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Updated: Sep 14, 2025
Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments
Published on: April 30, 2021
Organ-on-chip immunostaining method for three-dimensional identification and study of immune cells responding to
Francesco Noto1, Adele De Ninno2, Mario Falchi3
1Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy; Department of Translational Medical Sciences, University of Naples "Federico II", Naples, Italy.
Abstract:
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.
Insights
This study introduces a microfluidic system to model the tumor microenvironment, investigating how Decitabine (DNA methyl transferase inhibitor) affects melanoma cells and immune cell interactions for improved cancer drug testing.
Area of Science:
- Oncoimmunology
- Cancer Biology
- Microfluidics
Background:
- Epigenetic alterations, particularly DNA hypermethylation, are crucial in melanoma development and drug resistance.
- DNA hypomethylating agents show promise in restoring tumor suppressor genes and enhancing anti-tumor immunity.
- Microfluidic systems offer advanced platforms for studying complex 3D cell and tissue models in cancer research.
Purpose of the Study:
- To develop and utilize a microfluidic device to simulate a 3D tumor microenvironment (TME).
- To investigate the effects of Decitabine (DAC), a DNA methyl transferase inhibitor (DNMTi), on melanoma cells and immune cell interactions within the TME.
- To assess immune cell migration towards DAC-treated and untreated melanoma cells and characterize specific immune cell subpopulations.
Main Methods:
- Fabrication of custom microfluidic devices to create a 3D TME.
- Culturing melanoma cells and immune cells within the microfluidic system.
- On-chip immunostaining and Laser-Scanning Confocal Microscopy (LSCM) for 3D imaging and analysis of tumor-immune interactions.
- Evaluation of immune cell migration patterns in response to DAC treatment.
Main Results:
- The study successfully reproduced a 3D TME using microfluidic devices.
- Decitabine (DAC) treatment influenced the crosstalk between melanoma and immune cells.
- Specific immune cell subpopulations were identified and characterized, revealing insights into tumor-immune interactions.
- Preferential migration of immune cells towards treated melanoma cells was observed.
Conclusions:
- The developed microfluidic protocol provides a valuable tool for studying tumor-immune cell dynamics under drug treatment.
- This approach facilitates in-depth characterization of cellular interactions within a simulated TME.
- The protocol holds significant potential for pre-clinical drug testing and development in melanoma and other cancers.
