STSM at Toxalim DR2 INRAE Institute Toulouse - Report from Martina Stampar

During my Short-Term Scientific Mission (STSM) at the Toxalim DR2 INRAE Institute in Toulouse, France, I worked on non-genotoxic carcinogens (NGTxCs)—chemicals that induce cancer without directly damaging DNA. In addition to advancing my theoretical understanding of these substances, I gained practical expertise in developing and analyzing advanced in vitro 3D co-culture models. Throughout this project, I worked under the mentorship of Dr. Marc Audebert. My primary focus during the STSM was to establish and characterize a 3D co-culture system using three different liver cell lines: HepaRG (human liver cells), h-TERT-HSC (hepatic stellate cells), and TPH-1 (monocytic cells).

The aim was to create a more complex, physiologically relevant model to study NGTxC-induced toxicity. Using magnetic beads (NanoShuttle-Grainer), I integrated these three cell types. The magnetic beads facilitate cell aggregation, promoting the formation of spheroids that more closely mimic the structural and functional complexity of human liver tissue. These beads are biocompatible, composed of gold, iron oxide, and Poly-L-Lysine, and they do not affect cell proliferation, viability, metabolism, oxidative stress, or the phenotype. Furthermore, they do not interfere with experimental techniques such as immunohistological labeling, fluorescence detection, HR microscopy, Western Blot, or PCR/qPCR.

Once the multi-cellular co-culture system was established, I characterized it in terms of cell viability, differentiation, and interactions, ensuring it was suitable for toxicological testing. The INRAE institute is a global leader in developing systems that replicate human physiology, particularly in the fields of toxicology and biomedical research. Their expertise in tissue engineering and in vitro technologies proved invaluable throughout this process.

After learning how to maintain the co-culture system, I focused on applying confocal microscopy to analyze fixed 2D and 3D cell samples. I first learned how to fix and stain both monolayer cells and multicellular spheroids specifically for confocal imaging. Using advanced staining techniques, I labeled the spheroids with specific markers and fluorochromes to visualize cellular morphology, protein localization, and interactions within the co-culture system. The host institution’s extensive experience with confocal microscopy enabled high-resolution imaging of cellular interactions and structural changes, allowing for detailed analysis of these complex biological systems.

Once the co-culture models were established and characterized, I exposed the spheroids to selected NGTxCs and non-carcinogenic chemicals (as controls) to evaluate their effects. The exposure lasted for 24 and 96 hours (with repeat treatments) using five different compounds: etoposide (genotoxic), colchicine (genotoxic), KBrO3, D-mannitol, and a control. For each treatment, four spheroids were exposed to five concentrations of each compound in 10-fold dilutions. All experiments were performed in triplicate.

To assess the impact of the treatments, I first observed structural changes in the treated spheroids using confocal microscopy. This high-resolution imaging provided insights into cellular morphology, protein localization, and the interactions between cells within the co-culture system. I used a panel of five cell markers—DAPI (for nuclei), pH3 (for mitotic cells), GammaH2A.X (for double strand breaks), HMOX1 (for oxidative stress response), and IL-6 (for inflammatory response)—to characterize cellular responses to the treatments.

Additionally, we collected samples for further transcriptomic analysis, which will take place at the National Institute of Biology (NIB). This analysis will assess changes in gene expression triggered by NGTxCs, identifying the molecular pathways involved in NGTxC-induced carcinogenesis. These insights are critical for understanding the cellular mechanisms driving NGTxC toxicity.

The outcomes of the STSM directly support the objectives of the COST Action IMPROVE by:

• Advancing scientific understanding of NGTxC mechanisms,
• Contributing to the development and validation of alternative testing strategies (NAMs),
• Strengthening international collaboration in predictive toxicology, and
• Promoting knowledge transfer and capacity building between partner institutions.

In conclusion, the STSM successfully met its scientific, technical, and collaborative goals. It contributed meaningfully to the development of New Approach Methodologies (NAMs) and established a strong basis for long-term international cooperation under the framework of the COST Action IMPROVE.