Clonal expansions of cytotoxic T cells infiltrating the αKO pancreatic tumors.

(A) Experimental design schema for single cell RNA sequencing of infiltrating CD8+ T cells in αKO tumors. Cells were implanted in the pancreas of two B6 mice and tumors were harvested at 12 days post-implantation. Single cell suspensions were enriched with CD8+ beads followed by concurrent single-cell transcriptional and T-cell receptor (TCR) profiling using 10x Genomics protocols. (B) Size distribution for CD8+ T cell clonal types in αKO tumors. Non-clonal: N≤2; small expansion: 3 ≤N<50; large expansion: N≥50. (C) Amino acid sequences of the CDR3 region for large expansion clonotypes. αKO1 tumor has 5 and αKO2 tumor has 4 large expansion clonotypes (full list shown in the Supplementary File 1). (D) UMAP of all CD8+ T cell clonotypes. Principle component analysis of gene expression profiles generated 4 cell clusters annotated based by ProjectTILs. EffectorMem: effector memory; Tex: exhausted; Tpex: precursor exhausted; NaiveLike: naïve. Each dot corresponds to a single cell. (E) Major markers used for cell type annotation by Projectils. (F) Clonal size distribution mapped to the gene expression UMAP showing that the largest clonotypes are mostly effector memory T cells. (G) The gene expression profile distribution of individual large expansion clonotypes are shown. The majority of cells for each clone mapped to the effector memory T cell functional group.

A genome-wide CRISPR gene-deletion screen identifies Pccb contributing to PIK3CA-mediated pancreatic tumor immune evasion. (A) A schema for the genetic screen. Mouse genome-scale CRISPR/Cas9 knockout library (mGeCKO v2A) containing three sgRNA each for 20611 genes was transduced into KrasG12D/+;Tp53R172H/+;Pik3ca-/- (αKO) cells. Pooled cells were implanted in the pancreas of eight C57BL/6J mice. (B) IVIS imaging was performed to monitor tumor progression in these mice. In two animals, tumors were still present at the end of the screen (annotated as tumor 1 & 2). (C) Harvested DNAs prepared from the tumors were subjected to next generation sequencing (NGS) to identify enriched sgRNAs barcodes. Sequences corresponding to only six genes in the mGeCKO v2A library have detectable NGS counts as shown in the table. In both tumors, sgRNA sequences mapping to the Pccb gene are most numerous (full list shown in the Supplementary File 2).

Ablation of PCCB reverses the immune recognition of αKO cells.

(A) Pccb was deleted in αKO cells by CRISPR/Cas9 to generate p-αKO cells. Western blotting confirmed PCCB and PIK3CA loss in p-αKO cells. Additionally, western blotting was performed for PTEN, phospho-Thr308 AKT, phospho-Ser473 AKT, and phospho-PRAS40, with Actin and HSP90 used as loading controls. (B) Orthotopic implantation of p-αKO tumors in the pancreas of C57BL/6J mice was monitored by IVIS imaging of the luciferase signal weekly for 8 weeks. (C) Kaplan-Meier survival curves for mice implanted with KPC, αKO, and p-αKO cells. Median survival KPC: 15.5 days (N=8); αKO, all alive (N=9); p-αKO, 40.5 days (N=12). ****P < 0.0001 for KPC vs. αKO; ***P=0.0009 for αKO vs. p-αKO; ****P < 0.0001 for KPC vs. p-αKO (log-rank test). (D) Percentage of B6 mice that are tumor-free at 8 weeks after implantation with KPC (0%), αKO (100%) and p-αKO (0%) cells.

T cells infiltrate p-αKO tumors with increased expression of immune checkpoints

(A) Pancreatic tissue sections prepared from C57BL/6J mice implanted with KPC, αKO, and p-αKO cells. Tumor sections were stained with H&E and IHC with CD3, CD4, and CD8 antibodies. Representative sections are shown. Scale bar: 60μm. (B) Quantification of tumor-infiltrating T cells in 5 representative tumor sections for each group (mean ± SD, n=3). CD3: ****P<0.0001 for KPC vs. αKO; **P=0.0063 for KPC vs. p-αKO; *P=0.0471 for αKO vs. p-αKO. CD4: ****P<0.0001 for KPC vs. αKO; *P=0.0164 for KPC vs. p-αKO; ns for αKO vs. p-αKO. CD8: ***P=0.0009 for KPC vs. αKO; ***P=0.0005 for KPC vs. p-αKO; and ns for αKO vs. p-αKO (two-tailed t test). ns: not significant. (C) αKO and p-αKO cells were implanted in the head of pancreas of C57BL/6J mice. Tumor sections were stained with checkpoint markers: PD1, CTLA4, LAG3, TIGIT and TIM3. Representative sections are shown. Scale bars: 60 μm. (D) Quantifications of cells positive for each checkpoint marker at representative tumor sections (mean ± SD, N=5). αKO vs. p-αKO: **P = 0.0023 for PD1; *P = 0.0371 for CTLA4; ***P = 0.0002 for LAG3; ns for TIGIT and TIM3.

Inhibition of PD-1/PD-L1 checkpoint interaction leads to elimination of most p-αKO tumors.

(A) PD1-/- mice (N = 9) were implanted with p-αKO cells in the pancreas and was monitored for 8 weeks. IVIS imaging was performed to assess tumor size at the end of the experiment. C57BL/6J mice (N = 22) were implanted with p-αKO cells in the pancreas and randomly divided into two groups. One group was injected with 12.5 mg/mg of PD1 neutralizing antibody weekly for 8 weeks, and the vehicle group was injected with the inVivo Pure dilution buffer. IVIS imaging was performed to assess tumor size at the end of the experiment. (B) Kaplan-Meier survival curves for PD1-/- mice implanted with p-αKO cells, and vehicle and anti-PD1 treated C57BL/6J mice implanted with p-αKO cells. Survival rate for PD1-/- group is 89% and median survival undefined; survival rate for vehicle injected group is 22% and median survival is 41 days; survival rate for anti-PD1 injected group is 69% and median survival undefined. *P = 0.0436 for vehicle vs. anti-PD1 antibody injection. (C) Tumor free survival percentage of PD1-/- (56%), vehicle injected (0%), and anti-PD1 antibody injected (31%) groups.

Increased number of anti-tumor cytotoxic T cells infiltrating p-αKO tumors implanted in PD1-null vs. WT mice.

WT and PD1-null mice in B6 genetic background were implanted with p-αKO cells in the pancreas (n=2 for each group). Tumors were harvested from these animals 12 days post implantation. T cell suspensions prepared from these tumors were subjected to scRNAseq and TCR sequencing analysis using the same protocol as Figure 1. (A) The bar graph shows size distribution of CD8+ T cell clonotypes found in WT vs. PD1-null tumors. (B) CDR3 sequences of clonotypes with size ≥50 (defined as large expansion). Tumors in WT mice had 6 clonotypes with size ≥50 cells and tumors in PD1-null mice had 18 clonotypes with size ≥50 cells. (C) UMAP showing gene expression profiles of all CD8+ T clonotypes found in both groups. EffectorMem: effector memory; Tex: exhausted; Tpex: precursor exhausted; Tn: naïve. Each dot corresponds to a single cell. (D) UMAP showing gene expression profiles of large expansion CD8+ T cell clonotypes. Each clonotype is color coded showing the functional mapping of individual cells. The dash line marks where effector memory T cells are located on the UMAP.

PCCB regulates PDAC survival in a TIL-dependent manner.

(A) Overall survival between patients with high PCCB/PIK3CA ratio vs. patients with low PCCB/PIK3CA of 177 PDAC patients from TCGA database. P=0.19. (D) Two PDAC clinical data GSE15471 and GSE16515 illustrating PCCB mRNA expression in tumor sections vs. adjacent normal tissues. ***P=0.0001 for GSE15471, N=36 for tumor, and N=36 for normal; and ****P<0.0001 for GSE16515, n=36 for tumor, and N=16 for normal. (C) The overall survival comparisons among PDAC patients with high infiltrating CD8 T cell level with high or low PCCB expression in TCGA dataset analyzed with TCGA database. High vs. low PCCB expression was defined as 80% percentile.

In vitro characterization of p-αKO cells.

(A) Growth rates of KPC, αKO and p-αKO cell lines in culture were measured from day 1 to day 4 (mean ± SD, n=3). ***P= 0.0009 for KPC vs. p-αKO; not significant (ns) for αKO vs. p-αKO (One-way ANOVA). (B) Mitochondria function was evaluated by measuring the oxygen consumption rate (OCR) with Seahorse. 1st injection: oligomycin; 2nd injection: FCCP; 3rd injection: antimycin A & rotenone. (C) Metabolic changes between p-αKO and αKO revealed by non-targeted LC-MS analysis. Each data point represents the Log2 fold change of p-αKO over αKO, with a threshold set at 1.5-fold changes. Metabolites with differences exceeding this threshold were depicted in red. (D) LC-MS exploring changes of propionyl-CoA, propionylcarnitine, and 2-methylcitrate. Data are represented as ion counts ± SD (N=3 for all groups). ns: not significant. (E) LC-MS exploring changes of metabolites of the TCA cycle. Data are represented as ion counts ± SD (N=3 for all groups). ns: not significant.

Flow cytometry of myeloid cells infiltrated in αKO vs. p-αKO.

(A) The gating strategy of the myeloid panel. (B) Flow cytometry analysis of the cells isolated from αKO and p-αKO tumors implanted in C57BL/6J mice. The numbers presented in the figures represent cell percentages relative to the total cell counts from the respective previous groups.

Gene set enrichment analysis (GSEA) of cytokine-related pathways in αKO cells (n=3) vs. p-αKO cells (n=3). These pathways include IL2-STAT5 signaling pathway, interferon α response, IL6-JAK-STAT3 signaling pathway, interferon γ response, TNFA signaling via NFKB, and TGF β signaling pathway. NES: normalized enrichment score.