Figure 6.

KRAS Coordinates with p53 Loss for the Recruitment of Myeloid, but Not Treg, Cells

(A) Schematic representation of the inducible iKras^G12D/+;Trp53^f/f PDAC cell model (iKRAS^G12D).

(B) Growth kinetics of the iKRAS^G12D cell line (red circles) expressing iRFP subcutaneously injected into FVB recipients. Doxycycline (Dox) was maintained (red circles) or removed at day 7 (open circles) to modulate mutant KRAS expression. Tumor growth was measured using in vivo imaging using the Pearl imager and expressed relative to initial iRFP fluorescence at day 1 post-injection. Cohort sizes n = 10 and the means are represented as ±SEM.

(C–E) Analysis of individual tumor infiltrates of myeloid and T cell subsets 48 h post-Dox withdrawal. Cohort sizes n = 8−12 and the means are represented as ±SEM.

(C) Analysis of CD11b⁺, CD11b⁺F4/80⁺, and CD11b⁺CXCR3⁺ tumor-associated myeloid cells analyzed by flow cytometry (open circles Dox OFF and red circles Dox ON).

(D) Flow cytometry analysis of CD4⁺ and CD8⁺ T cell infiltrate frequencies.

(E) Analysis of intra-tumoral CD4⁺ T cell post ex vivo restimulation for expression of TNF-α⁺ (left) and CD4⁺FOXP3⁺KLRG1⁺ (right).

(F) Schematic representation of experimental design. C57Bl6/J-derived ID8 ovarian cancer cell lines were deleted of Trp53 using CRISPR/Cas9 and injected intraperitoneally into syngeneic recipients.

(G–I) Flow cytometry analysis of ascites and omentum at equivalent endpoints from mice bearing ID8-p53^WT (black circles) and ID8-p53^KO (open circles) tumors.

(G) CD4⁺FOXP3⁺ Treg cells in the ascites. Cohort size n = 4; means are represented as ±SEM.

(H) Representative plot of CD4⁺ T cells expressing FOXP3 and CD44.

(I) CD4⁺FOXP3⁺ T cells in tumor-immune infiltrates in the omentum Cohort size n = 9–10; means are represented as ±SEM.

A two-way ANOVA was used to analyze in vivo growth kinetics, and an unpaired Student’s t test was used for all bar graphs. p values are ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. See also Figure S6.