De Ninno, A. | bioRxiv Science

bioRxiv2024

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 DNMT 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.

cancer biology↗

bioRxiv2022

Rapid assessment of susceptibility of bacteria and erythrocytes to antimicrobial peptides by single-cell impedance cytometry

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells, in a rapid manner and with single-cell resolution. We show that the electrical signatures of Bacillus megaterium (B. megaterium) cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in presence of both cell types.

biophysics↗