2D Monolayer Culture Research Articles

Effect of lipopeptide structure on gene delivery system properties: Evaluation in 2D and 3D in vitro models.

Development of efficient biodegradable, environmentally responsive, biocompatible and non-toxic delivery system is needed for efficient gene delivery. As well known, properties of the vehicle are determined by the structure of carrier components. The aim of the current study was to estimate in vitro transfection efficacy of aliphatic di-, tri- and tetrapeptide-based cationic lipoplexes loaded with siRNA in function of a number of cationic groups using 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Physicochemical properties and cytotoxicity of the liposomes were found to be dependent upon a number of amino acid derivatives in an amphiphilic polar head. Uptake of liposomes loaded with nucleic acid (lipoplexes) and their localization in HEK293T cells was studied by confocal microscopy. The liposomes based on lipotripeptides had the highest transfection efficiency which was 20-fold higher than those fabricated from lipotetrapeptides.

Paper‐Based Breast Tumor Model Reveals New Insights into the Hypoxic Regulation of Estrogen Receptor Alpha

Nearly 75% of breast tumors express estrogen receptor alpha (ERα). These tumors are treated with endocrine therapies to block ERα‐directed transcription and halt tumor progression. However, acquired resistance to these therapies is common, and associated with a poor clinical prognosis. Hypoxia has been implicated in acquired endocrine resistance through loss of ERα expression based on studies conducted using 2D monolayer cultures. However, the impact of hypoxia on ERα expression and estrogen signaling has not been evaluated in 3D culture models, which recapitulate the cell‐cell and cell‐matrix interactions in vivo.Here, we compare the impact of hypoxia on ERα expression and estrogen signaling between 2D monolayer cultures and 3D paper‐based cultures. Paper‐based cultures are an emerging 3D culture system that relies on paper scaffolds to generate tissue‐like constructs whose microenvironments can be controlled and measured. Using the ERα‐positive cell lines, T47D‐KBluc and MCF‐7, we have demonstrated that ERα levels in 3D paper‐based cultures are insensitive to hypoxia (1% O2), yet estrogen signaling decreases under hypoxic conditions. To understand this inconsistency with 2D cultures, we compare the role of hypoxia inducible factor 1α in regulating ERα and estrogen signaling, and compare ERα distribution and localization between the two culture formats.Finally, we generate a breast tumor model by stacking individual scaffolds, which were seeded with ERα‐positive cell lines within a collagen matrix. We confirm and measure oxygen gradients within the tumor model using an oxygen‐sensitive thin film sensor, and evaluate the impacts of hypoxic tumor gradients on ERα and estrogen signaling. This work will provide new insights into the role of hypoxia in acquired endocrine resistance and generate a breast tumor model that will be further used study acquired endocrine resistance in a physiologically relevant in vitro tumor model.Support or Funding InformationThe project described was supported by the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through Grant Award Number UL1TR001111, along with Eli Lilly and Company's Young Investigator Award. ZWL was supported by the NC Louis Stokes Alliance for Minority Participation, NSF #1202467.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Repeated UVA Irradiation on Human Skin Fibroblasts Embedded in 3D Tense Collagen Matrix.

Skin photoaging is caused by cumulative UVA exposure that leads to dermal matrix alterations associated with impaired fibroblast functions. In this study, we evaluated the effects of repeated UVA irradiation on mechanically stressed fibroblasts which were embedded in 3D tense collagen matrix. By comparison to 2D monolayer culture, we investigated the expressions of alpha-smooth muscle actin (α-SMA) cytoskeleton and α2 subunit of integrin receptors, as well as the collagen metabolism, focusing to MMP-1 and collagen type-I expressions. We found that UVA exposure reduces collagen levels in both culture conditions. However, concerning integrin α2 and α-SMA expression, UVA irradiation had no effect on 2D culture, whereas in tense 3D culture, it had an inhibitory effect. In UVA-irradiated 3D culture, fibroblasts acquired elongated shape and lost their dynamic interaction with collagen fibers through a decrease in integrin α2 and α-SMA. Fibroblast responses to UVA irradiation were different in 2D versus 3D environment, highlighting the importance of collagen environment in the regulation of mechanical activities. The behavior of fibroblast upon mechanical stimulation closely mimics stressed extracellular environment. The model of UVA-irradiated fibroblasts cultured in tense 3D collagen gel illustrated the in vivo situation of both mechanically stressed and photoaged human skin.

Impact of RUNX2 gene silencing on the gemcitabine sensitivity of p53‑mutated pancreatic cancer MiaPaCa‑2 spheres.

Recently, it has been well‑recognized that the response toward anticancer drugs differs between two‑ and three‑dimensional (2D and 3D) invitro cancer cell growth models. In the present study, we have demonstrated that, similar to the conventional 2D monolayer culture systems which often lack invivo physiological insights, RUNX2 gene silencing increases the gemcitabine (GEM) sensitivity of the 3D spheres generated from p53‑mutated pancreatic cancer MiaPaCa‑2 cells. According to our results, MiaPaCa‑2 cells, but not p53‑wild‑type pancreatic cancer SW1990 cells efficiently formed sphere structures in serum‑free sphere‑forming medium. Although GEM treatment caused a marked induction of TAp73/TAp63 in MiaPaCa‑2 spheres accompanied by the transcriptional activation of p53 family‑target genes such as p21WAF1 and NOXA, only 20% of cells underwent cell death. Under these experimental conditions, mutant p53 expression level was increased in response to GEM and RUNX2 remained unchanged at the protein level regardless of GEM exposure, which may suppress the pro‑apoptotic activity of TAp73/TAp63. Notably, RUNX2 gene silencing markedly augmented GEM‑mediated cell death of MiaPaCa‑2 spheres compared to that of non‑depleted ones. Expression analyses revealed that forced depletion of RUNX2 further stimulated GEM‑induced upregulation of TAp63 as well as its downstream target genes such as p21WAF1 and NOXA. In summary, our observations strongly indicated that, similarly to 2D monolayer culture, RUNX2 gene silencing increased GEM sensitivity of MiaPaCa‑2 spheres and highlighted the therapeutic potential of RUNX2 in pancreatic cancer with p53 mutation.

Sarcoma Spheroids and Organoids-Promising Tools in the Era of Personalized Medicine.

Cancer treatment is rapidly evolving toward personalized medicine, which takes into account the individual molecular and genetic variability of tumors. Sophisticated new in vitro disease models, such as three-dimensional cell cultures, may provide a tool for genetic, epigenetic, biomedical, and pharmacological research, and help determine the most promising individual treatment. Sarcomas, malignant neoplasms originating from mesenchymal cells, may have a multitude of genomic aberrations that give rise to more than 70 different histopathological subtypes. Their low incidence and high level of histopathological heterogeneity have greatly limited progress in their treatment, and trials of clinical sarcoma are less frequent than trials of other carcinomas. The main advantage of 3D cultures from tumor cells or biopsy is that they provide patient-specific models of solid tumors, and they overcome some limitations of traditional 2D monolayer cultures by reflecting cell heterogeneity, native histologic architectures, and cell–extracellular matrix interactions. Recent advances promise that these models can help bridge the gap between preclinical and clinical research by providing a relevant in vitro model of human cancer useful for drug testing and studying metastatic and dormancy mechanisms. However, additional improvements of 3D models are expected in the future, specifically the inclusion of tumor vasculature and the immune system, to enhance their full ability to capture the biological features of native tumors in high-throughput screening. Here, we summarize recent advances and future perspectives of spheroid and organoid in vitro models of rare sarcomas that can be used to investigate individual molecular biology and predict clinical responses. We also highlight how spheroid and organoid culture models could facilitate the personalization of sarcoma treatment, provide specific clinical scenarios, and discuss the relative strengths and limitations of these models.

Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco’s Modified Eagle’s Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

A comprehensive analysis of human dental pulp cell spheroids in a three-dimensional pellet culture system

ObjectiveThree-dimensional (3D) cell culture methods are of high importance to studies of biological processes. This is particularly the case with spheroid cultures, which create 3D cell aggregates without the use of exogenous materials. Compared to conventional monolayer cultures, cellular spheroid cultures have been demonstrated to improve multilineage potential and extracellular matrix production. To address this issue in depth, we present a more comprehensive analysis of 3D human dental pulp cell (hDPC) spheroids. DesignhDPC spheroids were fabricated by the pellet culture method and were cultured without adding any reagent to induce differentiation. The gene-expression profiles of the 3D and two-dimensional (2D) cultured hDPCs were compared by complementary DNA microarray analysis. Odontoblastic and osteoblastic differentiation marker gene expression was evaluated by quantitative real-time PCR (RT-qPCR). Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to examine the morphology of hDPC spheroids and extracellular matrix components. ResultsCompared with 2D monolayer culture, microarray analysis identified 405 genes and 279 genes with twofold or greater differential expression after 3 days and 28 days of 3D culture, respectively. In 3D hDPC spheroids, gene ontology analysis revealed upregulation of extracellular matrix-related genes and downregulation of cell growth-related genes. RT-qPCR analysis showed higher expression levels of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM revealed the morphological characteristics of the fibrillar collagen-rich matrix and cell-cell interactions. ConclusionsThe present findings provide clues to understanding the mechanisms of pellet-cultured hDPCs and contribute to future research in the comparative studies of different 3D culture methods.

Hepatocyte Aggregate Formation on Chitin-Based Anisotropic Microstructures of Butterfly Wings.

Scaffold nanotopography plays the most significant role in the mimicry of the in vivo microenvironment of the hepatocytes. Several attempts have been made to develop methods and substrates suited to growing hepatocytes into aggregates. Functional biomaterials, particularly biodegradable polymers, have been used in several studies aimed to develop improved scaffolds with ordered geometry and nanofibrous architecture for tissue engineering. However, there are still some limitation in their fabrication: it is not cost-efficient, is time-consuming, and exhibits some technological complications. The synthetic scaffolds are usually non-biodegradable and can be non-biocompatible compared to the naturally derived biomaterials. Here, we utilized a simple, cost-effective, and green method with two-step chemical treatment to get more selected hydrophilic butterfly wings from Morpho menelaus, Papilio ulysses telegonus, and Ornithoptera croesus lydius as a chitin-based natural scaffolds to growing hepatocyte aggregates. We established a three-dimensional (3D) in vitro model for culture of HepG2 cells and aggregate formation that maintained the hepatocytes function on these natural anisotropic microstructures. Cells cultured on these substrates show higher viability than those cultured on a two-dimensional (2D) culture plate. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay results revealed excellent viability of HepG2 cells on P. u. telegonus wings (fibrous area). The results also demonstrated appropriate cell activity, cell retention, and stable and functional expression in terms of albumin secretion and urea synthesis activity compared to the 2D monolayer culture of hepatocytes on the culture dish surface. With a slightly different degree, the other substrates also shown similar results. We anticipate that these natural anisotropic, biodegradable, and biocompatible substrates can maintain long-term hepatic culture as an in vitro 3D model for potential therapeutic applications and regenerative tissue applications. The model presented here provides a feasible alternative to the synthetic scaffolds and is expected to be more reliable for 3D organotypic liver culture models based on such scaffolds.

Doxorubicin resistance in breast cancer cells is mediated by extracellular matrix proteins

BackgroundCancer cell resistance to therapeutics can result from acquired or de novo-mediated factors. Here, we have utilised advanced breast cancer cell culture models to elucidate de novo doxorubicin resistance mechanisms.MethodsThe response of breast cancer cell lines (MCF-7 and MDA-MB-231) to doxorubicin was examined in an in vitro three-dimensional (3D) cell culture model. Cells were cultured with Matrigel™ enabling cellular arrangements into a 3D architecture in conjunction with cell-to-extracellular matrix (ECM) contact.ResultsBreast cancer cells cultured in a 3D ECM-based model demonstrated altered sensitivity to doxorubicin, when compared to those grown in corresponding two-dimensional (2D) monolayer culture conditions. Investigations into the factors triggering the observed doxorubicin resistance revealed that cell-to-ECM interactions played a pivotal role. This finding correlated with the up-regulation of pro-survival proteins in 3D ECM-containing cell culture conditions following exposure to doxorubicin. Inhibition of integrin signalling in combination with doxorubicin significantly reduced breast cancer cell viability. Furthermore, breast cancer cells grown in a 3D ECM-based model demonstrated a significantly reduced proliferation rate in comparison to cells cultured in 2D conditions.ConclusionCollectively, these novel findings reveal resistance mechanisms which may contribute to reduced doxorubicin sensitivity.

A Novel Polyamine-Targeted Therapy for BRAF Mutant Melanoma Tumors.

Mutant serine/threonine protein kinase B-Raf (BRAF) protein is expressed in over half of all melanoma tumors. Although BRAF inhibitors (BRAFi) elicit rapid anti-tumor responses in the majority of patients with mutant BRAF melanoma, the tumors inevitably relapse after a short time. We hypothesized that polyamines are essential for tumor survival in mutant BRAF melanomas. These tumors rely on both polyamine biosynthesis and an upregulated polyamine transport system (PTS) to maintain their high intracellular polyamine levels. We evaluated the effect of a novel arylpolyamine (AP) compound that is cytotoxic upon cellular entry via the increased PTS activity of melanoma cells with different BRAF mutational status. Mutant BRAF melanoma cells demonstrated greater PTS activity and increased sensitivity to AP compared to wild type BRAF (BRAFWT) melanoma cells. Treatment with an inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), further upregulated PTS activity in mutant BRAF cells and increased their sensitivity to AP. Furthermore, viability assays of 3D spheroid cultures of mutant BRAF melanoma cells demonstrated greater resistance to the BRAFi, PLX4720, compared to 2D monolayer cultures. However, co-treatment with AP restored the sensitivity of melanoma spheroids to PLX4720. These data indicate that mutant BRAF melanoma cells are more dependent on the PTS compared to BRAFWT melanoma cells, resulting in greater sensitivity to the PTS-targeted cytotoxic AP compound.

Abstract A161: Comparison of EGFR inhibitor response of patient-derived NSCLC lines grown as monolayers, simple spheroids, and mixed cell complex spheroids

Abstract Background: Human cancer cell lines are important tools in cancer research and drug discovery. NCI has established cell lines from patient-derived xenografts. These lines form multicellular spheroids under low adhesion culture conditions. In the tumor microenvironment, stromal (fibroblast, endothelial cells) and cancer cells engage in crosstalk mediated by soluble factors and cell-cell contact; therefore, we generated complex spheroids by mixing tumor cells, endothelial, and mesenchymal stem cells. We investigated the growth inhibitory activity of EGFR inhibitors, erlotinib and osimertinib, in eight patient-derived NSCLC lines. Materials and Methods: Erlotinib and osimertinib were tested in 9-point concentration response starting at 30uM and 5uM, respectively, and diluting by half-logs. Tumor cells were cultured in 96-well plates as monolayers, in 96-well U-bottom ultra-low adhesion (ULA) plates as 3D simple spheroids, and tumor cells were cocultured with human bone marrow-derived mesenchymal stem cells (hMSC) and human endothelial cells (HUVEC) in 96-well ULA plates as 3D complex spheroids. At 72h post plating, the cells were exposed to the test drugs for 72h and 96h. Cell viability was measured using luminescence by 2D CellTiter-Glo® in 2D monolayer cultures and 3D CellTiter-Glo® in 3D simple and complex spheroids. Cell viability was determined relative to a vehicle-treated control and IC50s were calculated from concentration response curves. To determine the localization of HUVEC and hMSC in complex spheroids, GFP-labeled HUVEC and REP-labeled hMSC were used. Images were captured at several time points using a Leica DMi8 microscope. Results: The response of 4 lung adenocarcinoma lines and 1 lung neuroendocrine line with 3 sublines exposed to the EGFR inhibitors erlotinib and osimertinib was assessed. All lines cultured as monolayer were unresponsive to erlotinib (>30 µM) and osimertinib (>5 µM). Simple spheroids formed by TLG0904F1496 and TLG0703F948 tumor cells; complex spheroids formed by TLG0703F948, TLG0904F1496 and its three clones were sensitive to erlotinib with IC50s below the clinical Cmax, indicating the importance of the 3D culture format. When tested separately as monolayers, hMSC and HUVEC were resistant to erlotinib with an IC50s of >30 and 13 µM, respectively. We observed varied patterns of HUVEC and hMSC localization in the complex spheroids. The TLG0904F1496 and three clones formed spheroids with a tight cluster of HUVEC and hMSC in the center while the other four lines have HUVEC and hMSC dispersed throughout the spheroid. Conclusion: Patient-derived NSCLC line response to anticancer drugs varied with culture conditions. Osimertinib showed activity in the T790 mutant TLG0703F948 line in 3D complex spheroids only. The differential response to EGFR inhibitors in 3D simple and 3D complex spheroids, versus 2D culture, suggests that the tumor microenvironment may play a role in EGFR inhibitor activity and needs to be explored further using imaging techniques. Citation Format: Gurmeet Kaur, John W. Connelly, Annamaria Rapisarda, James H. Doroshow, Beverly A. Teicher. Comparison of EGFR inhibitor response of patient-derived NSCLC lines grown as monolayers, simple spheroids, and mixed cell complex spheroids [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A161.

Elevation of sensitivity to anticancer agents of human lung adenocarcinoma A549 cells by knockdown of claudin-2 expression in monolayer and spheroid culture models

Claudins, tight junctional proteins, regulate the paracellular permeability of ions and small molecules. Claudin-2 is highly expressed in human lung adenocarcinoma cells and is involved in the up-regulation of cell proliferation. However, the effect of claudin-2 on cellular sensitivity to anticancer agents has not been clarified. The cytotoxicity of anticancer agents such as cisplatin, gefitinib and doxorubicin (DXR) was increased by claudin-2 knockdown in A549 cells. Claudin-2 knockdown also significantly decreased the expression level of multidrug resistance-associated protein/ABCC2. The expression levels of other drug efflux transporters were unchanged. The intracellular accumulation of 5-chloromethylfluorescein diacetate (CMFDA) and DXR, substrates of ABCC2, was increased by claudin-2 knockdown, whereas the efflux was decreased. MK-571, an inhibitor of ABCC2, enhanced the cytotoxicity of anticancer agents. Claudin-2 knockdown decreased the levels of p-c-Jun and nuclear Sp1. SP600125, an inhibitor of c-Jun, and mithramycin, an inhibitor of Sp1, decreased the level of ABCC2. The promoter activity of ABCC2 was decreased by claudin-2 knockdown, SP600125 and mithramycin treatments, suggesting that claudin-2 is involved in the up-regulation of ABCC2 expression at the transcriptional level. Claudin-2 knockdown increased the paracellular permeability of DXR in a 2D monolayer culture model. In addition, the accumulation of DXR into spheroids was enhanced by claudin-2 knockdown, resulting in a reduction in cell viability. We suggest that claudin-2 may be a novel therapeutic target in lung adenocarcinoma, because claudin-2 knockdown increased the accumulation of anticancer agents in cancer cells and spheroids.

3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes

Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs.

Potentiation of Bio Repositories In Personalized Medicine: Tumor Cells Establishment

The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help bio repositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

Organotypic three-dimensional cancer cell cultures mirror drug responses in vivo: lessons learned from the inhibition of EGFR signaling.

Complex three-dimensional (3D) in vitro models that recapitulate human tumor biology are essential to understand the pathophysiology of the disease and to aid in the discovery of novel anti-cancer therapies. 3D organotypic cultures exhibit intercellular communication, nutrient and oxygen gradients, and cell polarity that is lacking in two-dimensional (2D) monolayer cultures. In the present study, we demonstrate that 2D and 3D cancer models exhibit different drug sensitivities towards both targeted inhibitors of EGFR signaling and broad acting cytotoxic agents. Changes in the kinase activities of ErbB family members and differential expression of apoptosis- and survival-associated genes before and after drug treatment may account for the differential drug sensitivities. Importantly, EGFR oncoprotein addiction was evident only in the 3D cultures mirroring the effect of EGFR inhibition in the clinic. Furthermore, targeted drug efficacy was strongly increased when incorporating cancer-associated fibroblasts into the 3D cultures. Taken together, we provide conclusive evidence that complex 3D cultures are more predictive of the clinical outcome than their 2D counterparts. In the future, 3D cultures will be instrumental for understanding the mode of action of drugs, identifying genotype-drug response relationships and developing patient-specific and personalized cancer treatments.

Three-dimensional hydrogel is suitable for targeted investigation of amoeboid migration of glioma cells.

Glioblastoma (GBM) invasion and migration are key biological behaviors leading to refractoriness to current therapies and infiltration into the non-tumor brain parenchyma. GBM cell migration is strongly dependent on tumor architecture in vivo, which is absent in traditional two-dimensional (2D) monolayer culture. The present study applied a three-dimensional (3D) hydrogel model to rebuild the tumor architecture in vitro. Treatment with NSC23766, a specific inhibitor of Ras-related C3 botulinum toxin substrate 1 (Rac1), inhibited the mesenchymal invasiveness however triggered the amoeboid motility called mesenchymal-amoeboid transition (MAT). Notably, NSC23766 stimulated U87 GBM cell migration in the 3D hydrogel. However, this compound inhibited cell motility in 2D monolayer culture without tumor architecture for MAT, suggesting the advantage of 3D hydrogel to investigate tumor cell invasion. Due to the inverse interaction of Rac1 and Ras homolog family member A (RhoA) signaling in the transition between mesenchymal and amoeboid morphology, simultaneous treatment of NSC23766 and Y27632 (selective Rho associated coiled-coil containing protein kinase 1 inhibitor), abolished U87 GBM cell migration through inhibiting MAT and amoeboid-mesenchymal transition. In addition, Y27632 induced integrin expression which gave rise to the focal adhesion to facilitate the mesenchymal invasion. The results of the present study demonstrated that the 3D hydrogel was a preferable model in vitro to study tumor cell invasion and migration. The combined inhibition of Rac1 and RhoA signaling would be a promising strategy to suppress GBM invasion.

Top-down proteomics for assessing the maturation of stem cell-derived cardiomyocytes

Background: Recent advancements in human pluripotent stem cell (hPSC) technology and cardiomyocyte (CM) differentiation have empowered research in cardiac development and diseases. However, the relative immaturity of stem cell-derived CMs is well documented. Since the expression of sarcomeric protein isoforms, as well as their post-translational modifications (PTMs), change as CMs mature, we sought to develop a method utilizing powerful top-down high-resolution mass spectrometry (MS) to assess the relative maturity of CMs focusing on the expression of sarcomeric protein isoforms and their PTMs. Methods: hPSCs were differentiated to CMs using a small molecule directed protocol and cultured in either 2D monolayer or 3D engineered cardiac tissue (ECT). Sarcomeric proteins were extracted from the CMs and analyzed using a top-down proteomics platform coupling reverse phase chromatography to high-resolution MS. Results: We identified and quantified the major sarcomeric protein isoforms and their PTMs in CMs from 2D monolayer or 3D ECT culture. Slow skeletal troponin I (ssTnI) was highly expressed while cardiac troponin I (cTnI) was under detection limit in 2D-CMs at both day 30 and 60, indicating the relative immaturity of 2D-CMs by day 60. However, the relative expression of the ventricular regulatory light chain versus the atrial isoform in CMs increased by about 7-fold at day 60 compared to the 2D-CMs at day 30, confirming that CMs exhibit maturation with longer 2D culture as anticipated. Tropomyosin phosphorylation decreased in CMs with longer 2D culture, which may serve as a novel marker for assessing CM maturation. CMs cultured in 3D ECT for 42 days after 30 days in 2D culture exhibited a 2-fold increase in cTnI expression relative to ssTnI, compared to day-72 CMs maintained in 2D culture, suggesting that 3D culture can promote CM maturation. These results demonstrate the power of top-down proteomics to assess the maturation of hPSC-derived CMs.

Three-Dimensional Cell Culture and Drug Testing in a Microfluidic Sidewall-Attached Droplet Array.

Three-dimensional (3D) cell culture provides an effective way over conventional two-dimensional (2D) monolayer culture to more closely imitate the complex cellular organization, heterogeneity, and interactions as well as tissue microenvironments in vivo. Here we present a novel droplet-based 3D cell culture method by using droplet array attached on the sidewall of a PDMS piece. Such an arrangement not only avoids cells from adhering on the chip surface for achieving 3D cell culture in nanoliter-scale droplets, but also facilitates performing multiple operations to cells in droplets, including cell suspension droplet generation, drug treatment, and cell staining with a capillary-based liquid handling system, as well as in situ observation and direct scanning with a confocal laser scanning microscope. We optimized the system by studying the effects of various conditions to cell culture including droplet volume, cell density and fabrication methods of the PDMS pieces. We have applied this system in the 3D culture of HepG2 cells and the stimulation testing of an anticancer drug, doxorubicin, to 3D cell spheroids.

Role of Biorepositories in Personalized Medicine: Isolation and Processing of Primary Tumor Cells for the initiation Tumor Cultures

The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help bio repositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.

Abstract 5793: Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model

Abstract The purpose of this study was to examine the induction of epithelial to mesenchymal transition (EMT) in a prostate cancer cell line by measuring classical biomarker expression in three-dimensional (3D) spheroid cultures compared to traditional 2D monolayers in an effort to develop a more biologically relevant assay. Using Ultra-Low Attachment (ULA) microplates, we grew spheroids from a human prostate cancer cell line (DU 145). Numerous studies have implicated a role for EMT in carcinoma invasion and metastasis. EMT is characterized by rearrangement of the extracellular matrix (ECM) and differential regulation of ECM proteins. We induced EMT using TGF-beta and phorbol-12-myristate-13-acetate (PMA) and compared expression levels of specific biomarkers, such as E-cadherin, fibronectin, and IL-6, using AlphaLISA and LANCE (TR-FRET) assay technologies. We confirmed that treatment of DU 145 cells with TGF-beta is sufficient for inducing changes in both EMT biomarker expression and characteristic cellular morphology in monolayer cultures. However, in 3D spheroid cultures, we observed only a partial EMT response to the same TGF-beta treatment as demonstrated by changes in the expected biomarker expression pattern. Using the small molecule, PMA, we see significant differences in the levels of IL-6 secretion after EMT induction between cells grown in monolayer and those grown in spheroids. Cellular proliferation, growth and vitality were assessed using ATPlite luminescence assays and confocal microscopy of live-stained cells with a high content imaging system. Though we observe increased proliferation in monolayer cultures compared to 3D spheroids, the changes observed in protein expression patterns cannot be sufficiently explained by differences in cell number or viability. These data illustrate the differences in protein expression levels and in cellular tolerance for compound treatment between a human prostate cancer cell line grown in monolayers and those same cells grown in 3D spheroids. Citation Format: Jen Carlstrom, Jeanine M. Hinterneder, Lindsay Nelson, Stephen Hurt. Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5793. doi:10.1158/1538-7445.AM2017-5793