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Abstract
Background Currently, diagnosis with pancreatic ductal adenocarcinoma (PDAC) renders an almost intrinsically poor patient prognosis. Despite complete surgical resection and intense neoadjuvant and/or adjuvant treatment the great majority of patients will ultimately relapse and die from the disease. Further, PDAC has been characterized as highly immune resistant. It is speculated that radiation, chemotherapy, or chemoradiation cause the release of tumor antigens and inflammatory cytokines eventually leading to increased immunogenicity of PDAC.
Methods We used computational quantitative multiplex immune fluorescence (qmIF) (n=31) and the NanoString assay (n=34) to quantitatively analyze the effect of neoadjuvant chemoradiation (CRT) on the tumor immune microenvironment (TIME) of PDAC.
Results When comparing non-treated (NT) to neoadjuvant chemoradiation (CRT) tumors, the proportion of tumor within the overall tissue sample was markedly lower in treated tumors (figure 1; Mann Whitney U, U=25, p<0.0001). Additionally, the overall density of Ki67+ cells throughout all tissue was significantly lower in samples that received CRT (figure 1; Mann Whitney U, U=52, p=0.0067). An overall influx of CD3+ cells was noted in CRT samples. T cell influx was accompanied by upregulation of inflammatory genes. When considering T cell subsets, an increase in the CD8+ (Cytotoxic) and CD4+FOXP3+ (Treg) cell densities in the tumor of CRT samples was found. CD4+FOXP3- (T helper) cell density was found to be increased in the tumor, stroma, and overall tissue in CRT samples (figure 2). When comparing samples from patients who lived longer than 2 years to samples from patients who did not within the CRT group, a notably higher ratio of Tregs to CD3+ cells was observed in patients who lived less than 2 years (Mann Whitney U, U=0, p=0.0006). When used as a predictor, the ratio of Tregs to CD3+ cells also correlated closely to patient survival (figure 3); Mantel-Cox, p=0.0121).
Representative tissue segmentation and multiplex immune fluorescence (mIF) image of a non-treated patient and a patient who received neoadjuvant chemoradiation and analysis of total tumor density. Tissue segmentation images for A) NT and B) CRT. Blue cells are DAPI (nuclei) positive. Red areas represent tumor tissue, blue areas represent stromal tissue. Processing of slides done in inFormTM (PerkinElmer/Akoya). Multiplex view of the same C) NT and D) CRT images stained using mIF for DAPI (nuclei, blue), Ki67 (tumor, proliferative cells, red), CD3 (T cells, cyan), CD4 (T helper cells, orange), CD8 (cytotoxic T cells (CTLs), magenta), FOXP3 (T regulatory cells (Tregs), yellow), CD68 (macrophages, green). White bars = 100μm. E) Comparison of the proportion of total cells in the tumor tissue to the overall tissue sample between treatment groups (p
Density of immune cells in stroma, tumor, and total tissue comparing non-treated to neoadjuvant chemoradiation treated tissue. A) CD3+ cells in tumor (p=0.0006), B) CD3+ cells in stroma (p=0.0078), and C) CD3+ cells in total (p=0.0013). D) CD3+CD8+ cells in tumor (p=0.0079) , E) CD3+CD8+ cells in stroma (p=0.0758), and F) CD3+CD8+ cells in total (p=0.0076). G) CD3+CD4+FOXP3- cells in tumor (p=0.0010), H) CD3+CD4+FOXP3- cells in stroma (p=0.0216), and I) CD3+CD4+FOXP3- cells in total (p=0.0078). J) CD3+CD4+FOXP3+ cells in tumor (p=0.0089), K) CD3+CD4+FOXP3+ cells in stroma (p=0.4211), and L) CD3+CD4+FOXP3+ cells in total (p=0.1464). (*≤0.05, **≤0.01 ***≤0.001, ****≤0.0001)
Treg/CD3 differences in CRT Patients Survival was analyzed in n=14 patients using qmIF using Kaplan Meier analysis based on median Treg/CD3 value cutoff. Survival was significantly lower in patients with a high CD4+FOXP3+/CD3+ ratio (p=0.0121).
Conclusions We find that CRT greatly alters the TIME of PDAC, altering distributions of tumor cells within the microenvironment and inducing an overall influx of T cells, including cytotoxic, helper, and Treg T cell subsets. In patients receiving CRT, it appears as though the proportion of T cells infiltrating the tumor that are Tregs is closely associated with patient outcome, with a higher proportion of Treg infiltration correlating with a poor outcome. This data suggests that therapies targeting regulatory T cells should be explored in combination with chemo-radiotherapy in PDAC.
Ethics Approval The study was approved by Columbia University’s Ethics Board, approval number AAAQ7337.
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