Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate.

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

Cancer-associated fibroblasts mediate cancer progression and remodel the tumouroid stroma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are highly differentiated and heterogeneous cancer-stromal cells that promote tumour growth, angiogenesis and matrix remodelling. METHODS: We utilised an adapted version of a previously developed 3D in vitro model of colorectal cancer, composed of a cancer mass and the surrounding stromal compartment. We compared cancer invasion with an acellular stromal surround, a "healthy" or normal cellular stroma and a cancerous stroma. For the cancerous stroma, we incorporated six patient-derived CAF samples to study their differential effects on cancer growth, vascular network formation and remodelling. RESULTS: CAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. These processes correlated with the upregulation of hepatocyte growth factor (HGF), metallopeptidase inhibitor 1 (TIMP1) and fibulin-5 (FBLN5). Vascular remodelling of previously formed endothelial structures occurred through the disruption of complex networks, and was associated with the upregulation of vascular endothelial growth factor (VEGFA) and downregulation in vascular endothelial cadherin (VE-Cadherin). CONCLUSIONS: These results support, within a biomimetic 3D, in vitro framework, the direct role of CAFs in promoting cancer invasion, and their key function in driving vasculogenesis and angiogenesis.

Cancer invasion regulates vascular complexity in a three-dimensional biomimetic model.

INTRODUCTION: There is a growing appreciation for including a complex, vascularised stroma in three-dimensional (3D) tumour models to recapitulate the native tumour microenvironment in situ. METHODS: Using a compartmentalised, biomimetic, 3D cancer model, comprising a central cancer mass surrounded by a vascularised stroma, we have tested the invasive capability of colorectal cancer cells. RESULTS: We show histological analysis of dense collagen I/laminin scaffolds, forming necrotic cores with cellular debris. Furthermore, cancer cells within this 3D matrix form spheroids, which is corroborated with high EpCAM expression. We validate the invasive growth of cancer cells into the stroma through quantitative image analysis and upregulation of known invasive gene markers, including metastasis associated in colon cancer 1, matrix metalloproteinase 7 and heparinase. Tumouroids containing highly invasive HCT116 cancer masses form less complex and less branched vascular networks, recapitulating 'leaky' vasculature associated with highly metastatic cancers. Angiogenic factors regulating this were vascular endothelial growth factor A and hepatocyte growth factor active protein. Where vascular networks were formed with less invasive cancer masses (HT29), higher expression of vascular endothelial cadherin active protein resulted in more complex and branched networks. To eliminate the cell-cell interaction between the cancer mass and stroma, we developed a three-compartment model containing an acellular ring to test the chemoattractant pull from the cancer mass. This resulted in migration of endothelial networks through the acellular ring accompanied by alignment of vascular networks at the cancer/stroma boundary. DISCUSSION: This work interrogates to the gene and protein level how cancer cells influence the development of a complex stroma, which shows to be directly influenced by the invasive capability of the cancer.

Modeling Patient-Derived Glioblastoma with Cerebral Organoids.

The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a "normal" human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

Engineering a vascularised 3D in vitro model of cancer progression.

The hallmark of tumours is the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues, which include hypoxia, also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. We engineered compartmentalized biomimetic colorectal tumouroids with stromal surrounds that comprised a range of ECM densities, composition and stromal cell populations. Recapitulating tissue ECM composition and stromal cell composition enhanced cancer cell invasion. Manipulation of ECM density was associated with an altered migration pattern from glandular buds (cellular aggregates) to epithelial cell sheets. Laminin appeared to be a critical component in regulating endothelial cell morphology and vascular network formation. Interestingly, the disruption of vascular networks by cancer cells was driven by changes in expression of several anti-angiogenic genes. Cancer cells cultured in our biomimetic tumouroids exhibited intratumoural heterogeneity that was associated with increased tumour invasion into the stroma. These findings demonstrate that our 3D in vitro tumour model exhibits biomimetic attributes that may permit their use in studying microenvironment clues of tumour progression and angiogenesis.

Efficacy of DOPE/DC-cholesterol liposomes and GCPQ micelles as AZD6244 nanocarriers in a 3D colorectal cancer in vitro model.

AIM: In this work, we use cationic organic nanocarriers as chemotherapy delivery platforms and test them in a colorectal cancer 3D in vitro model. MATERIALS & METHODS: We used 3beta-(N-[N',N'-dimethylaminoethane]carbamoyl])cholesterol (DC-chol) and dioleoylphosphatidylethanolamine (DOPE) liposomes and N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) micelles, to deliver AZD6244, a MEK inhibitor, to HCT116 cells cultured as monolayers and in 3D in vitro cancer models (tumoroids). RESULTS: Nanoparticle-mediated drug delivery was superior to the free drug in monolayer experiments and despite their therapeutic effect being hindered by poor diffusion through the cancer mass, GCPQ micelles were also superior in tumoroids. CONCLUSION: These results support the role of nanoparticles in improving drug delivery and highlight the need to include 3D cancer models in early phases of drug development.

Initial validation of equilibrium contrast imaging for extracellular volume quantification using a three-dimensional engineered tissue model.

BACKGROUND: To test the principles underpinning equilibrium contrast imaging estimation of tissue extracellular volume (ECV) fraction, using a three-dimensional (3D) engineered tissue model with known cellular and extracellular volumes. METHODS: Six 3D tissue models (tumoroids) consisting of cell cultures within a collagen containing hydrogel were constructed after culture centrifugation and direct measurement of the cell component volume. Measured tumoroid ECV ranged from 0.89 to 1. ECV was calculated after measuring the T1 relaxation time at 3 Tesla using inversion recovery relaxometry (TI 100-1500 ms) within the tumoroids and surrounding medium before and 375 min after spiking the medium with Gadolinium (to achieve a concentration of 1.4 mM/L). Linear regression model prediction of directly measured ECV (ECVm ) by EQ-MRI measured ECV (ECVeq ); and Bland-Altman agreement between measures was assessed. RESULTS: The fractional cellular volume measured by EQ-MRI (ECVeq ) within the tumoroids ranged from 0.821 to 0.963. ECVeq was a good predictor of ECVm (R2 = 0.77, P = 0.02). The regression line Y-axis intercept (when X = 0) was 0.045 ± 0.019 with a slope of 1.28 ± 0.35. Bland-Altman comparison demonstrated 95% limits of agreement between -0.002 and 0.114 with a bias (SD) of 0.056 (0.03). CONCLUSION: This study supports the principles of ECV estimation using equilibrium contrast MRI, but future development of this model may allow validation over a wider, more physiological ECV range and a greater understanding of the effect of tissue extracellular protein burden on ECV.

The efficacy of cetuximab in a tissue-engineered three-dimensional in vitro model of colorectal cancer.

The preclinical development process of chemotherapeutic drugs is often carried out in two-dimensional monolayer cultures. However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics. With this objective in mind, we have developed and established a physiologically relevant three-dimensional in vitro model of colorectal cancer based on the removal of interstitial fluid from collagen type I hydrogels. We employed the RAFT™ (Real Architecture For 3D Tissue) system for producing three-dimensional cultures to create a controlled reproducible, multiwell testing platform. Using the HT29 and HCT116 cell lines to model epidermal growth factor receptor expressing colorectal cancers, we characterized three-dimensional cell growth and morphology in addition to the anti-proliferative effects of the anti-epidermal growth factor receptor chemotherapeutic agent cetuximab in comparison to two-dimensional monolayer cultures. Cells proliferated well for 14 days in three-dimensional culture and formed well-defined cellular aggregates within the concentrated collagen matrix. Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01). Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status. In summary, these results confirm the use of a three-dimensional in vitro cancer model as a suitable drug-screening platform for in vitro pharmacological testing.

A 3D in vitro cancer model as a platform for nanoparticle uptake and imaging investigations.

In order to maximize the potential of nanoparticles (NPs) in cancer imaging and therapy, their mechanisms of interaction with host tissue need to be fully understood. NP uptake is known to be dramatically influenced by the tumor microenvironment, and an imaging platform that could replicate in vivo cellular conditions would make big strides in NP uptake studies. Here, a novel NP uptake platform consisting of a tissue-engineered 3D in vitro cancer model (tumoroid), which mimics the microarchitecture of a solid cancer mass and stroma, is presented. As the tumoroid exhibits fundamental characteristics of solid cancer tissue and its cellular and biochemical parameters are controllable, it provides a real alternative to animal models. Furthermore, an X-ray fluorescence imaging system is developed to demonstrate 3D imaging of GNPs and to determine uptake efficiency within the tumoroid. This platform has implications for optimizing the targeted delivery of NPs to cells to benefit cancer diagnostics and therapy.