Optimization of Methylcellulose-Based 3D Culture Reveals Microenvironment-Dependent Regulation of ERα Activity in Luminal Breast Cancer Cells

Bidirectional phenotypic transition in macrophages influences triple-negative breast cancer cell immune evasive patterns and response to chemotherapies in a co-culture spheroid model

Tumor-associated macrophage (TAM) infiltration is a critical characteristic of triple-negative breast cancer (TNBC) related to drug resistance and poor prognosis. Integrating macrophages into TNBC spheroids is crucial to improve the accuracy of 3D in vitro models that mimic the complexity of the tumor microenvironment (TME) and assess treatment response. However, this remains challenging since the reciprocal effects of these two cell types on each other are not fully understood. In this study, we used the TNBC cell line, MDA-MB-231, and polarized M1-like or M2-like macrophages derived from THP-1 monocytes to establish 3D co-culture spheroids to examine bidirectional interactions between these cells and responses to chemotherapy. Drug efficacy, epithelial-mesenchymal transition (EMT) in cancer cells, macrophage phenotypes, and RNA sequencing, including pathway enrichment analysis, were performed in 3D spheroids. CIBERSORTx deconvolution of RNA sequencing results facilitated the separation of cell types within mixtures to estimate their corresponding cell fractions. We observed that M2 macrophages increased the viability of MDA-MB-231 cells in 3D spheroids, while both M1 and M2 macrophages increased the chemosensitivity of 3D spheroids to doxorubicin and paclitaxel. Interestingly, instead of maintaining their phenotypes, both M1 and M2 macrophages lost some polarization and formed a mixed M1-M2 phenotype when co-cultured with MDA-MB-231 cells in 3D spheroids, a phenomenon further supported by RNA-seq deconvolution analysis. However, the fraction of M1-like macrophages shifting to M2-like was much lower than the fraction of M2-like macrophages shifting to M1-like in the 3D co-cultures. Compared with 2D cultures, an expected mesenchymal transition, numerous differentially expressed genes (DEGs) and various pathways, including both tumor-promoting and tumor-suppressing genes, were observed in 3D spheroid MDA-MB-231 cells. However, both M1- and M2-like macrophages induced only partial EMT phenotype changes of cancer cells in co-cultures. Furthermore, a coexistence of pro-inflammatory and anti-inflammatory DEGs was observed in both M1 and M2-like co-cultured cancer spheroids. In conclusion, our findings present an effective 3D co-culture system of breast cancer cells and integrated macrophages for studying dynamic cellular phenotype changes and reciprocal interactions in a heterogeneous environment to mimic aspects of the TME and enhance the accuracy of preclinical in vitro treatment response studies.

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.

Spheroid cultures reveal cancer stem cell enrichment and distinct chemotherapeutic response in triple-negative breast cancer

Three-dimensional (3D) cancer spheroids mimic key morphological and biological features of solid tumors, providing a valuable model for evaluating drug responses. Here, we investigated whether intrinsic cancer stem cell composition and tumor subtype determine spheroid formation capacity, transcriptional profiling, and therapeutic response in breast cancer cell lines. Using the liquid overlay technique, we optimized conditions for reproducible spheroid generation using 10 human cancer cell lines derived from breast cancer, glioblastoma, and malignant melanoma. Spheroid morphology and growth dynamics were assessed over a 20-day period, while breast cancer stem cell abundance was evaluated in 2D monolayer cultures and 3D spheroids using immunohistochemistry and flow cytometry. RNA sequencing (RNA-seq) transcriptomic profiling and drug responses to bortezomib+nedaplatin following 24- and 72-hour exposure were compared between both culture systems. Optimized conditions yielded compact spheroids (5/10 cell lines), loose aggregates (3/10), and no spheroid formation (2/10). Compact spheroids (mean ± SEM, 576 ± 18 μm) and those derived from triple-negative breast cancers (TNBCs; 550 ± 19 μm) were significantly smaller than loose aggregates (822 ± 36 μm) and non-TNBC-derived spheroids (934 ± 41 μm), respectively. RNA-seq demonstrated that transcriptional variation was primarily driven by intrinsic differences between cell lines rather than culture conditions. However, spheroid-forming cells induced distinct gene expression patterns enriched in extracellular matrix remodeling, developmental pathways, and stemness-related genes. In total, 1478 differentially expressed genes were associated with spheroid formation. TENM4 (Teneurin Transmembrane Protein 4) was the only consistently deregulated gene in both spheroid-forming breast cancer models, showing up to 4.9-fold and 2.6-fold higher expression in HCC1806 and BT-474 spheroids, respectively. Consistent with the transcriptomic findings, immunohistochemical analysis demonstrated increased TENM4 protein expression, predominantly localized to the spheroid cores. Drug screening further demonstrated increased chemoresistance in 3D spheroids, with up to 7.5-fold higher viability in TNBC spheroids following bortezomib+nedaplatin treatment. Taken together, these findings highlight molecular and cellular determinants of spheroid formation in breast cancer cell lines, including stemness-associated features and upregulation of TENM4. Our results further demonstrate that intrinsic tumor subtype shapes spheroid morphology, transcriptional profiles, and drug response, providing novel insight into the mechanisms underlying 3D growth and chemoresistance that may help inform future therapeutic strategies.

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.

A 3D-bioprinted osteogenic niche model reveals bone microenvironment-driven malignant phenotypes in prostate cancer.

The 3D co-culture model provides a biomimetic platform for simulating the PCa bone-metastatic microenvironment and may serve as a useful tool for preclinical drug screening and evaluation of combination treatment strategies for bone-metastatic prostate cancer.

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