Organ-on-chip immunostaining method for three-dimensional identification and study of immune cells responding to drug-treated tumor cells.

Epigenetic deregulation is implied in cancer initiation and resistance to antitumor drugs. In melanoma, aberrant DNA hypermethylation is frequently observed, resulting in the silencing of several genes involved in cell cycle regulation, apoptosis, tumor growth and drug resistance. DNA hypomethylating agents have been recently evaluated in both preclinical and clinical studies as a strategy to restore tumor suppressor genes and to increase immune recognition by tumors, highlighting their potential in pre-clinical models of melanoma. Advanced microfluidic system for the culture of complex three-dimensional cell, tissue and organ models have proven utility for oncoimmunology studies and drug testing. Here we present a protocol employing ad hoc fabricated microfluidic devices to reproduce a three-dimensional (3D) tumor microenvironment (TME) to study two aspects of the crosstalk between immune and cancerous cells under the effect of Decitabine (DAC), a DNA methyl transferase inhibitor (DNMTi). First, we evaluated the preferential migration of immune cells towards treated and non-treated melanoma cells inside the chip. Next, we identified a specific subpopulation of migrated immune cells, with an on-chip immunostaining protocol resulting in the acquisition and evaluation of 3D images on a Laser-Scanning Confocal Microscopy (LSCM) station for in-depth characterization of tumor-immune interactions. This protocol may find broad application for pre-clinical drug testing in cancer studies.