Cytokine expression profile in human pancreatic carcinoma cells and in surgical specimens: implications for survival

Abstract

Cytokine shedding by tumor cells into the local microenvironment modulates host immune response, tumor growth, and metastasis. The study aimed to verify the hypothesis that the immunological microenvironment of pancreatic carcinoma exists in a prevalently immunosuppressive state, influencing survival. We analyzed expression profiles of pro-inflammatory (IL-1beta, IL-2, IL-6, IL-8, IL-12 p40, IL-18 and IFN-gamma) and anti-inflammatory (IL-10, IL-11, IL-13 and TGF-beta isoforms) cytokines. The study was performed both in vitro, in five pancreatic carcinoma cell lines (real time RT-PCR), and in specimens from 65 patients, comparing tumoral versus non-tumoral pancreatic tissues (real time RT-PCR and immunohistochemistry). Furthermore, cytokines were measured in supernatants and sera (from patients and controls) by ELISA. All cell lines expressed IL-8, IL-18, TGF-beta1, TGF-beta2 and TGF-beta3, but not IFN-gamma and IL-2 transcripts. Expression of IL-1beta, IL-6, IL-10, IL-11, IL-13 and IL-12 mRNA was variable. All the above cytokines were detected as soluble proteins in supernatants, except IL-13. Tumor tissues overexpressed IL-1beta, IL-6, IL-8, IL-10, IL-11, IL-12 p40, IL-18, IFN-gamma, TGF-beta1, TGF-beta2 and TGF-beta3 at the mRNA level and IL-1beta, IL-18, TGF-beta2 and TGF-beta3 also at the protein level. Conversely, non-tumor tissues had stronger RNA and protein expression of IL-13. Survival was significantly longer in patients with high IL-1beta and IL-11 and moderate IL-12 expression. Serum IL-8, IL-10, IL-12, IL-18, TGF-beta1 and TGF-beta2 were higher in patients than in controls, as opposed to IL-1beta and IL-13. Patients with low circulating levels of IL-6, IL-18 and TGF-beta2 survived longer. Pancreatic cancer is characterized by peculiar cytokine expression patterns, associated with different survival probabilities.

Fig. 1

Levels of pro- (IL-1β, IL-6, IL-8, IL-12 p40, IL-18) and anti-inflammatory (IL-10, IL-11, IL-13 and TGF-β isoforms) cytokine mRNA in PT-45, Capan-2, BxPC-3, PANC-1 and MIAPaCa-2 cell lines. mRNA levels were assessed via real-time RT-PCR, and normalized to β-actin mRNA levels. MNE values are shown

Fig. 2

Detection of pro-inflammatory cytokines in non-tumoral and tumoral pancreatic tissue specimens as determined by immunohistochemistry. Specimens are representative examples of nine non-tumoral and forty one tumoral pancreas tissue samples (original magnification 250×)

Fig. 3

Detection of anti-inflammatory cytokines in non-tumoral and tumoral pancreatic tissue specimens as determined by immunohistochemistry. Specimens are representative examples of nine non-tumoral and 41 tumoral pancreas tissue samples (original magnification 250×)

Fig. 4

Kaplan–Meier estimates of survival in pancreatic carcinoma patients, stratified by in situ cytokine expression pattern

Fig. 5

Secretion of cytokines by pancreatic carcinoma cell lines as determined by ELISA in supernatants

Fig. 6

Serum cytokine concentration in patients and controls by ELISA. Median, 10th, 25th, 75th and 90th percentiles are presented as vertical boxes with error bars. Dots indicate outliers

Fig. 7

Kaplan–Meier estimates of survival in pancreatic carcinoma patients, stratified by circulating serum cytokine pattern