A 3D-tumoroid model is established and Apelin (APLN), the ligand for APLNR, is identified as a basally upregulated angiogenic factor in NF2-deficient meningiomas, suggesting that APLN expression is regulated, at least in part, by mTORC1.
Abstract
NF2-associated meningiomas and schwannomas are vascular tumors, and while VEGF inhibition with bevacizumab has benefited some NF2-related schwannomas, most NF2-associated meningiomas remain nonresponsive.
Leveraging our transcriptomic data, we performed Gene Ontology (GO) analysis comparing NF2-deficient meningioma cells with NF2-expressing arachnoid cells (ACs). We then established a 3D in vitro angiogenesis model by co-culturing NF2-null meningioma cells with human umbilical vein endothelial cells (HUVECs). Endothelial sprouting was assessed by CD31/PECAM immunostaining. Effects of third-generation mTORC1-selective inhibitor RMC-6272 as well as APLN knock-out using CRISPR-Cas9 gene editing were also examined.
GO analysis identified vascular development among the top significantly upregulated pathways in NF2-deficient cells. In 3D co-culture, ECs formed radially sprouting tube-like networks from the spheroid surface, and our data support an angiogenesis phenotype driven by meningioma cells. Given these results along with hyperactivation of mTORC1 upon NF2-deficiency, we examined whether RMC-6272 disrupts meningioma-driven angiogenesis. RMC-6272 potently suppressed EC sprouting. Cross-referencing baseline transcriptomic data we identified Apelin (APLN), the ligand for APLNR, as a basally upregulated angiogenic factor in NF2-deficient meningiomas. qRT-PCR confirmed increased APLN expression in NF2-null immortalized and patient-derived meningioma lines, with reduced expression upon mTORC1 inhibition. Apelin-13 stimulation enhanced sprouting, whereas APLN deletion reduced endothelial sprouting.
Here we establish a 3D-tumoroid model and implicate tumor-derived Apelin as an important contributor to NF2-associated meningioma angiogenesis. Our data also suggest that APLN expression is regulated, at least in part, by mTORC1. T1ogether, these results provide a preclinical platform for investigating angiogenic vulnerabilities beyond VEGF in NF2-deficient meningiomas.
Background The complex interplay between tumor cells and the stromal components of the glioma microenvironment necessitates the development of sophisticated in vitro models capable of modelling key aspects of cellular interactions that occur beyond the limitations of conventional monocultures. Methodology The development and characterization of homo- and heterotypic 3D spheroid models incorporating CCF-STTG1 astrocytes, HMC3 microglia, and U87MG glioma cells was undertaken. The assessment of morphological, molecular, and functional properties was performed via flow cytometry, cytokine arrays, ECM analysis and invasion assays (Matrigel™/gelatin). Results Heterotypic spheroids have been observed to spontaneously self-assemble into a spatially polarized architecture, with microglia and glioma cells segregating into distinct compartments, a pattern suggestive of the cellular topology at the invasive front. The morphological, molecular, and functional properties of the generated 3D models recapitulated several established features associated with in vivo tumors, including growth, invasion, resistance to chemotherapy, and metabolic reprogramming alongside the expression of stemness markers, and key pro-invasive mediators (MMPs, SDF-1α, VEGF). Secretome profiling revealed a marked, non-additive upregulation of chemokines (IP-10, MIP-1α) and the emergence of novel correlations (HGF/SDF-1α, MCP-1/LIF), indicating potential modulation of paracrine networks involved in immune cell trafficking in the heterotypic setting. The initial formation of a rigid ECM matrix appears to be initiated by microglia, while the supply of fibronectin and laminin may be linked to astrocytes exhibiting some features of reactive gliosis, which could help organize invasion pathways. Conclusion These heterotypic 3D spheroid models offer a stroma-enriched, reproducible platform for the analysis of stromal contributions to glioma progression and for exploratory preclinical evaluation of therapeutic strategies.
Y. Nikitina, Alina Kazakova, A.A. Leonteva et al.· Frontiers in Bioengineering...· 0 citations
Objective: Breast cancer (BC) is the most frequently diagnosed cancer in women, with the luminal subtype representing most cases. Luminal tumors depend on the ERα-FOXA1-GATA3 transcriptional network, which is influenced by the tumor microenvironment. Although three-dimensional (3D) culture systems better mimic in vivo conditions than 2D models, luminal breast cancer cells often fail to form stable spheroids. This study aimed to optimize 3D culture conditions and examine how microenvironmental architecture influences the ERα–FOXA1–GATA3 network.Material and Methods: We cultured MCF7 and T47D cells under standard conditions to establish 3D models. We seeded cells on Petri dishes containing media supplemented with different concentrations of methylcellulose (MC) to promote spheroid formation. We monitored morphology microscopically. We analyzed the expression levels of ESR1, FOXA1, and GATA3 in 2D and 3D cultures by RT-qPCR. We evaluated ERα chromatin binding at selected target regions by ChIP-qPCR. We assessed statistical significance using Student's t-test.Results: Optimization of MC-based 3D culture conditions showed that medium viscosity affects both spheroid morphology and transcriptional regulation. Spheroids formed under 10% MC displayed increased expression of ESR1, FOXA1, and GATA3 compared with 2D cultures. CRISPR/Cas9-mediated knockout of ESR1 and FOXA1 disrupted spheroid organization. ChIP-qPCR analysis revealed viscosity-dependent changes in ERα chromatin binding, demonstrating that spheroid compactness influences ERα transcriptional activity. Data represent mean ± SD from three independent experiments.Conclusion: These findings indicate that the physical properties of the microenvironment influence ESR1 expression and ERα activity. Optimization of 3D culture conditions is therefore important for accurately studying ERα signaling in luminal breast cancer models.
Gozde Korkmaz, Elif Kansız· Istanbul University Journal...· 0 citations
INTRODUCTION
Prostate cancer (PCa) is one of the most prevalent malignancies in men and frequently progresses to bone metastasis. Understanding the interactions between PCa cells and the bone microenvironment, as well as their impact on therapeutic response, is therefore of critical clinical relevance.
OBJECTIVES
This study aimed to investigate the effects of the osteogenic microenvironment on prostate cancer cells by constructing three-dimensional (3D) bioprinted in vitro co-culture models, and to evaluate how the osteogenic niche influences tumor malignant phenotypes.
METHODS
This study employed extrusion-based 3D bioprinting (3DP) to construct in vitro co-culture models of the PCa osteogenic microenvironment. Two 3D-PCa models based on LNCaP and PC-3 cells were each co-cultured with osteogenically differentiated adipose-derived stem cells (ADSCs) to generate corresponding osteogenic niche models. Tumor phenotypes and drug responses were evaluated using functional assays, histological and immunofluorescence analyses, molecular profiling, and RNA sequencing.
RESULTS
The 3D bioprinted constructs exhibited structural stability and high reproducibility, providing a 3D growth environment that mimics key aspects of the in vivo tumor niche. The 3D-PCa models showed enhanced drug resistance, invasive potential, and adaptation to androgen-deprivation. Meanwhile, ADSCs exhibited robust osteogenic differentiation within 3D scaffolds. Under co-culture conditions, 3D-PCa cells exhibited enhanced malignant-like phenotypes, including increased proliferation and reduced drug sensitivity. Multi-level analyses further indicated that these phenotypic changes were associated with coordinated activation of epithelial-mesenchymal transition (EMT)-, hypoxia-, and mitogen-activated protein kinase (MAPK)-related signaling programs, accompanied by alterations in drug transporter expression and cell cycle distribution.
CONCLUSION
The 3D co-culture model provides a biomimetic platform for simulating the PCa bone-metastatic microenvironment. This system enables investigation of the effects of the osteogenic niche on tumor behavior and therapeutic responses, and may serve as a useful tool for preclinical drug screening and evaluation of combination treatment strategies for bone-metastatic prostate cancer.
Chengyi Zhong, M. Pang, Hang Sun et al.· Journal of Advanced Research· 0 citations
Vasculogenic mimicry (VM) predicts poor prognosis in non-small cell lung cancer (NSCLC). Hypoxia is a critical tumor microenvironmental regulator, yet its detailed mechanism linking to VM formation remains poorly defined. This study aimed to elucidate the role of hypoxic endothelial cells in promoting NSCLC aggressiveness and VM formation. We integrated clinical data analysis, single-cell RNA sequencing (scRNA-seq), transcriptomic profiling, and multiple in vitro and in vivo functional validations to consolidate our findings. Clinical NSCLC data revealed a positive correlation between endothelial expression of the hypoxia marker carbonic anhydrase IX (CA9) and VM activity. Conditioned medium from hypoxic HUVECs promoted NSCLC cell proliferation, migration, invasion, stemness, and VM network formation in vitro. Endothelial HIF-1α overexpression accelerated tumor growth and VM in vivo. Transcriptomics screening pinpointed the calcium channel gene Inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) as the core downstream effector in this hypoxic endothelial paracrine signaling. Follow-up mechanistic assays revealed that hypoxic endothelial supernatant and the ITPR3 inhibitor 2-aminoethoxydiphenyl borate (2-APB) activated calcium/endoplasmic reticulum (ER) stress signaling to promote malignant phenotypes in NSCLC, and these effects were further validated by ITPR3 knockdown. In conclusion, hypoxia-activated endothelial cells drive NSCLC aggressiveness and VM through a paracrine axis involving ITPR3-mediated ER calcium activation. This HIF-1α/ITPR3 axis represents a potential therapeutic target for disrupting the tumor-vascular niche in NSCLC.
Wenhao Ji, Shizhou Yang, Xiaojing Lai et al.· Biochemical Pharmacology· 0 citations
BACKGROUND
Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and can influence tumor progression and therapeutic response. However, whether functional heterogeneity of CAFs contributes to 5-fluorouracil (5-FU) resistance in gastric cancer remains unclear.
METHODS
Patient-derived organoids (PDOs) and matched CAFs were established from gastric cancer specimens and cocultured using a Transwell noncontact system. The effects of CAFs on PDO growth and chemotherapy response were evaluated. Candidate secreted factors were analyzed by qRT-PCR and ELISA. Gain- and loss-of-function experiments, together with transcriptomic and protein analyses, were performed to investigate the underlying mechanisms. A CAF-containing xenograft model was established to evaluate the therapeutic effects of VEGFA blockade combined with 5-FU treatment in vivo.
RESULTS
CAFs derived from different patients exhibited marked functional heterogeneity in regulating PDO growth and chemotherapy response. A VEGFA-associated functional CAF state significantly promoted PDO proliferation and enhanced resistance to 5-FU. Vascular endothelial growth factor A (VEGFA) was identified as a key secreted factor that was significantly upregulated in these growth-promoting CAFs. Exogenous VEGFA recapitulated the pro-growth and anti-apoptotic effects observed, whereas VEGFA blockade attenuated CAF-mediated chemoprotective effects and enhanced PDO sensitivity to 5-FU. In vivo, bevacizumab improved the response of CAF-containing tumors to 5-FU treatment, supporting the therapeutic relevance of targeting CAF-derived VEGFA. These effects were driven by the activation of the PI3K/AKT-mTOR survival pathway in PDOs.
CONCLUSION
A VEGFA-associated functional CAF state promotes gastric cancer organoid growth and chemoresistance through paracrine signaling, highlighting the CAF-VEGFA axis as a potential therapeutic target.
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
