Journal: Journal for immunotherapy of cancer

Article Title: Depletion of tumor-derived CXCL5 improves T cell infiltration and anti-PD-1 therapy response in an obese model of pancreatic cancer.

doi: 10.1136/jitc-2024-010057

Figure Lengend Snippet: Figure 1 (A) Schematic (Created in BioRender Walsh, R (2024) https://BioRender.com/o20p233) illustrating human adipose tissue conditioned media (hAT-CM) collection, storage, and application. Human adipose tissue is incubated in serum-free media at a 1:8 w/v ratio for 1 hour as a wash, then incubated for 24 hours in fresh serum-free media to collect adipose-associated factors. Media is collected and spun to remove cells and debris, then aliquoted, frozen, and diluted for application in functional assays. (B) Proliferation measured by flow cytometry for EdU incorporation into cells in vitro in response to stimulus with pooled hAT-CM diluted 1:3 in Serum Free DMEM (red triangles), Serum Free DMEM (black open squares), or 10% fetal bovine serum (FBS) supplemented DMEM (black open circles). Immortalized pancreatic ductal HPNE-G12D cells and Pancreatic Ductal Adenocarcinoma (PDAC) tumor cell lines (Panc1 and MiaPaCa2) were treated with diluted hAT-CM, serum- free media, or complete media for 24 hours. Then, EdU was spiked and mixed into the media to reach a final concentration of 10 µM for 6 hours to measure incorporation and active proliferation by flow cytometry to show that hAT-CM can induce proliferation of cancer cells in vitro: PI was used as a counter stain following fixation and permeabilization to quantify the total number of cells, and double positive EdU+PI+ events were quantified as a percentage of total PI+events. (C) Kaplan-Meier plot for CXCL5 high or low patients was generated using GEPIA showing significant differences in overall survival between the upper and lower half expressing patients in the PAAD-TCGA dataset±CI, p=0.024, n=89 patients for each dataset compared. (D) mRNA (left) and relative protein abundance (right) of normal (blue) and tumor (red) tissue shows increased mRNA expression and protein abundance of CXCL5 in tumor compared with adjacent normal tissue (CPTAC-cProSite). mRNA expression: n=39 for normal, n=138 for tumor; protein abundance: n=65 for normal, n=135 for tumor. The dashed line across each sample represents mean expression or abundance. (E) 24-hour stimulation of cells with hAT-CM shows CXCL5 in the media of the stimulated cells measured by ELISA normalized to serum free (SF) values, or compared with treatment with 1% FBS containing media; n=2 wells per treatment. Statistical analyses were performed using Brown-Forsythe and Welch corrected one-Way ANOVA in GraphPad Prism for (B) (EdU proliferation). CPTAC data (D) failed normality testing (S1D-E), therefore, the Mann- Whitney test was applied to CPTAC data to compare normal and tumor mRNA and protein abundance. P values <0.05 were considered significant and displayed up to four decimals. Error bars represent SD. ANOVA, analysis of variance.

Article Snippet: Cell lines MiaPaCa2, HPNE- G12D, Panc1, HEK- 293T, cell lines were acquired and used within 15 passages from purchase (ATCC).

Techniques: Incubation, Functional Assay, Flow Cytometry, In Vitro, Concentration Assay, Staining, Generated, Expressing, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal for immunotherapy of cancer

Article Title: Depletion of tumor-derived CXCL5 improves T cell infiltration and anti-PD-1 therapy response in an obese model of pancreatic cancer.

doi: 10.1136/jitc-2024-010057

Figure Lengend Snippet: Figure 2 Eight individual **hAT-CM samples (four male, three female, and one non-reported sex of varying BMIs/stages, see online supplemental table 1) or two replicates of serum-free media were applied to the indicated cells in separate wells in cell culture in six well plates. RNA was isolated from each well and subjected to RNA sequencing analysis to assess the response of HPNE-G12D, Panc1, and MiaPaCa2 cells to hAT-CM (n=8, diluted 1:3 in serum-free media) versus serum-free conditions (n=2). (A) Volcano plots displaying differential gene expression in response to hAT-CM treatment across three cell lines: HPNE-G12D, Panc1, and MiaPaCa2, versus serum-free incubation. (B) Differentially expressed genes were subjected to Upstream Analysis using Qiagen Ingenuity Pathway Analysis (IPA); IPA was used to predict upstream signaling pathways (cytokines, growth factors, receptors) activated across all three cell lines tested in response to hAT-CM. The z-scores of the 27 (*) overlapping signaling pathways with positive z-scores, indicating potential influence on the cellular transcriptomes, were plotted in a heatmap in order of clustering in IPA (C). The top two hits (TNF and IL-1β) are expanded out (D, E) to visualize averaged log2FC versus serum free conditions for all genes associated with the pathway and their differential expression across cell lines in a heatmap clustered by Clustergrammer (see online supplemental figure 2C for permanent links to interactive plots prepared using Average linkage and Cosine distance, and online supplemental table 3 for the gene list/values input into Clustergrammer); Red=positive FC, Blue=Negative FC. Range for (D)=−2.38:7.73 across 200 pathway associated genes, (E)=−1.86:7.73 across 136 IL-1β associated genes. 94 genes overlap across both groups. (F) P values of TNF and IL-1β upstream pathway activity from IPA are plotted for each cell line. **6/8 samples received no neoadjuvant therapy, while 2/8 were not reported or treatment history was not found. Abbreviations: BMI, body mass index; hAT-CM, human adipose tissue conditioned media; IPA, ingenuity pathway analysis.

Article Snippet: Cell lines MiaPaCa2, HPNE- G12D, Panc1, HEK- 293T, cell lines were acquired and used within 15 passages from purchase (ATCC).

Techniques: Cell Culture, Isolation, RNA Sequencing, Gene Expression, Incubation, Protein-Protein interactions, Quantitative Proteomics, Activity Assay