Fig 1: Tumor Regression, Cytokine Production, and Myeloid Infiltration Are Influenced by p53(A) Schematic representation of the experimental design. KC1 and KFC1 pancreatic-tumor-derived cell lines expressing iRFP were subcutaneously implanted into FVB, MHC-mismatched recipient mice.(B) Growth curve of KC1 (black circle) and KFC1 (red circle) cell lines injected into five FVB recipient mice per genotype. Tumor growth is measured as an increase in iRFP signal compared to the original fluorescence count on day 1 using the Pearl imager. Two-way ANOVA was used for statistical analysis, and the means are represented as ±SEM.(C) Individual tumors were harvested on day 7 post-injection and digested into single cells. Flow cytometry analysis was performed to measure F4/80+ immune tumor infiltrates, and the means are represented as ±SEM.(D) KC1 (black circle) and KFC1 (red circle) cells were orthotopically injected into the pancreas of FVB recipients (cohort size n = 3 per genotype). Graph shows percentage of pancreatic CD11b+ infiltrates 7 days post-operation, and the means are represented as ±SEM.(E) Schematic representation of the experimental design. KC1 PDAC cells were deleted for Trp53 by CRISPR, generating an isogenic pair (KC1-p53WT and KC1-p53KO).(F) A chemotaxis assay performed on BMDMs migrating towards KC1-p53WT or KC1-p53KO conditioned medium, or culture medium. The extent of BMDM migration was calculated as phase area density of technical replicate wells (n = 8) and the means are represented as ±SD.(G) Enzyme-linked absorbent assays (ELISAs) were performed with conditioned medium collected from KC1-p53WT (black) and KC1-p53KO (white) cells. ELISAs from left to right are CCL11, CXCL1, CXCL5, CCL3, M-CSF, and MCP1. Concentration was measured as pg/mL and the means are represented as ±SD.(H) Cytokine array of conditioned media from KC1-p53WT (left) and KC1-p53KO (right) cell lines. (Left) Boxed duplicate dots show changes in CXCL10 (green), CXCL11 (pink), TNF-α (blue), with a positive invariant control (orange). (Right) pixel quantification of each dot normalized to the positive control and the means are represented as ± SD.(I) 6 mice were injected with either the KC1-p53WT (black circle) or the KC1-p53KO (open circle) cells. The growth curve was measured by in vivo imaging of iRFP using the Pearl imager and graph shows fold increase in fluorescence from day 1 post-injection. Two-way ANOVA was used for statistical analysis, and the means are represented as ± SEM.(J–M) Tumors derived from KC1-p53WT (n = 5) or KC1-p53KO cells (n = 7) were digested into single-cells at day 3 post-injection. Digests were analyzed for infiltrating myeloid populations by flow cytometry. Graphs show tumors from individual mice, and the means are represented as ±SEM.(J) Flow cytometry analysis showing frequencies of tumor-infiltrating CD11b+ myeloid cells in mice harboring KC1-p53WT (black circles) or KC1-p53KO (open circles) tumors.(K) Frequency of proliferating intratumoral CD11b+ populations measured by Ki67 staining.(L) Frequency of CXCR3 expression on infiltrating CD11b+ cells.(M) Percentage of CD11b+ cells within tumor digests expressing surface CCR2.(N) Serum collected at endpoint (day 14) from FVB mice injected with the KC1-p53WT cell line (n = 8) or the KC1-p53KO cell line (n = 8) and analyzed for circulating levels of MCP1 by ELISA. The means are represented as ±SEM.Student’s unpaired t test was applied to experiments unless otherwise indicated and p values are ∗p < 0.05, ∗∗p < 0.01. See also Figure S2.