Conclusion
In PDAC, PMN infiltrates are associated with alterations of the tumor microarchitecture. As a causal relationship, we propose a reprogramming of stellate cells by PMN-derived azurocidin towards a phenotype, which affects the microarchitecture of the tumor.
Methods
DW-MRI was performed in 33 PDAC patients prior to surgery. In parallel, tissue specimen were examined histologically for growth pattern, azurocidin-positive PMN infiltrates, and the presence of alpha-smooth muscle actin (α-SMA) and metalloproteinase 9 (MMP9)-positive myofibroblastic cells. For confirmation of the histological findings, a tissue microarray of a second cohort of patients (n=109) was prepared and examined similarly. For in vitro studies, the pancreatic stellate cell line RLT was co-cultivated either with isolated PMN, PMN-lysates, or recombinant azurocidin and characterized by Western blot, flow cytometry, and proteome profiler arrays.
Results
Tumors with high PMN density showed restricted water diffusion in DW-MRI and histologic apparent alterations of the tumor microarchitecture (microglandular, micropapillary, or overall poorly differentiated growth pattern) as opposed to tumors with scattered PMN. Areas with altered growth pattern lacked α-SMA-positive myofibroblastic cells. Tissue microarrays confirmed a close association of high PMN density with alterations of the tumor microarchitecture and revealed a significant association of high PMN density with poor histologic grade of differentiation (G3). In vitro experiments provided evidence for direct effects of PMN on stellate cells, where a change to a spindle shaped cell morphology in response to PMN and to PMN-derived azurocidin was seen. Azurocidin incorporated into stellate cells, where it associated with F-actin. Down-regulation of α-SMA was seen within hours, as was activation of the p38-cofilin axis, up-regulation of MMP9, and acquisition of intracellular lipid droplets, which together indicate a phenotype switch of the stellate cells.