2250P - Exploring tumor microenvironment in lung cancer through patient-derived 3D models

Background

Tumor Microenvironment (TME) drives tumor growth, invasion, and metastasis. Studying its characteristics requires preclinical models to understand non-small cell lung cancer (NSCLC). PDTOs could help to explore tumor heterogeneity and mimic the organ's structure and functions to study NSCLC TME. Specific goals: (i) test and optimize different extracellular matrices for the development of NSCLC PDTO, (ii) develop 3D co-cultures of patient-derived lung tumorspheres and cancer-associated fibroblasts (CAFs) to investigate their interaction.

Methods

Long-term NSCLC primary cultures were established as PDTOs and tumorspheres. (i) 6 matrices were tested combining PEG hydrogel with heparin with/without RGD, and type I collagen (murine). Matrigel served as positive control. Cell viability was assessed with Calcein/PI and Cell Titer Glo 3D assays. Confocal microscopy evaluated PDTO size and matrix complexity. (ii) Co-culture methods between tumorspheres (SPH) and immortalized 154hTERT CAF cell line in 1:3 ratio for 48 hours were evaluated. Confocal microscopy and flow cytometry analyzed and distinguished populations with CD90 staining and Cell Trackers (Deep Red and Green).

Results

(i): All matrices supported organoid growth. PEG matrices showed optimal cell viability. Collagen matrices had the highest organoid count via confocal microscopy. (ii) SPH in non-adherent conditions, co-cultured with adhered monolayer CAFs, led to undesired SPH differentiation. A complete suspension culture achieved the expected invasion of tumorspheres with CAFs. CD90 was ineffective as a marker due to some CD90+ SPH. Varying concentrations of Cell Trackers stained CAFs, and a 0.25 μM concentration of Cell Tracker Green yielded the best CAF-SPH separation.

Conclusions

Our results showed that viable organoids can grow in the matrices tested. We have optimized conditions for the co-culture of tumorspheres and CAFs in 3D. Next, we will test organoid recovery techniques from ECMs to perform further studies and evaluate potential molecular mediators involved in SPH-CAF intercommunication as potential biomarkers to translate into the clinical practice. Support: CIBERONC (CB16/12/00350); AICO/2021/333 and CIACIF/2021/398 (GVA-FSE; GRISOLIAP/2021/030 (GVA-AMACMA).

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

FIHGUV, CIPF and CIBERONC.

Funding

CIBERONC (CB16/12/00350); AICO/2021/333 and CIACIF/2021/398 (GVA-FSE; GRISOLIAP/2021/030 (GVA-AMACMA).

Disclosure

All authors have declared no conflicts of interest.