N. E. Cieza Rubio1, R. L. Heimark1,2 1University Of Arizona,Department Of Surgery,Tucson, AZ, USA 2The Arizona Cancer Center,Tucson, AZ, USA
Introduction:
Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells. Aim: We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia.
Methods:
Model of Acute Pancreatitis: Pancreatitis was induced using a regimen of 7 hourly intraperitoneal injections of caerulein (50 g/kg) followed by a 48-hour rest period and a second regimen of 7 hourly intraperitoneal injections of caerulein before sacrifice at different time points (1, 12, 24, 48 and 72 hours after last caerulein injection).
Model of MDSCs Depletion: Wild type mice were treated with bi-daily intraperitoneal doses of CXCR2a (SB-265610) (4 mg/kg). Treatment started one day before induction of pancreatitis with caerulein and continued until animal euthanasia.
Positive Selection of gMDSCs: After preparation of a single-cell suspension from pancreas or spleen, CD11b+Gr1+ cells were indirectly magnetically labeled with Anti-Ly-6G-Biotin and Anti-Biotin Microbeads.The magnetically labeled Ly6G+ cells, also known as positively selected cell fraction, were retained within the column and collected once the column is removed from the magnetic field.
Cell surface marker analysis was performed by flow cytometry using the BD FACSCANTO II.
Results:
Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein and an early influx of MDSCs into the pancreas was observed flow cytometry and immunocytochemistry. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CCR2 antagonist (SB265610) in wild type mice the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation.
Conclusion:
MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.