Incucyte device

First, the proliferative effect of plasma of 3 HCC and 6 CRC patients 90 min post liver RFA was investigated on human HCC cell lines HepG2, Huh7 and Hep3B. A multiplex ELISA protein analysis identified potential plasma cytokines and growth factors upregulated by RFA. To confirm specific factors proliferative effects, recombinant proteins of the upregulated proteins observed post-RFA were incubated with human HCC cell lines, as measured by the IncuCyte device (Sartorius, USA). Next, primary mouse hepatocytes and immortalized human hepatocytes^{18 (link)} were subjected to moderate hyperthermia (43–45 °C) to mimic the thermal stress induced during ablation in normal parenchymal cells^{11 (link)}. Medium of both human and mouse heated hepatocytes (conditional medium) was added to the incubation medium of multiple tumor cell lines including HCC, CRC, and breast adenocarcinoma and tumor cell proliferation was monitored^{11 (link)}. The heated hepatocytes medium was then analyzed by protein array and multiplex ELISA to detect factors secreted post heating. Finally, given increased secretion of fibroblast growth factors including FGF2, FGF7, and FGFR3 post-heating in-vitro and in plasma of human and mice post-RFA treatment, we used an FGFR inhibitor to block the proliferation observed post-RFA in-vivo and post heating in-vitro.

The different cell lines were seeded in 96-well plates (3–5 × 103) (Greiner, Frickenhausen, Germany) in fresh media. After 12 h, cells were treated with the relevant concentration of the different drugs to obtain the IC50 on proliferation and cytotoxicity: metformin (2.5, 5, 10, 15, 20 and 25 mM), phenformin (50, 70, 100, 250, 500 and 1000 μM), VLX600 (0.1, 0.25, 0.5, 0.75, 1 and 2.5 μM) and rotenone (0.5, 1, 2.5, 5 μM) in quadruplicates. The wells were then imaged at 2h intervals (three images per well) for 48 h using an IncuCyte device (Sartorius, Göttingen, Germany). Proliferation was measured by percent confluency in phase contrast, and cytotoxicity was assessed using Cytotox Green (Sartorius, Göttingen, germany) following the protocol recommended by the manufacturer. Cytotox Green is a membrane-impermeant dye that fluoresces upon DNA binding and, therefore, stains dead cells. The IC50 on proliferation was determined by non-linear regression with GraphPad (San Diego, CA, USA) Prism 5–8. For ROS detection, cells were treated with 100 μM 2′7′-Dichlorofluorescin diacetate (DCFHDA) for 15 min. After treatment, cells were washed with PBS and DCFHDA fluorescence was determined as above.

Cells were cultured in 48-well plates and treated as indicated. The cells were then fixed with methanol/acetone (1:1) at −20 °C for 5 min, washed three times with PBS, and stained with propidium iodide (PI; final concentration, 1 μg/mL) at room temperature for 10 min. The plates were imaged on an IncuCyte device (Essen Bioscience, Ann Arbor, MI, USA) and analyzed using the IncuCyte ZOOM 2016B software. The processing definition of the IncuCyte program was set to recognize attached (live) cells by their red-stained nuclei. The percentage of live cells was normalized to that of untreated control cells (100%).