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

Hypoxia-activated endothelial cells drive stemness and vasculogenic mimicry in NSCLC via a HIF-1α/ITPR3 axis.

Hypoxia-activated endothelial cells drive NSCLC aggressiveness and VM through a paracrine axis involving ITPR3-mediated ER calcium activation, and this HIF-1α/ITPR3 axis represents a potential therapeutic target for disrupting the tumor-vascular niche in NSCLC.

Cellular adhesion-dependent 3D morphogenesis indicating brain tumor aggressiveness and chemosensitivity in spherical cavity culture

Malignancies of the nervous system remain a critical challenge in oncology, contributing substantially to cancer-related mortality in children and adults. Their aggressive behavior is influenced by adhesion heterogeneity of the tumor cells, as distinct patterns of cell-to-cell and cell-to-matrix interactions shape tumor organization, invasion, and resistance to conventional treatments. Here, we investigate tumor adhesion dynamics in a 3D spherical cavity culture platform that enables the rapid and high-throughput formation of uniform spheroids for parallel morphogenesis, adhesion profiling, and drug screening. The morphogenetic patterns of human neuroblastoma and patient-derived glioblastoma observed in 3D cavity culture closely correlated with tumor aggressiveness, ranging from adhesive layers in benign tumor subtype to compact spheroids in malignant cells and irregular aggregates in highly invasive, cancer stem-like counterparts. These morphologies corresponded to adhesion profiles: aggressive cells displayed elevated N-cadherin with variable integrin, while compact spheroids maintained a balance of both molecules. Blocking N-cadherin with ADH-1 disrupted spheroid integrity, and combining ADH-1 with DOX yielded synergistic cytotoxic effects in malignant phenotypes, highlighting N-cadherin upregulation in tumor cells with higher aggressiveness in 3D culture. Overall, our dynamic 3D spherical cavity culture enables reproducible 3D adhesion phenotyping, providing a simple method for malignancy assessment and supporting adhesion-targeted therapeutic strategies for personalized medicine.

Micro-vascularized Multicellular Pancreatic Ductal Adenocarcinoma (PDAC) spheroid: A novel 3D PDAC Research Model.

AIM To establish a multicellular three-dimensional (3D) pancreatic ductal adenocarcinoma (PDAC) spheroid model in a pancreas-derived extracellular matrix environment and determine how cancer-associated fibroblasts (CAFs) and endothelial CD93 affect spheroid architecture and endothelial organization. METHODS Porcine pancreatic decellularized extracellular matrix (dECM) was processed into a hydrogel and characterized. PANC-1 cells and mCherry-labelled human umbilical vein endothelial cells (HUVECs) were embedded with or without patient-derived CAFs. Microscopy, immunofluorescence staining, and image analysis were used to evaluate spheroid formation, cellular composition, HUVEC distribution, CD31/CD44 double-positive staining, and matrix-remodeling-associated proteins. CD93-overexpressing HUVECs were used to examine the effects of endothelial CD93. RESULTS The dECM hydrogel retained major matrix components and supported PDAC spheroid formation. HUVECs exhibited elongated and interconnected network-like distributions around and within PANC-1 aggregates. Compared with PANC-1/HUVEC co-cultures, CAF-containing tri-cultures formed more spheroids and showed greater mean and total spheroid volumes, higher cell density, and increased estimated PANC-1 and HUVEC abundance. The CD31/CD44 double-positive signal was 1.46-fold higher in tri-cultures. Endothelial CD93 overexpression increased spheroid number and cell density and produced a more intermixed cellular distribution, but reduced the number of large spheroids, mean spheroid volume, total spheroid burden, estimated PANC-1 abundance, and MMP2 and MMP9 immunofluorescence signals. The CD31/CD44 double-positive signal increased by 1.3-fold without a significant change in estimated HUVEC abundance. CONCLUSION This model enables analysis of tumor-endothelial-stromal interactions in a pancreas-derived dECM environment. CAFs and endothelial CD93 differentially altered spheroid architecture and the CD44-associated endothelial phenotype. The formation of continuous and functional microvascular structures remains to be established.

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

Angiogenesis inhibition in high grade glioma

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.