Reduction Of Metastasis With Plasma-Treated Liquid In Mice: The Role Of Myleoid Cells In The Tumor Microenvironment

Plasma-treated liquids have been proposed to be effective against tumor growth in vitro and in vivo. Recently, we have shown antitumor effects of DMEM exposed to the plasma of the kINPen MED in a murine, orthotopic pancreatic cancer model of diffuse peritoneal tumor metastasis [1]. Mice in the treatment group showed less metastasis formation, slower metastasis growth, increased apoptosis in tumor nodes, and better overall survival. At the same time, treatment was safe and did not affect other organs. In this study, we followed up on cellular and molecular events in the tumor microenvironment of peritoneal cancer lesions of treated compared to control mice. Special emphasize was given to myeloid cells. Tumor nodes were sectioned onto microscopy slides and stained with fluorescently labeled antibodies targeting subsets of myeloid cells. Congruent to the zone of apoptotic cell death, we identified a strong and significant influx of murine macrophages. As apoptosis was only on the tumor side exposed to the peritoneal cavity, macrophages likely influxed from this site. We next sought to identify their subtype with regard to the polarization paradigm. Almost all macrophages stained positive for CD206, a marker associated with M2 macrophages. Macrophages positive for inducible nitric oxide synthase (iNOS), a prominent marker for M1 macrophages, were nearly absent. Staining was confirmed with mouse tissues containing the respective population. This finding suggested that tumor growth was halted despite presence of M2 macrophages, which are thought to support tumor growth in general when becoming tumor-associated macrophages. Next, we stained for granulocytes influx. While we detected fewer granulocytes in tissues, we found indices for extracellular trap formation within tumor nodes, which are here linked to improved anticancer responses. Finally, we co-cultured murine PDA6606 pancreatic cancer cells with murine RAW-macrophages in vitro. Cancer cells strongly polarized macrophages to M2 but plasma treatment halted this to some extent. Plasma also increased migratory activity of macrophages but only when co-cultured with pancreatic cancer cells. Together with a rather inflammatory cytokine profile and cell surface marker expression with properties of M2 as well as M1, we propose a mixed response of murine RAW-macrophages in the tumor microenvironment upon plasma treatment. Download high-res image (365KB) Download full-size image Fig. 1: Tumor nodes were collected after 21 days with daily injection of NTP or medium (control), respectively and stained for F4/80 (A). The number of macrophages was significantly enhanced in treated tumors (B).