furthermore, at least one additional autocrine loop is required for IL33 upregulation (possibly also dependent on pSTAT3). CAF IL33 is secreted in response to oxidative stress, in which it signals to ST2 + immune cells ILC2s and Tregs, promoting an immunosuppressive TME and tumor growth. When stromal IL33 is removed, ILC2s and Tregs exhibit an altered secretory gene signature, and a shift in CAF and myeloid cell polarization is seen. This ultimately results in the recruitment and activation of CD8 + T cells, and the suppression of tumor growth.

Article Snippet: Primary antibodies were used at the following concentrations: human IL33 (1:50, #AF3625, R&D Systems), Cleaved Caspase 3 (1:100, #9664L, Cell Signaling), F4/80 (1:200, #70076, Cell Signaling), and myeloperoxidase (1:200, #AF3667, R&D Systems).

Techniques: Derivative Assay, Activation Assay

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: IL33 + stromal cells are abundant in human and mouse PDA. A, Human IHC staining of IL33 in matched adjacent normal (“Adj. Normal”) and PDA regions. S, stromal area; T, tumor area. B, UMAP visualization of human scRNA-seq dataset split into adjacent normal and PDA groups. n = number of patients in each dataset. C, Feature plot of IL33 transcription levels in human scRNA-seq. D, UMAP visualization of murine scRNA-seq dataset split into healthy, PanIN, and PDA groups. E, Dot plot representation of Il33 transcription levels across cell types in the murine scRNA-seq dataset. F, Co-IF staining of murine tissues [healthy (wildtype) aka WT, PanIN aka KC ( Ptf1a-Cre; LSL-Kras G12D ), and PDA aka KPC ( Ptf1a-Cre; Trp53 R172H/+ ;LSL-Kras G12D )]. IL33 (green), PDGFRα/β (red), E-Cadherin (white), DAPI (blue). IL33 CTCF was quantified per individual ROI; each ROI encompasses one PDGFRα/β + cell. N = 3 mice were quantified per group. N in the figure represents the number of ROIs measured per group. P values represent one-way ANOVA testing between groups. Line = mean CTCF.

Article Snippet: Primary antibodies were used at the following concentrations: IL33 (1:50, #AF3625, R&D Systems), PDGFRα/β (1:100, #AB32570, Abcam), E-Cadherin (1:50, #14472S, Cell Signaling), pSTAT3 (Y705; 1:100, #9145S, Cell Signaling), Ki67 (1:100, #AB15580, Abcam), α-SMA (1:1,000, #A2547, Sigma-Aldrich), CD8 (1:400, #98941, Cell Signaling), Granzyme-B (1:500, #17215, Cell Signaling), CD4 (1:100, #25229, Cell Signaling), CD44 (1:100, #MA1-10225, Invitrogen), and Foxp3 (1:100, #12653, Cell Signaling).

Techniques: Immunohistochemistry, Staining

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Stromal IL33 promotes PDA growth. A, Genetic scheme of Pdgfra-CreER T2/+ ;Il33 f/f murine model. Tamoxifen induces activation of the Cre-ERT2 fusion protein, allowing recombination to occur. B, Experimental design for the Pdgfra-CreER T2/+ ;Il33 f/f orthotopic tumor model. OT, orthotopic, CreER = Pdgfra-CreER T2/+ , CreER;Il33 f/f = Pdgfra-CreER T2/+ ;Il33 f/f . C, Western blot of PDGFRα + cells sorted from CreER and CreER;Il33 f/f orthotopic tumors. Two tumors/mice were pooled in each lane. D, Relative and absolute tumor sizes from CreER and CreER;Il33 f/f orthotopic tumors. (E + F) Immunostainings of CreER and CreER;Il33 f/f tumors: E, = Co-IF staining of Ki67 (green), PDGFRα/β (red), E-Cadherin (white), and DAPI (blue), F, = IHC staining of Cleaved Caspase-3 (CC3). In quantification, each dot represents one animal. G, Treatment schedule for the Pdgfra-CreER T2/+ ;Il33 f/f orthotopic tumor model adapted for scRNA-seq. H, UMAP visualization of orthotopic scRNA-seq dataset split into CreER and CreER;Il33 f/f groups. I, Waterfall plot depicting differential pathway enrichment in tumor cells based on the Hallmark collection of annotations. Positive normalized enrichment scores are enriched in the control group. Pathways of interest are bolded. padj = Bonferroni-corrected P value. J, Violin plot depicting expression of Il1rl1 (ST2) in select leukocytes from scRNA-seq. K, Experimental design for Il1rl1 +/+ and Il1rl1 −/− orthotopic tumor experiment. L, Relative and absolute tumor sizes from Il1rl1 +/+ and Il1rl1 −/− orthotopic tumors. Tumor weight/body weight ratios are relative to the control group. Histogram data are mean ± standard deviation. Experiments with two conditions were compared using a two-tailed Student t test.

Article Snippet: Primary antibodies were used at the following concentrations: IL33 (1:50, #AF3625, R&D Systems), PDGFRα/β (1:100, #AB32570, Abcam), E-Cadherin (1:50, #14472S, Cell Signaling), pSTAT3 (Y705; 1:100, #9145S, Cell Signaling), Ki67 (1:100, #AB15580, Abcam), α-SMA (1:1,000, #A2547, Sigma-Aldrich), CD8 (1:400, #98941, Cell Signaling), Granzyme-B (1:500, #17215, Cell Signaling), CD4 (1:100, #25229, Cell Signaling), CD44 (1:100, #MA1-10225, Invitrogen), and Foxp3 (1:100, #12653, Cell Signaling).

Techniques: Activation Assay, Western Blot, Staining, Immunohistochemistry, Control, Expressing, Standard Deviation, Two Tailed Test

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Loss of stromal IL33 alters the ST2 + immune cell secretome, resulting in a shift in CAF differentiation. A, Gene expression of activation markers split by CreER and CreER;Il33 f/f from scRNA-seq. B, Waterfall plot depicting differential pathway enrichment in fibroblasts based on the Hallmark collection of annotations. Negative normalized enrichment scores are enriched in the experimental group. Pathways of interest are bolded. No genesets were enriched in the control group with Bonferroni-corrected P value (padj) of < 0.05. C, Chord diagram visualizing differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) predicted to interact with fibroblasts. Edge widths are proportional to predicted interaction strength. D, UMAP visualization of fibroblasts from the CreER and CreER;Il33 f/f scRNA-seq datasets. E, Gene expression of markers representing CAF subtypes. F, Il33 expression in each CAF population split by experimental group. G, Histogram depicting the frequency of each CAF population across the CreER and CreER;Il33 f/f scRNA-seq datasets. H, Chord diagram visualizing differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) fibroblast-derived ligands and their predicted interaction partners. Edge widths are proportional to predicted interaction strength.

Article Snippet: Primary antibodies were used at the following concentrations: IL33 (1:50, #AF3625, R&D Systems), PDGFRα/β (1:100, #AB32570, Abcam), E-Cadherin (1:50, #14472S, Cell Signaling), pSTAT3 (Y705; 1:100, #9145S, Cell Signaling), Ki67 (1:100, #AB15580, Abcam), α-SMA (1:1,000, #A2547, Sigma-Aldrich), CD8 (1:400, #98941, Cell Signaling), Granzyme-B (1:500, #17215, Cell Signaling), CD4 (1:100, #25229, Cell Signaling), CD44 (1:100, #MA1-10225, Invitrogen), and Foxp3 (1:100, #12653, Cell Signaling).

Techniques: Expressing, Activation Assay, Control, Derivative Assay

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Inactivation of stromal IL33 enables cytotoxic T-cell activity. A, IHC staining of F4/80 in CreER and CreER ; Il33 f/f tumors. B, scRNA-seq gene expression of curated proinflammatory and immunosuppressive markers, grouped by cell type and split by experimental group. C, Chord diagram visualizing ligands differentially enriched (Bonferroni-corrected P value < 0.05 and fold-change ≥0.25) in CreER and CreER;Il33 f/f tumors that interact with CD8 + T cells. Edge widths are proportional to predicted interaction strength. Chemokines are bolded. D and E, Co-IF staining of CreER and CreER;Il33 f/f tumors: ( D ) = CD8 (green), Granzyme-B (red), E-Cadherin (white) and DAPI (blue), ( E ) = CD4 (yellow), Foxp3 (magenta), and DAPI (cyan). For staining quantification, each dot represents one animal, and values were compared using a two-tailed Student t test. Histogram data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: IL33 (1:50, #AF3625, R&D Systems), PDGFRα/β (1:100, #AB32570, Abcam), E-Cadherin (1:50, #14472S, Cell Signaling), pSTAT3 (Y705; 1:100, #9145S, Cell Signaling), Ki67 (1:100, #AB15580, Abcam), α-SMA (1:1,000, #A2547, Sigma-Aldrich), CD8 (1:400, #98941, Cell Signaling), Granzyme-B (1:500, #17215, Cell Signaling), CD4 (1:100, #25229, Cell Signaling), CD44 (1:100, #MA1-10225, Invitrogen), and Foxp3 (1:100, #12653, Cell Signaling).

Techniques: Activity Assay, Immunohistochemistry, Expressing, Staining, Two Tailed Test, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Expression of fibroblast IL33 is extrinsically induced by epithelial KRAS G12D and requires JAK1/2-STAT3 activation throughout tumorigenesis. A, Genetic scheme of the iKRAS G12D mouse. Doxycycline induces reversible expression of KRAS G12D in pancreatic epithelial cells. B, Diagram representing the various iKRAS G12D treatment models and collection points across tumorigenesis. Cae, caerulein; OT, orthotopic. C, UMAP visualization of iKRAS G12D scRNA-seq dataset. Projection on the left is colored by cell type (all datasets merged). Projections on the right are split by iKRAS G12D “ON” and “OFF” status and are colored by timepoint. D, Feature plot representation of Il33 expression levels split by iKRAS G12D “ON” and “OFF” status (all timepoints merged). E, Violin plots depicting fibroblast Il33 expression level per timepoint and split by iKRAS G12D “ON” and “OFF” status. Wilcoxon rank sum tests were performed between iKRAS G12D “ON” and “OFF” pairings per each timepoint, and Bonferroni adjusted P values are displayed above violins. F, GSEA enrichment plots of the Hallmark “IL6_JAK_STAT3_SIGNALING” pathway based on fibroblast iKRAS G12D “ON” and “OFF” differential gene expression analysis within each timepoint. G, Treatment scheme for iKRAS G12D “ON” model + JAK1/2 inhibitor. H, Co-IF staining of IL33 (green), PDGFRα/β (red), E-Cadherin (white), DAPI (blue). IL33 CTCF was quantified per individual ROI; each ROI encompasses one PDGFRα/β + cell. N = 3 mice were quantified per group. N in the figure represents the number of ROIs measured per group. P values represent a two-tailed Student t test. Line = Mean CTCF. I, Genetic scheme of Pdgfra-CreER T2/+ ; Stat3 f/f ( CreER;Stat3 f/f ) murine model. Tamoxifen induces activation of the Cre-ERT2 fusion protein, allowing recombination to occur. J, Diagram representing the treatment schedule for the CreER;Stat3 f/f orthotopic tumor model. K, Expression levels of Il33 in CAFs from J as measured by RT-qPCR. Values are normalized to Ppia (Cyclophilin A) and relative to the CreER group. Two-tailed Student t test was performed to compare groups; data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: IL33 (1:50, #AF3625, R&D Systems), PDGFRα/β (1:100, #AB32570, Abcam), E-Cadherin (1:50, #14472S, Cell Signaling), pSTAT3 (Y705; 1:100, #9145S, Cell Signaling), Ki67 (1:100, #AB15580, Abcam), α-SMA (1:1,000, #A2547, Sigma-Aldrich), CD8 (1:400, #98941, Cell Signaling), Granzyme-B (1:500, #17215, Cell Signaling), CD4 (1:100, #25229, Cell Signaling), CD44 (1:100, #MA1-10225, Invitrogen), and Foxp3 (1:100, #12653, Cell Signaling).

Techniques: Expressing, Activation Assay, Staining, Two Tailed Test, Quantitative RT-PCR, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Tumor cell-initiated autocrine signaling drives IL33 upregulation in pancreatic fibroblasts. A, Ex vivo culture scheme for iKRAS G12D ; Trp53 R172H/+ (cell line 9805) CM generation and healthy pancreatic fibroblasts (cell line CD1WT). B, Western blot of CD1WT whole cell lysates after 24 hours of treatment with DMEM, iKRAS G12D “OFF” CM, iKRAS G12D “ON” CM, or concurrent iKRAS G12D “ON” CM and JAK1/2i. C, RT-qPCR of CD1WT after treatment with DMEM, JAK1/2i (4 hours, 0.3 μmol/L), iKRAS G12D “ON” CM (24 hours), or pretreatment of iKRAS G12D “ON” CM for 20 hours followed by spike-in of JAK1/2i (0.3 μmol/L) for an additional 4 hours (24 hours total iKRAS G12D “ON” CM treatment). Groups were compared with ordinary one-way ANOVA. D, Western blot of CD1WT whole cell lysates after 24 hours of treatment with DMEM, iKRAS G12D “ON” CM, rIL6 (left) or rLIF (right). E, Representative western blot of CD1WT whole cell lysates after treatment with DMEM, iKRAS G12D “OFF” CM, or iKRAS G12D “ON” CM for increasing intervals of time. Densitometry quantification for IL33 normalized to loading control (α-tubulin) and pSTAT3 normalized to total STAT3 are shown. Quantification is relative to the 0-hour timepoint. Ordinary one-way ANOVA was performed to compare each timepoint to the control. F, RT-qPCR of CD1WT after treatment with DMEM, iKRAS G12D “OFF” CM, or iKRAS G12D “ON” CM for increasing intervals of time. Values are log 10 transformed to better visualize large changes in gene expression level. Ordinary one-way ANOVA was performed to compare each timepoint to the 0-hour timepoint. Only comparisons with P value < 0.05 are shown. G, Experimental scheme to block autocrine signaling in CD1WT. CD1WT were treated with DMEM, iKRAS G12D “ON” CM, or rLIF for 18 hours, and then, the resulting CM was set aside. Cells were washed with PBS and then given back their original 18-hour CM or given GolgiStop (1.3 μL/2 mL) + fresh DMEM, iKRAS G12D “ON” CM, or rLIF media. Cells were incubated for an additional 6 hours before harvesting CD1WT RNA and protein. H, RT-qPCR of CD1WT after autocrine blocking experiment. Two-tailed Student t test was performed to compare groups of interest (all tested comparisons shown). I, Western blot of CD1WT whole cell lysates after autocrine blocking experiment. In all experiments with iKRAS G12D CM, doxycycline is used as a vehicle control. In all experiments with JAK1/2i (ruxolitinib), DMSO was used as a vehicle control. All replicates represent complete, independent experiments. RT-qPCR values are normalized to Ppia (Cyclophilin A) and relative to the untreated DMEM group. Histogram data are mean ± standard deviation.

Article Snippet: Primary antibodies were used at the following concentrations: IL33 (1:50, #AF3625, R&D Systems), PDGFRα/β (1:100, #AB32570, Abcam), E-Cadherin (1:50, #14472S, Cell Signaling), pSTAT3 (Y705; 1:100, #9145S, Cell Signaling), Ki67 (1:100, #AB15580, Abcam), α-SMA (1:1,000, #A2547, Sigma-Aldrich), CD8 (1:400, #98941, Cell Signaling), Granzyme-B (1:500, #17215, Cell Signaling), CD4 (1:100, #25229, Cell Signaling), CD44 (1:100, #MA1-10225, Invitrogen), and Foxp3 (1:100, #12653, Cell Signaling).

Techniques: Ex Vivo, Western Blot, Quantitative RT-PCR, Control, Transformation Assay, Expressing, Blocking Assay, Incubation, Two Tailed Test, Standard Deviation

Journal: Cancer Discovery

Article Title: Oncogenic KRAS-Dependent Stromal Interleukin-33 Directs the Pancreatic Microenvironment to Promote Tumor Growth

doi: 10.1158/2159-8290.CD-24-0100

Figure Lengend Snippet: Tumor cell KRAS G12D initiates upregulation of fibroblast IL33, promoting immunosuppression in PDA. Working model. During PanIN and PDA, KRAS G12D -dependent tumor cell-derived signaling factors initiate fibroblast autocrine signaling, including the JAK1/2-STAT3 pathway. This fibroblast reprogramming results in the upregulation of IL33. Furthermore, at least one additional autocrine loop is required for IL33 upregulation (possibly also dependent on pSTAT3). CAF IL33 is secreted in response to oxidative stress, in which it signals to ST2 + immune cells ILC2s and Tregs, promoting an immunosuppressive TME and tumor growth. When stromal IL33 is removed, ILC2s and Tregs exhibit an altered secretory gene signature, and a shift in CAF and myeloid cell polarization is seen. This ultimately results in the recruitment and activation of CD8 + T cells, and the suppression of tumor growth.

Article Snippet: Primary antibodies were used at the following concentrations: IL33 (1:50, #AF3625, R&D Systems), PDGFRα/β (1:100, #AB32570, Abcam), E-Cadherin (1:50, #14472S, Cell Signaling), pSTAT3 (Y705; 1:100, #9145S, Cell Signaling), Ki67 (1:100, #AB15580, Abcam), α-SMA (1:1,000, #A2547, Sigma-Aldrich), CD8 (1:400, #98941, Cell Signaling), Granzyme-B (1:500, #17215, Cell Signaling), CD4 (1:100, #25229, Cell Signaling), CD44 (1:100, #MA1-10225, Invitrogen), and Foxp3 (1:100, #12653, Cell Signaling).

Techniques: Derivative Assay, Activation Assay