Figure 3. Genetic silencing of tumor cell-intrinsic Cxcl1 overcomes T-cell exclusion and controls tumor growth in a CD8⁺ T-cell dependent manner in-vivo.

A, Violin plot representing difference in primary tumor weights in C57/BL6 mice orthotopically-injected with KPC^EV or KPC-Cxcl1^KO tumor-cells (n=20/group; left), with representative images of tumors from each group showing phenotypic reproducibility (n=5 each; right); B, Kaplan-Meier curves showing overall survival of KPC-Cxcl1^KO and KPC^EV orthotopically-injected mice (n=15; median 48 vs. 21 days); C, Bubble plot visualizing differentially upregulated pathways (using KEGG and Reactome knowledgebases) in 3-week whole-tumor transcriptomes from KPC^EV compared with KPC-Cxcl1^KO orthotopic tumors via RNA-sequencing (n=3 biologic replicates); D, Volcano plot depicting significantly enriched genes related to immune regulation in KPC^EV (Cxcl1, Vegfa, Il6, Csf2), and KPC-Cxcl1^KO (Cxcl10, Gzmb, Cxcr3, Cxcl9, Cd96, Cd3d, Cd4, Ciita, H2-Eb1); E, Pie charts showing relative proportions of immune-cell fractions of macrophages (Mϕ), PMN-MDSC, CD4⁺ T-cells, and CD8⁺ T-cells using CIBERSORT immune deconvolution from transcriptomes in KPC^EV vs. KPC-Cxcl1^KO tumors (n=3 biologic replicates per group); F-G, viSNE contour plots of flow cytometric immunophenotyping in concatenated single-cell suspensions from KPC^EV or KPC-Cxcl1^KO orthotopic tumors (left), with adjacent violin plots (right) representing absolute cell counts of PMN-MDSC, CXCR2⁺ PMN-MDSC, monocytic MDSC (moMDSC), M2-like macrophage (F), CD4⁺ T-cells and CD8⁺ T-cells (G), and central memory T-cells, effector memory T-cells, degranulating CD8⁺ T-cells (H) from each biologic replicate (n=6–8/group); I, Schematic of experimental design utilizing CD8⁺ T-cell neutralizing antibodies (CD8neuAb) in C57Bl/6 mice or CD8α^−/− transgenic mice (B6.Cd8^atm1Mak; CD8^KO) for orthotopic injections; J, Representative ultrasound images from mice in each treatment group showing tumor growth dynamics in vivo; K-L, tumor growth curves (K) and Kaplan-Meier survival estimates (L) from mice across 5 groups in T-cell depletion experiments (n=10 mice/group), with median survival (MS) of each cohort indicated in parentheses.