Addition of Losartan to FOLFIRINOX and Chemoradiation Reduces Immunosuppression-Associated Genes, Tregs, and FOXP3+ Cancer Cells in Locally Advanced Pancreatic Cancer

Abstract

Purpose: Adding losartan (LOS) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) resulted in 61% R0 surgical resection in our phase II trial in patients with locally advanced pancreatic cancer (LAPC). Here we identify potential mechanisms of benefit by assessing the effects of neoadjuvant LOS on the tumor microenvironment.

Experimental design: We performed a gene expression and immunofluorescence (IF) analysis using archived surgical samples from patients treated with LOS+FFX+CRT (NCT01821729), FFX+CRT (NCT01591733), or surgery upfront, without any neoadjuvant therapy. We also conducted a longitudinal analysis of multiple biomarkers in the plasma of treated patients.

Results: In comparison with FFX+CRT, LOS+FFX+CRT downregulated immunosuppression and pro-invasion genes. Overall survival (OS) was associated with dendritic cell (DC) and antigen presentation genes for patients treated with FFX+CRT, and with immunosuppression and invasion genes or DC- and blood vessel-related genes for those treated with LOS+FFX+CRT. Furthermore, LOS induced specific changes in circulating levels of IL-8, sTie2, and TGF-β. IF revealed significantly less residual disease in lesions treated with LOS+FFX+CRT. Finally, patients with a complete/near complete pathologic response in the LOS+FFX+CRT-treated group had reduced CD4+FOXP3+ regulatory T cells (Tregs), fewer immunosuppressive FOXP3+ cancer cells (C-FOXP3), and increased CD8+ T cells in pancreatic ductal adenocarcinoma lesions.

Conclusions: Adding LOS to FFX+CRT reduced pro-invasion and immunosuppression-related genes, which were associated with improved OS in patients with LAPC. Lesions from responders in the LOS+FFX+CRT-treated group had reduced Tregs, decreased C-FOXP3 and increased CD8+ T cells. These findings suggest that LOS may potentiate the benefit of FFX+CRT by reducing immunosuppression.

Figure 1.. Differential gene expression between the 3 treatment groups (losartan+FFX+CRT n=17; FFX+CRT n=19; and untreated, n=9).

(A) Study design showing the sources of human PDAC tissues. (B) PCA plots showing the clustering of all 3 groups. (C) Volcano plots of highly upregulated and downregulated genes between losartan+FFX+CRT versus untreated and FFX+CRT versus untreated. P values generated by DESeq2 (Wald test) and adjusted p values based on FDR set at 0.05 (BH method).

Figure 2.. FFX+CRT and losartan+FFX+CRT enhance the expression of genes linked to the maturation of blood vessels, leukocyte transendothelial migration, T cell activation, cytolytic activity of T cells and NK cells, and dendritic cells.

Effect of FFX+CRT and losartan+FFX+CRT on the expression of genes associated with (A) blood vessel maturation and integrity, (B) leukocyte transendothelial migration, (C) T cell activation, (D) cytolytic activity of T cells and NK cells and (E) dendritic cells. P values generated by DESeq2 (Wald test) and adjusted p values based on FDR set at 0.05 (BH method).

Figure 3.. The addition of losartan to FFX+CRT decreased the expression of pro-invasion-associated and immunosuppression genes and increased the expression of tumor suppressor genes.

(A) Pro-invasion genes. (B) Tumor suppressor genes. P values generated by DESeq2 (Wald test) and adjusted p values based on FDR set at 0.05 (BH method). (C-D) Heatmaps of Spearman rank correlation of genes associated with RORA and NFATC4 in tumor samples treated with (C) losartan+FFX+CRT and (D) FFX+CRT. Gene sets include the following: tumor suppressors (RORA, CYLD, FEZ1), blood vessels (NFATC4, AKT3, PECAM1, CDH5, JAM3), dendritic cells (IL3RA, FLT3LG, CSF1), MHC II (HLA-DRB3, LAMP2), T cell activation (STAT4, CD6, TNFSF8), inflammation inhibition (NCF4, SERPING1, NFKBIA, A2M) and epithelial (EPCAM, CDH1). (E) Quantitative analysis of residual disease in resected PDAC lesions (immunofluorescence of cytokeratin-19+ cells located in the tumor bed). (F) Losartan+FFX+CRT compared to FFX+CRT induced a greater down-regulation of macrophage related genes. (G) Losartan+FFX+CRT and FFX+CRT induced a significant down-regulation CEACAM1, while the addition of losartan to FFX+CRT induced a greater down-regulation of TIGIT and FOXP3. P values generated by DESeq2 (Wald test) and adjusted p values based on FDR set at 0.05 (BH method).

Figure 4.. Gene set stratification of overall survival (OS) for patients treated with losartan+FFX+CRT and FFX+CRT.

(A-D) Patients treated with losartan+FFX+CRT. Gene sets composed of (A-C) immunosuppression and invasion / proliferation, (B) immunosuppression, (C) invasion / proliferation genes, (D) DCs, blood vessel maturation, transendothelial migration and tumor suppression related genes stratified OS. (E-H) For patients treated with FFX+CRT, gene sets (E, F) negatively or (G, H) positively associated with OS. P values based on t-test. For the complete list of gene sets refer to Supplementary Table 4.

Figure 5.. Immunofluorescence (IF) staining and quantitative analysis in PDAC lesions in losartan+FFX+CRT-treated patients.

(A, C) Representative IF staining of cytokeratin-19+ cancer cells (cyan), CD4+ T cells (green), FOXP3+ cells (red) and CD4+FOXP3+ Tregs (yellow merge, bottom right) in (A) losartan+FFX+CRT non-responders (NR, CAP score 2/3) and (C) responders (R, CAP score 0/1). White box shows inset area. White arrows indicate FOXP3+Tregs. Nuclei are stained with DAPI (blue). Scale bar is 60 μM. (B) Corresponding quantitative analysis of FOXP3+ Tregs in PDAC lesions from panels A and C (p = 0.029). Non-responders in light blue and responders in dark blue. (D) Corresponding distance analysis of FOXP3+Tregs to cancer cells in losartan+FFX+CRT-treated patients from panels A and C (*p = 0.0058). (E) Representative IF staining of CD8+ T cells (green) in losartan+FFX+CRT non-responders (NR) and responders (R). Scale bar is 50 μM. (F) Corresponding quantitative analysis in PDAC lesions from panel E showing the number of CD8+ T cells in non-responders (NR, light blue) compared to responders (R, dark blue) (p = 0.024). (G) Corresponding distance analysis of CD8+ T cells to residual cancer cells from panel E. (H) Representative IF staining of CD4+ T cells (red) and cytokeratin-19+ cancer cells (green) in losartan+FFX+CRT non-responders (NR) and responders (R). Scale bar is 50 μM. (I) Corresponding quantitative analysis in PDAC lesions from H of the number of CD4+ T cells. (J) Corresponding distance analysis of CD4+ T cells to cancer cells from panel H (*p = 0.016). For barplots, ANOVA test followed by Fisher’s Least Significant Difference (LSD) test was performed and for distance metrics, Kruskal-Wallis test was done (alpha = 0.05).

Figure 6.. Immunofluorescence (IF) staining and quantitative analysis of FOXP3+ cancer cells (C-FOXP3) in PDAC lesions.

(A-B) Representative images of IF staining of FOXP3+ cells (red), CD4+ T cells (white), cytokeratin-19+ cancer cells (CK, green), and C-FOXP3 (merged yellow, far right panels) in losartan+FFX+CRT-treated (A) non-responders (NR, CAP score 2/3) and (B) responders (R, CAP score 0/1). White arrows indicate FOXP3+Tregs. Nuclei are stained with DAPI (blue). Scale bar is 50 μM. (C, E) Corresponding quantitative analysis in PDAC lesions of number of FOXP3+Tregs in (C) losartan+FFX+CRT non-responders (NR, light blue) and responders (R, dark blue) (p = 0.0377) and (E) FFX+CRT non-responders (NR, light green) and responders (R, dark green). P-value based on the Fisher’s Least Significant Difference (LSD) test. (D, F) Correlation analysis of the number of C-FOXP3+ cells and FOXP3+Tregs in (D) losartan+FFX+CRT (R = 0.84, p = 2.6e-05) and (F) FFX+CRT (R = 0.6, p = 0.1). R correlation and p-value generated by fitting linear model using lm function in R. (G-I) Cytokine levels of serial plasma samples from FFX+CRT (green) and losartan+FFX+CRT (blue) for (G) IL-8, (H) TGF-β and (I) sTie2. (I) For sTie2, losartan+FFX+CRT group stratified based on pathological response for non-responders (NR, light blue) and responders (R, dark blue). In IL-8 and TGF-β, *slope indicates statistical significance in treatment x time interaction term in mixed effect model and in Tie2 plot, * indicates overall significant difference between FFX+CRT versus losartan+FFX+CRT responders (independent of time).