magnetic-based commercial cd8 t cell negative selection kit  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: PTEN and BRCA1 tumor suppressor loss associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in a murine model of ovarian cancer

doi: 10.1101/2022.06.27.497846

Figure Lengend Snippet: Flow cytometric analysis of immune cell proportions revealing proportion of isolated (A) CD8+ cytotoxic T cells and (B) F480+ macrophages used for T cell-macrophage co-culture assay assessing IFNγ producing T cells after exposure to macrophages derived from ascites of ID8 -Trp53 -/- ; Pten -/- or ID8- Trp53 -/- ; Brca1 -/- injected mice.

Article Snippet: Splenic T cells were enriched using a magnetic-based commercial CD8 T cell negative selection kit (StemCell Technologies, BC, Canada; ) as per the manufacturer’s protocol.

Techniques: Isolation, Co-culture Assay, Derivative Assay, Injection

Journal: bioRxiv

Article Title: PTEN and BRCA1 tumor suppressor loss associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in a murine model of ovarian cancer

doi: 10.1101/2022.06.27.497846

Figure Lengend Snippet: (A) Kaplan-Meier survival analysis using clinical data for patients with PTEN intact (n=10) had significantly higher disease specific survival (DSS) than patients with PTEN absent (n=57; p=0.0376). Fluorescent immunostaining of CD8+ cytotoxic T cells and CD68+ macrophages within tumors of patients with (B) complete absence of PTEN (PTEN absent; n=9) and (C) complete presence of PTEN (PTEN intact; n=51) in stromal and epithelial compartments of patient tumors using immunohistochemistry. Immunofluorescent staining of CD8 and CD68 expressing cells were quantified in different tissue compartments. (D) Infiltration patterns of CD68+ macrophages between different compartments in tumors with PTEN absence and PTEN intact were compared, revealing a significantly higher amount of CD68+ macrophages in the stroma compared to the epithelium in tumors with PTEN absence (p=0.0005). Average of duplicate or triplicate cores for each sample was taken and Mann-Whitney non-parametric test was used to determine statistical significance of immune cell infiltration. Log-rank test was applied to determine statistical significance of Kaplan-Meier survival analysis, using R statistical software. ** p<0.005, *** p<0.001, **** p<0.0001. P-value <0.05 was considered statistically significant. ns: not significant.

Article Snippet: Splenic T cells were enriched using a magnetic-based commercial CD8 T cell negative selection kit (StemCell Technologies, BC, Canada; ) as per the manufacturer’s protocol.

Techniques: Immunostaining, Immunohistochemistry, Staining, Expressing, MANN-WHITNEY, Software

Journal: bioRxiv

Article Title: PTEN and BRCA1 tumor suppressor loss associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in a murine model of ovarian cancer

doi: 10.1101/2022.06.27.497846

Figure Lengend Snippet: (A) Kaplan-Meier survival analysis of mice injected with either ID8- Trp53 -/- , ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- cells. (n = 10-15) for each treatment group. (B) In vivo bioluminescent imaging of mice injected with ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- luciferase-tagged cells tracking tumor progression once/week over 4 weeks. Total RNA from untreated tumors of different genotypes was subject to NanoString gene expression profiling using the panCancer immune gene panel displayed as a (C) heat map showing differential expression pattern of groups of genes involved in various immune functions, (D) 2-fold differentially expressed genes and (E) 0.5-fold differentially expressed genes. Proportion of cells derived from ascites of mice injected with ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- cells expressing (F) CD45+CD3+CD8+CD69+, (G) CD45+CD11b+F4/80+CD206+, and (H) CD45+CD11b+GR1+. (I) IL-6, (K) IL-10 and (G) CXCL-10 cytokine levels derived from the ascites fluid of untreated mice injected with ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- cells. Log-rank (Mantel-Cox) test was applied to derive significant differences in (A). Mann-Whitney non-parametric test was used for (F-L) * p<0.05, ** p<0.005, *** p<0.001, **** p<0.0001. Gene expression data analysis was performed using nSolver Advanced Analysis Software. Mean ± SD.

Article Snippet: Splenic T cells were enriched using a magnetic-based commercial CD8 T cell negative selection kit (StemCell Technologies, BC, Canada; ) as per the manufacturer’s protocol.

Techniques: Injection, In Vivo, Imaging, Luciferase, Expressing, Derivative Assay, MANN-WHITNEY, Software

Journal: bioRxiv

Article Title: PTEN and BRCA1 tumor suppressor loss associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in a murine model of ovarian cancer

doi: 10.1101/2022.06.27.497846

Figure Lengend Snippet: Total RNA from untreated tumors of different genotypes was subject to NanoString gene expression profiling using the panCancer immune gene panel displayed as (A) gene scores representing various immune cell types and (B) heatmap of 1.5-fold significantly differentially expressed genes between tumors generated from untreated ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- cells using NanoString panCancer immune gene panel. Proportion of cells derived from ascites of mice injected with ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- cells expressing (C) CD45+CD3+CD8+, (D) CD45+CD11b+F480+PDL1+. (E) CXCL10 and (F) CCL5 cytokine levels within 48-hour conditioned media from ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- cells. (G) Heatmap displaying macrophage associated gene expression between ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- derived tumors. Cytokine experiments were derived from duplicate experiments of triplicate wells. Data analysis was performed using nSolver Advanced Analysis Software. Mann-Whitney non-parametric test was used. * p<0.05 ** p<0.005, *** p<0.001, **** p<0.0001. Mean ± SD. ns: not significant.

Article Snippet: Splenic T cells were enriched using a magnetic-based commercial CD8 T cell negative selection kit (StemCell Technologies, BC, Canada; ) as per the manufacturer’s protocol.

Techniques: Expressing, Generated, Derivative Assay, Injection, Software, MANN-WHITNEY

Journal: bioRxiv

Article Title: PTEN and BRCA1 tumor suppressor loss associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in a murine model of ovarian cancer

doi: 10.1101/2022.06.27.497846

Figure Lengend Snippet: (A) Co-culture of ascites-derived macrophages of mice injected with ID8 -Trp53 -/- ; Pten -/- or ID8- Trp53 -/- ; Brca1 -/- cells. Baseline proportions of CD80+ M1 macrophages and CD206+ M2 macrophages added to healthy T cells displayed. Histogram showing representative average of IFNγ producing CD8+ T cells from triplicate wells 48h post-stimulation with macrophages derived from ascites of either cell genotype (using n=5 mice for each genotype). Averages of triplicate wells used within this assay were displayed as a bar graph. (B) t-SNE plots generated from a concatenation of myeloid markers expressed on ascites derived from ID8 -Trp53 -/- ; Pten -/- cells using Flowjo® FlowSOM and Phenograph Plug-in features (n=5 for each treatment group: untreated, carboplatin, and carboplatin + STING agonist). Corresponding cell proportions for each marker within ascites from individual mice displayed as bar graphs below. (C) Proportion of Live+CD45+CD3+CD8+CD69+ activated T cells within the ascites generated from untreated, carboplatin or carboplatin + STING agonist treated ID8 -Trp53 -/- ; Pten -/- or ID8- Trp53 -/- ; Brca1 -/- cells; analyzed using one-way ANOVA. Mann-Whitney non-parametric test was used for (A). * p<0.05 ** p<0.005, *** p<0.001, **** p<0.0001. Mean ± SD. ns: not significant.

Article Snippet: Splenic T cells were enriched using a magnetic-based commercial CD8 T cell negative selection kit (StemCell Technologies, BC, Canada; ) as per the manufacturer’s protocol.

Techniques: Co-Culture Assay, Derivative Assay, Injection, Generated, Marker, MANN-WHITNEY