Reconstructing the glioblastoma microenvironment in heterotypic 3D spheroids: a multicellular model to study tumor-stromal crosstalk

3D co-culture model of tumor spheroid and endothelial cells unveils ccRCC aberrant vasculature and distinct sensitivity to targeted treatments

Beyond the traditional dish: advanced 3D ex vivo culture platforms for tumor modelling.

The tumor microenvironment (TME) and its complex, dynamic interactions play a pivotal role in cancer development, progression and therapy response. However, faithful recapitulation of the diverse cellular and structural components of the TME in vitro remains a major challenge in cancer research. Traditional 2D cancer cell cultures fail to preserve TME interactions and tissue organization that critically impacts tumor behavior, while advanced 3D systems, including organoids, 3D-bioprinted structures and microfluidic platforms capture only selected aspects of TME complexity and host physiology. Recent advances in the culture of primary tumor specimens with minimal disruption to tissue architecture has given rise to a rapidly evolving set of model systems that benefit TME research. These ex vivo cultures (EVCs), as we collectively refer to them here, are established from fresh primary tumor tissue and preserve the native tumor architecture, extracellular matrix composition, immune and stromal compartments and their multilayered crosstalk within a physiologically relevant context. This review outlines the historical evolution of 2D and 3D model systems in oncology, followed by a comprehensive overview of current EVC methodologies. Furthermore, we address their applications in fundamental cancer research, personalized medicine and drug discovery, while highlighting their advances and challenges for the future.

Adaptation of Non-Invasive Cancer Cells to 3D Collagen I Microenvironment Induces Transcriptional Reprogramming Accompanied by a More Complex RNA Landscape

Nowadays, most cancer research still depends on traditional cell culture in Petri dishes or cell culture flasks which do not have the ability to mimic physiological-like conditions in vitro. However, the behavior of cancer cells strongly relies on the interaction with their extracellular microenvironment. Consequently, current advanced approaches focus on three-dimensional (3D) cell culture to overcome such limitations and to enable a better understanding of fundamental processes including cancer development, progression, apoptosis and invasion. However, transcriptional adaptation to and temporal stability within an in vitro 3D microenvironment still appear to be remarkably understudied. In our study, we compared the cellular behavior and whole transcriptome gene expression of three frequently used non-invasive cancer cell lines (HCT-116, A549 and T47D), embedded within a collagen I (Coll I)-based 3D microenvironment to its counterparts grown as simple monolayers in a time-dependent manner. Thereby, changes in morphology and doubling time became apparent between both cultivation systems, and RNA sequencing-based transcriptome-wide analysis revealed a remarkable increase in transcriptional complexity under 3D conditions. In line with the 3D-dependent phenotype, unidirectional shifts for genes involved in cell cycle regulation (e.g., CCNB1, CCNB2), cell–matrix interaction (e.g., ADAM8, ITGA2) and metabolic signaling (e.g., HK2, ENO2) were identified over time, being either activated or repressed. Interestingly, all three cell lines cultured in Coll I matrices displayed a highly distinct RNA content and composition, along with a significantly increased number of expressed protein-coding genes (increase of 3–6%) as well as long non-coding RNAs (increase of 26–48%), suggesting a more multifaceted transcription profile under 3D conditions. Our work clearly highlights that an in vitro 3D Coll I-based cell culture system has an incisive cell-specific impact on the whole transcriptome on a qualitative and quantitative level. This tremendous transcriptional reprogramming implies essential changes in gene regulatory networks and affects phenotypic cancer cell behavior, which should be considered when focusing on downstream applications.

A 3D Tumor Spheroid Platform to Model Fusobacterium nucleatum-Induced Tumorigenesis.

Tumor-Mimetic Microenvironment Primes Cancer Stemness and Drug Resistance through Nucleoskeleton-Mediated Chromatin Remodeling.

The tumor microenvironment (TME) critically regulates cancer progression by providing biochemical and biophysical cues that shape cellular behavior. However, how defined physical microenvironments govern cancer stemness and chemoresistance through mechanotransduction remains poorly understood. Here, we systematically engineered eight tumor-mimetic microenvironments by integrating serum, oxygen, and 3D compacted culture to investigate their effects on A549 non-small cell lung cancer cells. Among all conditions, cells cultured under 3D culture (PM4C) exhibited reduced cellular stiffness, enhanced expression of cancer stemness markers (EpCAM and CD44), and significantly increased resistance to cisplatin in both in vitro and nude mouse xenograft models. Transcriptomic analysis revealed that differentially expressed genes in the PM4C group were predominantly enriched in cell adhesion, mechanotransduction, stemness, and cisplatin resistance pathways. Metabolomic profiling further revealed a substantial accumulation of anaerobic metabolites associated with the maintenance of stemness. Mechanistically, the PM4C microenvironment remodeled matrix production, cell-ECM interactions, and cytoskeletal organization while inducing epigenetic reprogramming (reduced H3K9 acetylation), collectively promoting a stem-like and chemoresistant phenotype. These findings establish a direct mechanistic link between TME and cancer cell stemness, demonstrating that TME can reprogram stemness and drug responsiveness through mechano-epigenetic regulation. This work provides a mechanobiological framework for engineering physiologically relevant tumor organoids and offers new strategies for developing TME-targeted drugs and therapies.

Evaluation of extracellular vesicles released by 3D spheroids and 2D conventional cell culture of the SKOV3 ovarian cancer cell line

Cancer cells communicate with the surrounding tumour microenvironment by secreting extracellular vesicles (EVs), referred to as exosomes, which play a critical role in cancer progression by transferring bioactive cargo including proteins, lipids, mRNAs, miRNAs, and DNA fragments to healthy cells, enabling distant organ colonization and metastasis. SKOV3, a widely used human ovarian adenocarcinoma cell line, is traditionally cultured under anchorage-dependent (2D monolayer) conditions; however, suspension culturemore faithfully mimics the three-dimensional, non-adherent microenvironment encountered in vivo, giving rise to altered cell signalling, spheroid formation, and distinct exosome release profiles. This research compares exosomes secreted by SKOV3 cells under 2D and 3Dconditions, isolated through ultrafiltration and Size Exclusion Chromatography (SEC), and characterised using Scanning Transmission Electron Microscopy (STEM), Dynamic Light Scattering (DLS), Raman spectroscopy, and Flow Cytometry. Multivariate analysis across FastGrow and McCoy’s media identified the principal components governing exosome physical properties, while optimised SEC runs demonstrated how Sepharose 2B and 6B columns yield purer exosomal fractions. Additional investigations included refractive index analysis, storage effects, Surface-Enhanced Raman Scattering (SERS) of Rhodamine 6G and Crystal Violet, BCA protein estimation, and spheroid growth analysis. This work presents an accessible, reproducible isolation protocol to lower the technical barrier across laboratory settings, laying the groundwork for non-invasive exosome-based cancer screening.