Pancreatic adenocarcinoma exerts systemic effects on the peripheral blood myeloid and plasmacytoid dendritic cells: an indicator of disease severity?

Abstract

Background: Dendritic cells (DCs) isolated from tumor bearing animals or from individuals with solid tumors display functional abnormalities and the DC impairment has emerged as one mechanism for tumor evasion from the control of the immune system. Ductal pancreatic adenocarcinoma (PDAC), the most common pancreatic cancer, is recognized as a very aggressive cancer type with a mortality that almost matches the rate of incidence.

Methods: We examined the systemic influence ductal pancreatic adenocarcinoma (PDAC) exerted on levels of peripheral blood DCs and inflammatory mediators in comparison to the effects exerted by other pancreatic tumors, chronic pancreatitis, and age-matched controls.

Results: All groups examined, including PDAC, had decreased levels of myeloid DCs (MDC) and plasmacytoid DCs (PDC) and enhanced apoptosis in these cells as compared to controls. We found elevated levels of PGE2 and CXCL8 in subjects with PDAC, and chronic pancreatitis. Levels of these inflammatory factors were in part restored in PDAC after tumor resection, whereas the levels of DCs were impaired in the majority of these patients approximately 12 weeks after tumor removal. Our results prove that solid pancreatic tumors, including PDAC, systemically affect blood DCs. The impairments do not seem to be tumor-specific, since similar results were obtained in subjects with chronic pancreatitis. Furthermore, we found that PDAC patients with a survival over 2 years had significant higher levels of blood DCs compared to patients with less than one year survival.

Conclusions: Our findings points to the involvement of inflammation in the destruction of the blood MDCs and PDCs. Furthermore, the preservation of the blood DCs compartment in PDAC patients seems to benefit their ability to control the disease and survival.

Figure 1

Characterization and quantification of MDCs and PDCs. (A) PBMCs isolated from individuals with pancreatic duct adenocarcinoma (PDAC) (1 week pre surgery) and healthy age matched volunteers were analyzed for MDCs and PDCs levels by flow cytometry. The PBMCs stained with different direct conjugated mabs to distinguish the MDC and PDC from the rest of the cells. The gating was set to exclude debris (R1) and second gate (R2) was set on Lineage (FITC) negative and HLA DR (PerCP) positive cells. The MDCs were identified as Lin-HLA DR+ CD11c+ cells (R3) and PDCs as Lin-HLA DR+ CD123+ cells (R4). Top panel shows the dot plots from a healthy volunteer and the bottom panel shows the dot plots from an individual with PDAC. (B) Percentage of MDCs (top panel) and PDCs (lower panel) prior tumor resection in healthy controls, PDAC, none resectable pancreatic tumor (NRPT), billary duct adenocarcinoma (BDAC), ampullary carcinoma (AC), endocrine carcinoma (EC), and chronic pancreatitis (CP). (C) Correlation of MDC and PDCs numbers (% out of PBMCs) between controls and PDAC. Control: Solid line and black circles r² = 0.50. PDAC: striped line and white circles r² = 0.098. Statistically significant differences between individuals with pancreatic disease and healthy controls are indicated as; * = p < 0.05, ** = p < 0.005, *** = p < 0.001

Figure 2

Resection of pancreatic tumor mass restored the MDCs and PDCs in some subjects. (A) Percentage of MDCs (top) and PDCs (bottom) of total PBMCs in healthy age match controls and pre and post tumor resection in individuals with pancreatic duct adenocarcinoma (PDAC) (B) Pre and post tumor resection levels of MDCs (top) and PDCs (bottom) in individuals with PDAC. (C) Dot plots showing the percentage of MDCs (CD11c+) and PDCs (CD123+) pre and post tumor resection in a subject with PDAC with fully recovered DC populations 12 weeks post surgery. Percent of MDCs (top) and PDCs (bottom) of total PBMCs in healthy age match controls and pre and post resection in individuals with (D) ampullary carcinoma (AC), (E) endocrine carcinoma (EC) and (F) chronic pancreatitis (CP). Statistically significant differences between individuals with pancreatic disease and healthy controls are indicated as; * = p < 0.05, ** = p < 0.005, *** = p < 0.001

Figure 3

The non DC in lin-HLA DR+ cell population is increased in individuals with different types of pancreatic cancers and chronic pancreatitis. Percentage of non DC (lin-HLA DR+ CD11c-CD123-) within the lin-HLA DR+ cell population in healthy age match controls and pancreatic duct adenocarcinoma (PDAC), none resectable pancreatic tumor (NRPT), billary duct adenocarcinoma (BDAC), ampullary carcinoma (AC), endocrine carcinoma (EC), and chronic pancreatitis (CP) prior surgery (A) and after surgery (B). The proportion of non DC was estimated as the mean percentage of the total amount of lin-HLA-DR+ cells. Statistically significant differences between individuals with pancreatic disease and healthy controls are indicated as; * = p < 0.05, ** = p < 0.005, *** = p < 0.001.

Figure 4

Increased spontaneous apoptosis among the MDCs and PDCs in individuals with PDAC and other types pancreatic cancers. (A) Percentage apoptotic MDCs (top panel) and PDCs (lower panel) of total MDCs and PDCs prior tumor resection in controls, pancreatic duct adenocarcinoma (PDAC), none resectable pancreatic tumor (NRPT), billary duct adenocarcinoma (BDAC), ampullary carcinoma (AC), endocrine carcinoma (EC), and chronic pancreatitis (CP). Percent apoptotic MDCs (top) and PDCs (bottom) of total MDCs and PDCs in healthy age match controls and pre and post tumor resection in individuals with PDAC (B), AC (C), EC (D) and CP (E). Correlation between percent apoptotic MDCs and level of MDCs (left) and correlation between percent apoptotic PDCs and level of PDCs (right) in individuals with PDAC pre tumor resection (F) and post tumor resection (G). The numbers of Annexin V positive MDCs and PDCs were measured as the percentage of Annexin V+ cells out of the total amount of MDCs or PDCs. Statistically significant differences between individuals with pancreatic disease and healthy controls are indicated as; * = p < 0.05, ** = p < 0.005, *** = p < 0.001.

Figure 5

Elevated levels of inflammatory cytokines in individuals with pancreatic duct adenocarcinoma. PGE₂ levels in plasma from pancreatic duct adenocarcinoma (PDAC), none resectable pancreatic tumor (NRPT), billary duct adenocarcinoma (BDAC), ampullary carcinoma (AC), endocrine carcinoma (EC), and chronic pancreatitis (CP) pre (A) and post (B) tumor resection compared to levels in healthy age matched controls. CXCL8 levels in plasma from PDAC, NRPT, BDAC, AC, EC, and CP pre (C) and post (D) tumor resection compared to levels in healthy age matched controls. CXCL8 levels in plasma from PDAC pre and post (E) tumor resection compared to levels in healthy age matched controls. Statistically significant differences between individuals with pancreatic disease and healthy controls are indicated as; * = p < 0.05, ** = p < 0.005, *** = p < 0.001.

Figure 6

Long-time survivors have more DCs compared to short-time survivors. (A) Percent DCs of total PBMCs in healthy age match controls and pre tumor resection in PDAC patients surviving less than one year (short-time survivor) and more than two years survival (long-time survivor). (B and C) Comparison of the one year survival between the PDAC patient group with the lowest (N = 12) and highest (N = 12) levels of MDCs (≤0.25% and >0.25%) and PDCs (≤0.14% and >0.14%), respectively. (D) Percent DCs of total PBMCs in PDAC patients with more than two years survival (long-time survivor) and in patients with ampullary carcinoma (AC) and endocrine carcinoma (EC) patients. Statistically significant differences between individuals with pancreatic disease and healthy controls are indicated as; * =p < 0.05, *** = p < 0.001.