Tri-culture Model Research Articles

Abstract 696: Spatially patterned, 3D in vitro models of cancer metastasis to bone for elucidating key drivers of metastasis and drug discovery

Abstract Bone is one of the most common sites of cancer metastasis, affecting more than 70% of patients with advanced breast cancer and prostate cancer. Despite its prevalence, there are still no effective treatments for bone metastasis, leading to very poor survival rates and the mechanism by which certain cancer cells preferentially spread to the bones remains poorly understood. Thus, there is a critical need to develop in vitro experimental models that can mimic key aspects of bone metastasis development to elucidate driving mechanisms of this disease and expedite the discovery of novel drug candidates. To address this unmet need, here we report a spatially patterned, 3D in vitro model which mimics cancer metastasis to bone by incorporating interactions with multiple tissue types. The model is composed of an outer ring of tissue engineered bone derived from 3D osteogenic differentiation of human mesenchymal stem cells (hMSCs), and a center containing cancer cells. This interface between these two tissues mimics the way cancer cells invade into the bones through the bone marrow/bone interface and allows us to track the invasion of fluorescently labeled cancer cells into bone using confocal imaging. Cancer cells and hMSCs were also labeled with orthogonal luciferases (FireflyLuc and NanoLuc), allowing for simultaneous quantification of cancer cell proliferation and osteoblast survival using BLI. We tested the ability of this model to recapitulate known characteristics of in vivo bone metastases including tissue-specific invasion, cancer aggressiveness, cancer-induced bone resorption, and in vivo drug response. Using multiple established breast cancer (MDA-MB-231 and MCF-7) and prostate cancer (LNCaP and PC-3) cell lines, we demonstrate that such spatially patterned coculture models mimic the preferential invasion of cancer cells to bone, but not cartilage or muscle (negative controls) and the rate of invasion in this 3D model correlated with the level of cancer cell aggressiveness. To mimic the effect of cancer invasion on bone remodeling, we established a triculture model in which osteoclasts were seeded over the tissue engineered bone. Micro-computed tomography imaging validated that this model could recapitulate cancer-induced bone resorption, and the degree of bone resorption also correlated with cancer cell aggressiveness. Using parathyroid hormone as a model drug, we demonstrate this 3D model recapitulates drug responses consistent with what has been observed using in vivo mouse models. Such spatially patterned 3D models can provide a scalable platform for drug screening with substantially reduced time and cost compared to animal models. Furthermore, integrating these 3D cancer metastasis models with high-dimensional methods, such as single-cell RNAseq could expedite the discovery of novel druggable drivers of bone metastasis. Citation Format: Eva C. González Díaz, Michelle Tai, Callan E. Monette, Joy Wu, Fan Yang. Spatially patterned, 3D in vitro models of cancer metastasis to bone for elucidating key drivers of metastasis and drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 696.

Recapitulated Crosstalk between Cerebral Metastatic Lung Cancer Cells and Brain Perivascular Tumor Microenvironment in a Microfluidic Co-Culture Chip.

Non‐small cell lung carcinoma (NSCLC), which affects the brain, is fatal and resistant to anti‐cancer therapies. Despite innate, distinct characteristics of the brain from other organs, the underlying delicate crosstalk between brain metastatic NSCLC (BM‐NSCLC) cells and brain tumor microenvironment (bTME) associated with tumor evolution remains elusive. Here, a novel 3D microfluidic tri‐culture platform is proposed for recapitulating positive feedback from BM‐NSCLC and astrocytes and brain‐specific endothelial cells, two major players in bTME. Advanced imaging and quantitative functional assessment of the 3D tri‐culture model enable real‐time live imaging of cell viability and separate analyses of genomic/molecular/secretome from each subset. Susceptibility of multiple patient‐derived BM‐NSCLCs to representative targeted agents is altered and secretion of serpin E1, interleukin‐8, and secreted phosphoprotein 1, which are associated with tumor aggressiveness and poor clinical outcome, is increased in tri‐culture. Notably, multiple signaling pathways involved in inflammatory responses, nuclear factor kappa‐light‐chain‐enhancer of activated B cells, and cancer metastasis are activated in BM‐NSCLC through interaction with two bTME cell types. This novel platform offers a tool to elucidate potential molecular targets and for effective anti‐cancer therapy targeting the crosstalk between metastatic cancer cells and adjacent components of bTME.

A parallelized, perfused 3D triculture model of leukemia for in vitro drug testing of chemotherapeutics

Leukemia patients undergo chemotherapy to combat the leukemic cells (LCs) in the bone marrow. During therapy not only the LCs, but also the blood-producing hematopoietic stem and progenitor cells (HSPCs) may be destroyed. Chemotherapeutics targeting only the LCs are urgently needed to overcome this problem and minimize life-threatening side-effects. Predictive in vitro drug testing systems allowing simultaneous comparison of various experimental settings would enhance the efficiency of drug development. Here, we present a three-dimensional (3D) human leukemic bone marrow model perfused using a magnetic, parallelized culture system to ensure media exchange. Chemotherapeutic treatment of the acute myeloid leukemia cell line KG-1a in 3D magnetic hydrogels seeded with mesenchymal stem/stromal cells (MSCs) revealed a greater resistance of KG-1a compared to 2D culture. In 3D tricultures with HSPCs, MSCs and KG-1a, imitating leukemic bone marrow, HSPC proliferation decreased while KG-1a cells remained unaffected post treatment. Non-invasive metabolic profiling enabled continuous monitoring of the system. Our results highlight the importance of using biomimetic 3D platforms with proper media exchange and co-cultures for creating in vivo-like conditions to enable in vitro drug testing. This system is a step towards drug testing in biomimetic, parallelized in vitro approaches, facilitating the discovery of new anti-leukemic drugs.

In Vitro-In Vivo Correlation of Blood-Brain Barrier Permeability of Drugs: A Feasibility Study Towards Development of Prediction Methods for Brain Drug Concentration in Humans.

In vitro human blood-brain barrier (BBB) models in combination with central nervous system-physiologically based pharmacokinetic (CNS-PBPK) modeling, hereafter referred to as the "BBB/PBPK" method, are expected to contribute to prediction of brain drug concentration profiles in humans. As part of our ongoing effort to develop a BBB/PBPK method, we tried to clarify the relationship of in vivo BBB permeability data to those in vitro obtained from a human immortalized cell-based tri-culture BBB model (hiBBB), which we have recently created. The hiBBB models were developed and functionally characterized as previously described. The in vitro BBB permeabilities (Pe, × 10-6cm/s) of seventeen compounds were determined by permeability assays, and in vivo BBB permeabilities (QECF) for eight drugs were estimated by CNS-PBPK modeling. The correlation of the Pe values with the QECF values was analyzed by linear regression analysis. The hiBBB models showed intercellular barrier properties and several BBB transporter functions, which were enough to provide a wide dynamic range of Pe values from 5.7 ± 0.7 (rhodamine 123) to 2580.4 ± 781.9 (rivastigmine). Furthermore, the in vitro Pe values of the eight drugs showed a good correlation (R2 = 0.96) with their in vivo QECF values estimated from human clinical data. We show that in vitro human BBB models provide clinically relevant BBB permeability that can be used as input for CNS-PBPK modeling. Therefore, our findings will encourage the development of a BBB/PBPK method as a promising approach for predicting brain drug concentration profiles in humans.

Triculture Model of In Vitro BBB and its Application to Study BBB‐Associated Chemosensitivity and Drug Delivery in Glioblastoma (Adv. Funct. Mater. 10/2022)

Blood–Brain Barrier In article number 2106860, Kangwon Lee, Nakwon Choi, Hong Nam Kim, and co-workers fabricate a brain-on-a-chip platform to replicate the complicated glioblastoma multiforme microenvironment and the anatomical features and functionality of the in vivo blood–brain barrier, and demonstrate the influence of the glioblastoma multiforme microenvironment on tumor behavior and drug delivery.

Immune cell mediated cabozantinib resistance for patients with renal cell carcinoma.

Renal cell carcinoma (RCC) is the third most common genitourinary cancer in the USA. Despite recent advances in the treatment for advanced and metastatic clear cell RCC (ccRCC), the 5-year relative survival rate for the distant disease remains at 12%. Cabozantinib, a tyrosine kinase inhibitor (TKI), which is one of the first-line therapies approved to treat advanced ccRCC as a single agent, is now being investigated as a combination therapy with newer immunotherapeutic agents. However, not much is known about how cabozantinib modulates the immune system. Here, we present a high throughput tri-culture model that incorporates cancer cells, endothelial cells, and patient-derived immune cells to study the effect of immune cells from patients with ccRCC on angiogenesis and cabozantinib resistance. We show that circulating immune cells from patients with ccRCC induce cabozantinib resistance via increased secretion of a set of pro-angiogenic factors. Using multivariate partial least square regression modeling, we identified CD4+ T cell subsets that are correlated with cabozantinib resistance and report the changes in the frequency of these populations in ccRCC patients who are undergoing cabozantinib therapy. These findings provide a potential set of biomarkers that should be further investigated in the current TKI-immunotherapy combination clinical trials to improve personalized treatments for patients with ccRCC.

TMOD-21. A NOVEL IN-VITRO METHOD TO MODEL MACROPHAGES IN GLIOBLASTOMA

Abstract BACKGROUND The GBM tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemoresistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Others have addressed this problem by employing 3D bioprinting to incorporate astrocytes and macrophages in an extracellular matrix; however, they used serum-containing media and classically polarized anti-inflammatory macrophages. Serum has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, highlighting the importance of excluding it from these models. Additionally, tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. METHODS THP-1 monocytes and normal human astrocytes (NHAs) were transitioned into serum-free HL-1 and neurobasal-based media, respectively. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft(PDX) lines, using a transwell insert. CD206 expression was used to validate polarization and a cytokine array was used to characterize the cells. RESULTS There was no difference in proliferation rates at 72 hours for THP-1 monocytes grown in serum-free HL-1 media compared to serum-containing RPMI 1640 (p > 0.95). Macrophages polarized via transwell inserts expressed the lymphocyte chemoattractant protein, CCL2, whereas resting(M0), pro-inflammatory(M1), and anti-inflammatory(M2) macrophages did not. Additionally, these macrophages expressed more CXCL1 and IL-1ß relative to M1 macrophages. We have also demonstrated a method to maintain a tri-culture model of GBM PDX cells, NHAs, and TAMs in a serum-free media that supports the growth/maintenance of all cell types. CONCLUSIONS We have demonstrated a novel method by which we can polarize macrophages towards a tumor-supportive phenotype that differs in cytokine expression from traditionally polarized macrophages. This higher-fidelity method of modeling TAMs in GBM can aid in the development of targeted therapeutics that may one day enter the clinic in hopes of improving outcomes in GBM.

Triculture Model of In Vitro BBB and its Application to Study BBB‐Associated Chemosensitivity and Drug Delivery in Glioblastoma

AbstractPhysiologically, brain tumors interact with surrounding vascular and glial cells, and change their responses to survive in brain tissue‐specific microenvironments. A major difficulty in brain tumor treatment is caused by the organism's high resistance to pharmaceutical drugs and poor blood–brain barrier (BBB) penetration. Therefore, mimicking the physiological environment of brain tumors on in vitro platforms can aid in predicting the cellular response to drugs. Here, an engineered 3D human glioblastoma in vitro platform that is integrated with a tricultured BBB is presented. First, the barrier function of the constructed BBB model and its reversibility are characterized, after administrating BBB‐opening agents through the microvasculature. The brain tumor cells that are cocultured in the BBB show a more aggressive growth pattern and high drug resistance, as well as secreting high concentrations of inflammatory cytokines. Finally, the delivery of BBB‐nonpenetrating drugs are promoted by chemically opening the BBB. The results of this study indicate that the platform can potentially study the physiology of the BBB, and monitor drug responses based on the interaction of the brain tumor and BBB.

Role of enteric glial cells in the toxicity of phycotoxins: Investigation with a tri-culture intestinal cell model

Role of enteric glial cells in the toxicity of phycotoxins: Investigation with a tri-culture intestinal cell model

Design of experiment based optimization of an in vitro direct contact triculture blood brain barrier model for permeability screening

The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte and pericyte synergistic cell signaling interactions that underlie the brain microvessel endothelial cells (BMEC). In vivo relevant direct contact between astrocytes, pericytes, and BMECS, to our knowledge, has not been established in conventional Transwell® based in vitro screening models of the BBB. We hypothesize that a design of experiments (DOE) optimized direct contact layered triculture model will offer more in vivo relevance for screening in comparison to indirect models. Plating conditions including the seeding density of all three cell types, matrix protein, and culture time were assessed utilizing a DOE approach. A second set of DOE methods assessed the influence of medium additives on barrier properties. The optimized model was further assessed for p-glycoprotein function using a substrate and inhibitor along with a set of BBB paracellular and transcellular markers at varying permeation rates. The optimization revealed that length of culture post endothelial cell plating correlated highest with paracellular tightness. In addition, seeding density of the endothelial cell layer influenced paracellular tightness at earlier times of culture, and its impact decreased as culture is extended. At optimal conditions, the model revealed P-gp function along with the ability to differentiate between BBB positive and negative permeants. We have demonstrated that the implementation of DOE based optimization for biologically based systems is an expedited method to establish multi-component in vitro cell models. The direct contact BBB triculture model reveals that the physiologically relevant layering of the three cell types is a practical method of culture to establish a screening model compared to indirect plating methods that incorporate physical barriers between cell types. Additionally, the ability of the model to differentiate between BBB positive and negative permeants suggests that this model may be an enhanced screening tool for potential neuroactive compounds.

Bioengineering a humanized 3D tri-culture osteosarcoma model to assess tumor invasiveness and therapy response

Bioengineering a humanized 3D tri-culture osteosarcoma model to assess tumor invasiveness and therapy response

The Impact of Astrocytes and Endothelial Cells on Glioblastoma Stemness Marker Expression in Multicellular Spheroids.

Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is extremely malignant and lethal. GBM tumors are highly heterogenous, being comprised of cellular and matrix components, which contribute to tumor cell invasion, cancer stem cell maintenance, and drug resistance. Here, we developed a heterotypic 3D spheroid model integrating GBM cells with astrocytes and endothelial cells (ECs) to better simulate the cellular components of the tumor microenvironment and investigate their impact on the stemness marker expression of GBM cells, which has not been previously investigated. We used U87 GBM cells, C8-D1A mouse astrocytes, and human umbilical vein ECs to construct co- and tri-culture spheroid models in low-attachment U-well plates. We characterized the expression of known stemness markers NESTIN, SOX2, CD133, NANOG, and OCT4 in these models and compared it to respective mixed monoculture spheroids (control) using qRT-PCR and immunostaining. We incorporated GBM cells and astrocytes/ECs in 1:1, 1:2, 1:4, and 1:9 ratio and observed spontaneous self-assembled spheroids in all coculture conditions. We observed changing spheroid size dynamics over 7 days and an increased expression in stemness markers in GBM-astrocyte and GBM-EC coculture spheroids in 1:4 and 1:9 coculture conditions, respectively. In a triculture model employing GBM cells, astrocytes, and ECs in a 1:4:9 ratio, we found an increased expression of all the stemness markers. We elucidated the impact of astrocytes and ECs on GBM stemness marker expression. This multicellular spheroid model may provide an important tool for investigating the crosstalk between cell types in GBM. The online version contains supplementary material available at 10.1007/s12195-021-00691-y.

Regulatory T cells provide chondroprotection through increased TIMP1, IL-10 and IL-4, but cannot mitigate the catabolic effects of IL-1β and IL-6 in a tri-culture model of osteoarthritis

ObjectiveTo gain insight into Treg interactions with synovial tissues in early OA, an equine tri-culture model of OA was used to test the hypothesis that Tregs, in the absence of T Helper 17 ​cells, are sufficient to resolve inflammation elicited by IL-1β. MethodsTo model normal and OA joints, synoviocytes were co-cultured with chondrocytes in a transwell system and ± stimulated with IL-1β. Tregs were activated and enriched, then added to co-cultures, creating tri-cultures. At culture end, synoviocytes and chondrocytes were analyzed for gene expression, Treg Foxp3 expression was reexamined by flow cytometry, and conditioned media were evaluated by ELISA. ResultsTregs increased IL-10 and IL-4 in tri-culture media and increased TIMP1 gene expression in synoviocytes and chondrocytes. Tregs increased IL-6 in conditioned media and Il6 gene expression in synoviocytes, which was additive with IL-1β. In chondrocytes, addition of Tregs decreased Col2b gene expression while Acan gene expression was decreased by IL-1β and addition of Tregs. IL-17A was detected in tri-cultures. CCL2 and CCL5 were increased in tri-cultures. ConclusionsIn a tri-culture model of OA, addition of Tregs resulted in conditions conducive to chondroprotection including increased concentration of IL-10 and IL-4 in conditioned media and increased gene expression of TIMP1 in both chondrocytes and synoviocytes. However, there was increased concentration of the catabolic cytokine IL-6, and decreased gene expression of Col2b and Acan in IL-1β-stimulated chondrocytes. These results suggest that blocking IL-6 could enhance Treg function in mitigating OA progression.

Modelling TGFβR and Hh pathway regulation of prognostic matrisome molecules in ovarian cancer

Modelling TGFβR and Hh pathway regulation of prognostic matrisome molecules in ovarian cancer

Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer's disease.

Aberrant inflammation in the central nervous system (CNS) has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a novel approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular crosstalk along the neuroinflammatory axis in vitro.We used the tri-culture system to model neuroinflammation in Alzheimer’s Disease with hPSCs harboring the APPSWE+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APPSWE+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in AD and presents a broadly applicable platform to study neuroinflammation in human disease.

Biotransformations and cytotoxicity of eleven graphene and inorganic two-dimensional nanomaterials using simulated digestions coupled with a triculture in vitro model of the human gastrointestinal epithelium.

Engineered nanomaterials (ENMs) have already made their way into myriad applications and products across multiple industries. However, the potential health risks of exposure to ENMs remain poorly understood. This is particularly true for the emerging class of ENMs know as 2-dimensional nanomaterials (2DNMs), with a thickness of one or a few layers of atoms arranged in a planar structure. The present study assesses the biotransformations and in vitro cytotoxicity in the gastrointestinal tract of 11 2DNMs, namely graphene, graphene oxide (GO), partially reduced graphene oxide (prGO), reduced graphene oxide (rGO), hexagonal boron nitride (h-BN), molybdenum disulphide (MoS2), and tungsten disulphide (WS2). The evaluated pristine materials were either readily dispersed in water or dispersed with the use of a surfactant (Na-cholate or PF108). Materials dispersed in a fasting food model (FFM, water) were subjected to simulated 3-phase (oral, gastric, and small intestinal) digestion to replicate the biotransformations that would occur in the GIT after ingestion. A triculture model of small intestinal epithelium was used to assess the effects of the digested products (digestas) on epithelial layer integrity, cytotoxicity, viability, oxidative stress, and initiation of apoptosis. Physicochemical characterization of the 2DNMs in FFM dispersions and in small intestinal digestas revealed significant agglomeration by all materials during digestion, most prominently by graphene, which was likely caused by interactions with digestive proteins. Also, MoS2 had dissolved by ~75% by the end of simulated digestion. Other than a low but statistically significant increase in cytotoxicity observed with all inorganic materials and graphene dispersed in PF108, no adverse effects were observed in the exposed tricultures. Our results suggest that occasional ingestion of small quantities of 2DNMs may not be highly cytotoxic in a physiologically relevant in vitro model of the intestinal epithelium. Still, their inflammatory or genotoxic potential after short- or long-term ingestion remains unclear and needs to be studied in future in vitro and in vivo studies. These would include studies of effects on co-ingested nutrient digestion and absorption, which have been documented for numerous ingested ENMs, as well as effects on the gut microbiome, which can have important health implications.

Co-culture methods to study neuronal function and disease.

Co-culture methods to study neuronal function and disease.

A novel tri-culture model for neuroinflammation.

Neuroinflammation is believed to play a primary role in the pathogenesis of most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and schizophrenia. Currently, suitable in vitro neuroinflammation models for studying cellular interactions and inflammatory mechanisms at the neurovascular unit are still scarce. In this study, we established an experimentally flexible tri-culture neuroinflammation model combining murine microglial cells (N11), mouse neuroblastoma Nuro2A cell lines and brain microvascular endothelial MVEC(B3) cells in a transwell co-culture system stimulated with lipopolysaccharides. Neuroinflammation was induced in this tri-culture model as manifested by activated N11 cells via toll-like receptor 4, resulting in increased release of proinflammatory mediators (nitric oxide, interleukin-6 and tumour necrosis factor-α) through the activation of nuclear factor-κB signalling pathway. The released inflammatory cytokines from N11 in turn, damaged the tight junction in microvascular endothelial MVEC(B3) cells, increased permeability of endothelial barrier, and induced tau phosphorylation and up-regulated caspase-3 expression in mouse neuroblastoma Nuro2A cell lines, leading to neuroinflammation injury. In summary, this tri-culture inflammation model mimics the microenvironment, the cellular crosstalk and the molecular events that take place during neuroinflammation. It provides a robust in vitro model for studying neuroinflammation mechanisms and screening for potential therapeutics to treat various neurodegenerative diseases.

Neuroinflammation and EIF2 Signaling Persist despite Antiretroviral Treatment in an hiPSC Tri-culture Model of HIV Infection.

Neuroinflammation and EIF2 Signaling Persist despite Antiretroviral Treatment in an hiPSC Tri-culture Model of HIV Infection.

A Novel Scaffold-Based Hybrid Multicellular Model for Pancreatic Ductal Adenocarcinoma-Toward a Better Mimicry of the in vivo Tumor Microenvironment.

With a very low survival rate, pancreatic ductal adenocarcinoma (PDAC) is a deadly disease. This has been primarily attributed to (i) its late diagnosis and (ii) its high resistance to current treatment methods. The latter specifically requires the development of robust, realistic in vitro models of PDAC, capable of accurately mimicking the in vivo tumor niche. Advancements in the field of tissue engineering (TE) have helped the development of such models for PDAC. Herein, we report for the first time a novel hybrid, polyurethane (PU) scaffold-based, long-term, multicellular (tri-culture) model of pancreatic cancer involving cancer cells, endothelial cells, and stellate cells. Recognizing the importance of ECM proteins for optimal growth of different cell types, the model consists of two different zones/compartments: an inner tumor compartment consisting of cancer cells [fibronectin (FN)-coated] and a surrounding stromal compartment consisting of stellate and endothelial cells [collagen I (COL)-coated]. Our developed novel hybrid, tri-culture model supports the proliferation of all different cell types for 35 days (5 weeks), which is the longest reported timeframe in vitro. Furthermore, the hybrid model showed extensive COL production by the cells, mimicking desmoplasia, one of PDAC’s hallmark features. Fibril alignment of the stellate cells was observed, which attested to their activated state. All three cell types expressed various cell-specific markers within the scaffolds, throughout the culture period and showed cellular migration between the two zones of the hybrid scaffold. Our novel model has great potential as a low-cost tool for in vitro studies of PDAC, as well as for treatment screening.