PhD Scientific Days 2025

Poster Session II. - E: Pathological and Oncological Sciences

The Role of Dimensionality and Extracellular Matrix Formation in 3D Bioprinted Human Cell Cultures

Name of the presenter

Moldvai Dorottya

Institute/workplace of the presenter

Semmelweis University, Department of Pathology and Experimental Cancer Research

Authors

Dorottya Moldvai¹, Dániel Sztankovics¹, Péter Sasvári², Fanni Bugyi³, Titanilla Dankó¹, Ildikó Krencz¹, Risa Miyaura¹, Viktória Varga¹, Fatime Szalai¹, Kornélia Baghy¹, Gábor Barna¹, Anna Sebestyén¹

1: Semmelweis University, Department of Pathology and Experimental Cancer Research
2: Semmelweis University, Department of Physiology
3: MTA TTK, HUN-REN Research Centre for Natural Sciences

Text of the abstract

Introduction

Modernization plans of the EMA and the FDA include the partial elimination of animal experiments in drug development by 2030, thereby accelerating the need for alternative preclinical platforms. Different model systems have distinct limitations: animal studies poorly mimic human tissues, while recently applied in vitro assays fail to recapitulate tissue behavior. It was described that cellular metabolic activities and other phenotypic attributes may differ markedly across model systems, necessitating their careful consideration during preclinical development.

Aims and methods

Different model systems were generated from ZR75.1 and T47D human breast carcinoma cell lines: conventional 2D monolayers; 3D cultures (hanging-drop spheroids, gel-embedded spheroids, 3D bioprinted tissue-mimetic structures (TMS)); and xenograft tumors in SCID mice. Differences in metabolic and signaling activities were characterized by proteomic analyses, followed by hierarchical clustering and gene set enrichment analyses. In situ protein expression patterns were examined via IHC and WESTM Simple. Cell cycle phase distributions were determined by flow cytometry using propidium iodide staining. Sensitivities to the metabolic inhibitors (rapamycin, Atpenin-A5, ATR-101, 3-BP, BPTES) were assessed.

Results

Distinct proteomic signatures were revealed for each culture condition, enabling clear discrimination even in the absence of treatment. The 3D TMSs were found to recapitulate the cell cycle profile observed in xenograft tumors, including a pronounced G₁-phase accumulation. Tissue-like cell–cell and cell–extracellular matrix interactions were found exclusively in the 3D TMSs. Significantly reduced sensitivity to the tested metabolic inhibitors was exhibited by the 3D TMSs relative to 2D monolayers, which was attributed to altered metabolic adaptability and reduced mTOR signaling within the 3D microenvironment.

Conclusions

These observations underscore that culture dimensionality and extracellular context fundamentally influence tumor cell metabolism and intracellular signaling. The effects on drug responsiveness and resistance mechanisms highlight the necessity of developing preclinical models that closely emulate in situ tumor behavior.

Funding: 2024-2.1.1-EKÖP-2024-00004, EFOP-3.6.3-VEKOP-16-2017-00009, TKP2021-EGA-24, NKFI-PD-146373, NKFI-K-142799