Indole-3-carbinol-induced death in cancer cells involves EGFR downregulation and is exacerbated in a 3D environment

Indole-3-carbinol (I3C) is a promising anticancer dietary compound, which inhibits breast cancer in animal models. The objective of the current study was to characterize I3C-induced cell death in a panel of human breast tumorigenic cells (MCF7, MDA-MB-468, MDA-MB-231 and HBL100) in comparison with normal fibroblasts. Since epithelial cells are protected from cell death by a three-dimensional environment, 3D cell culture (collagen I gel and spheroids) was employed to investigate susceptibility to I3C. Cell viability in the presence of 256 μM I3C, a concentration close to the physiologically achievable range, was in the order fibroblasts = HBL100>MDA-MB-231>MCF7>MDA-MB-468 in monolayer culture. However, 3D culture conditions increased the susceptibility of MCF7 and MDA-MB-468 cancer cells towards I3C. I3C induced cell death in breast cancer MCF7, MDA-MB-468 and MDA-MB–231 cells via the mitochondrial apoptotic pathway. I3C significantly reduced levels of epidermal growth factor receptor (EGFR) in MDA-MB-468 after 6 h and in MDA-MB-231 and HBL100 cells after 30 h. Downregulation of EGFR in MDA-MB468 and MDA-MB-231 cells using an EGFR inhibitor resulted in apoptosis. EGFR modulation using EGF or an EGFR inhibitor markedly influenced viability and response to I3C in MDA-MB-468 cells in 3D conditions. EGFR expression was modulated by 3D conditions. Therefore, I3C-induced EGFR reduction in these cells is likely to be responsible for I3C-induced apoptosis.     

Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential

While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell–cell and cell–matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or “mesenspheres”, of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells.     

Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.     

Multi compartmental 3D breast cancer disease model–recapitulating tumor complexity in in-vitro

Breast cancer is the most common ailment among women. In 2020, it had the highest incidence of any type of cancer. Many Phase II and III anti-cancer drugs fail due to efficacy, durability, and side effects. Thus, accelerated drug screening models must be accurate. In-vivo models have been used for a long time, but delays, inconsistent results, and a greater sense of responsibility among scientists toward wildlife have led to the search for in-vitro alternatives. Stromal components support breast cancer growth and survival. Multi-compartment Transwell models may be handy instruments. Co-culturing breast cancer cells with endothelium and fibroblasts improves modelling. The extracellular matrix (ECM) supports native 3D hydrogels in natural and polymeric forms. 3D Transwell cultured tumor spheroids mimicked in-vivo pathological conditions. Tumor invasion, migration, Trans-endothelial migration, angiogenesis, and spread are studied using comprehensive models. Transwell models can create a cancer niche and conduct high-throughput drug screening, promising future applications. Our comprehensive shows how 3D in-vitro multi compartmental models may be useful in producing breast cancer stroma in Transwell culture.     

Establishment and characterization of lung co-culture spheroids for paclitaxel loaded Eudragit® RL 100 nanoparticle evaluation

3D cell cultures are regarded as a better and more relevant approach for screening drugs and therapeutics, particularly due to their likeness with the in vivo conditions. Spheroids offer an intermediate platform between in vitro and in vivo models, for conducting tumor-based investigations. In this study, a simple setup was developed for consistent generation of lung co-culture spheroids, which were developed using the cancer cell lines A549, NCI H460, and fibroblast cells WI-38. The potential of these spheroids for evaluating the toxicity of Eudragit® RL 100 nanoparticles (ENP) was explored. Monodisperse ENP, having the size range of 140–200 nm was prepared using the nanoprecipitation method. These were loaded with the poorly water-soluble anticancer drug paclitaxel. The evaluation of toxicity and uptake of drug-loaded ENP revealed that 2D monolayers were more sensitive to treatment than 3D spheroids. Within spheroids, co-cultures were more resistant to the treatment than monocultures. Overall, our findings demonstrated that the lung co-culture spheroids were a suitable model for accelerating the efficacy and toxicity-related investigations of novel drug delivery systems.     

High-Throughput 3D Screening Reveals Differences in Drug Sensitivities between Culture Models of JIMT1 Breast Cancer Cells

The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional monolayers on plastic. However, many cellular features are impaired in these artificial conditions, and large changes in gene expression compared to tumors have been reported. Three-dimensional cell culture models have become increasingly popular and are suggested to be better models than two-dimensional monolayers due to improved cell-to-cell contact and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput three-dimensional drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in two dimensions, in poly(2-hydroxyethyl methacrylate) induced anchorage-independent three-dimensional models, and in Matrigel three-dimensional cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating, or they were allowed to grow in three-dimensional cultures for 4 days before the drug treatment. Large variations in drug responses were observed between the models indicating that comparisons of culture model-influenced drug sensitivities cannot be made based on the effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in two-dimensional cultures, while the responses of cells grown in poly(2-hydroxyethyl methacrylate) resembled those of the two-dimensional cultures. Furthermore, comparing the gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study, we also suggest that three-dimensional cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode and be used as alternatives to traditional two-dimensional screens for better comparability to the in vivo state.     

Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.     

3D Spheroids’ Sensitivity to Electric Field Pulses Depends on Their Size

Dramatic differences of cells behavior exist between cells cultured under classical 2D monolayers and 3D models, the latter being closer to in vivo responses. Thus, many 3D cell culture models have been developed. Among them, multicellular tumor spheroid appears as a nice and easy-to-handle 3D model based on cell adhesion properties. It is composed of one or several cell types and is widely used to address carcinogenesis, or drugs screening. A few and recent publications report the use of spheroids to investigate electropermeabilization process. We studied the response of spheroids to electrical field pulses (EP) in terms of their age, diameter or formation technique. We found that small human HCT-116 colorectal spheroids are more sensitive to electric field pulses than larger ones. Indeed, the growth of spheroids with a diameter of 300 μm decreased by a factor 2 over 4 days when submitted to EP (8 pulses, lasting 100 μs at a 1,300 V/cm field intensity). Under those electrical conditions, 650 μm spheroids were not affected. These data were the same whatever the formation method (i.e. hanging drop and nonadherent techniques). These observations point out the fact that characteristics of 3D cell models have to be taken into account to avoid biased conclusions of experimental data.     

Proteomic Analysis of Stromal and Epithelial Cell Communications in Human Endometrial Cancer Using a Unique 3D Co‐Culture Model

Epithelial and stromal communications are essential for normal uterine functions and their dysregulation contributes to the pathogenesis of many diseases including infertility, endometriosis, and cancer. Although many studies have highlighted the advantages of culturing cells in 3D compared to the conventional 2D culture system, one of the major limitations of these systems is the lack of incorporation of cells from non‐epithelial lineages. In an effort to develop a culture system incorporating both stromal and epithelial cells, 3D endometrial cancer spheroids are developed by co‐culturing endometrial stromal cells with cancerous epithelial cells. The spheroids developed by this method are phenotypically comparable to in vivo endometrial cancer tissue. Proteomic analysis of the co‐culture spheroids comparable to human endometrial tissue revealed 591 common proteins and canonical pathways that are closely related to endometrium biology. To determine the feasibility of using this model for drug screening, the efficacy of tamoxifen and everolimus is tested. In summary, a unique 3D model system of human endometrial cancer is developed that will serve as the foundation for the further development of 3D culture systems incorporating different cell types of the human uterus for deciphering the contributions of non‐epithelial cells present in cancer microenvironment.     

Cancer spheroids derived exosomes reveal more molecular features relevant to progressed cancer

Cancer cell spheroids have been shown to be more physiologically relevant to native tumor tissue than monolayer 2D culture cells. Due to enhanced intercellular communications among cells in spheroids, spheroid secreted exosomes which account for transcellular transportation should exceed those from 2D cell culture and may display a different expression pattern of miRNA or protein. To test this, we employed a widely used pancreatic cancer cell line, PANC-1, to create 3D spheroids and compared exosomes generated by both 2D cell culture and 3D PANC-1 spheroids. We further measured and compared exosomal miRNA and GPC-1 protein expression with qRT-PCR and enzyme-linked immunosorbent assay, respectively. It showed that PANC-1 cells cultured in 3D spheroids can produce significantly more exosomes than PANC-1 2D cells and exosomal miRNA and GPC-1 expression derived from spheroids show more features relevant to the progression of pancreatic cancer. These findings point to the potential importance of using spheroids as in vitro model to study cancer development and progression.     

3D in vitro co-culture models based on normal cells and tumor spheroids formed by cyclic RGD-peptide induced cell self-assembly

Objectives

To design novel 3D in vitro co-culture models based on the RGD-peptide-induced cell self-assembly technique.

Results

Multicellular spheroids from M-3 murine melanoma cells and L-929 murine fibroblasts were obtained directly from monolayer culture by addition of culture medium containing cyclic RGD-peptide. To reach reproducible architecture of co-culture spheroids, two novel 3D in vitro models with well pronounced core–shell structure from tumor spheroids and single mouse fibroblasts were developed based on this approach. The first was a combination of a RGD-peptide platform with the liquid overlay technique with further co-cultivation for 1–2 days. The second allowed co-culture spheroids to generate within polyelectrolyte microcapsules by cultivation for 2 weeks. M-3 cells (a core) and L-929 fibroblasts (a shell) were easily distinguished by confocal microscopy due to cell staining with DiO and DiI dyes, respectively.

Conclusions

The 3D co-culture spheroids are proposed as a tool in tumor biology to study cell–cell interactions as well as for testing novel anticancer drugs and drug delivery vehicles.

     

Sensitization of HT29 colorectal cancer cells to vemurafenib in three‐dimensional collagen cultures

The extracellular matrix to which cancer cells adhere affects cellular sensitivity to anticancer drugs. We sought to examine the changes in sensitivity of colorectal cancer cells carrying the BRAF V600E mutation to vemurafenib cultured in three‐dimensional (3D) collagen‐I gels, while also identifying the signaling pathways involved in these changes. HT29 colorectal cancer cells were cultured in conventional tissue culture (TC) plastic plates or in collagen‐I gels. The HT29 cells demonstrated approximately 10‐fold higher sensitivity to vemurafenib in 3D‐collagen‐I gels compared with those cultured on conventional TC plastic plates. Furthermore, in cells cultured on TC plastic, vemurafenib was found to augment tyrosine phosphorylation of focal adhesion kinase (FAK), while 3D‐cultured cells expressed lower levels of FAK and vemurafenib did not affect its tyrosine phosphorylation, suggesting that FAK contributes to vemurafenib resistance. However, pharmacological inhibition of FAK did not sensitize the cells to vemurafenib. Also, the level of tyrosine‐phosphorylated epidermal growth factor receptor (EGFR)/ERBB2 family proteins was found to be lower in cells cultured in 3D‐collagen gel compared with those in cells cultured on TC plastic. Afatinib, an inhibitor of the EGFR/ERBB family of kinases, sensitized the cells to higher concentrations of vemurafenib, implying their participation in vemurafenib resistance. Adhesion to collagen‐I gel but not to the collagen‐I‐coated plastic surface sensitized the cells, suggesting that the rigidity of the media rather than adherence to collagen‐I may be important for cellular sensitivity to vemurafenib.     

Generation of renewable mouse intestinal epithelial cell monolayers and organoids for functional analyses

Background

Conditional reprogramming has enabled the development of long-lived, normal epithelial cell lines from mice and humans by in vitro culture with ROCK inhibitor on a feeder layer. We applied this technology to mouse small intestine to create 2D mouse intestinal epithelial monolayers (IEC monolayers) from genetic mouse models for functional analysis.

Results

IEC monolayers form epithelial colonies that proliferate on a feeder cell layer and are able to maintain their genotype over long-term passage. IEC monolayers form 3D spheroids in matrigel culture and monolayers on transwell inserts making them useful for functional analyses. IEC monolayers derived from the Cystic Fibrosis (CF) mouse model CFTR ?F508 fail to respond to CFTR activator forskolin in 3D matrigel culture as measured by spheroid swelling and transwell monolayer culture via Ussing chamber electrophysiology. Tumor IEC monolayers generated from the Apc^Min/+ mouse intestinal cancer model grow more quickly than wild-type (WT) IEC monolayers both on feeders and as spheroids in matrigel culture.

Conclusions

These results indicate that generation of IEC monolayers is a useful model system for growing large numbers of genotype-specific mouse intestinal epithelial cells that may be used in functional studies to examine molecular mechanisms of disease and to identify and assess novel therapeutic compounds.

     

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases,Drug Metabolism,and Toxicity: Emerging Culture Paradigms and Applications

Recent research has shown that the maintenance of relevant liver functions ex vivo requires models in which the cells exhibit an in vivo‐like phenotype, often achieved by reconstitution of appropriate cellular interactions. Multiple different models have been presented that differ in the cells utilized, media, and culture conditions. Furthermore, several technologically different approaches have been presented including bioreactors, chips, and plate‐based systems in fluidic or static media constituting of chemically diverse materials. Using such models, the ability to predict drug metabolism, drug toxicity, and liver functionality have increased tremendously as compared to conventional in vitro models in which cells are cultured as 2D monolayers. Here, the authors highlight important considerations for microphysiological systems for primary hepatocyte culture, review current culture paradigms, and discuss their opportunities for studies of drug metabolism, hepatotoxicity, liver biology, and disease.     

Vitamin D, tamoxifen and β-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium

Reciprocal interactions between tumor cells and endothelial cells constitute the most important stage of tumor metastasis. There is growing evidence suggesting that β-estradiol and vitamin D modulate the progression of steroid-sensitive breast cancers. In keeping with those results, the purpose of the study reported here was to determine the cytotoxic and antiproliferative activity of tamoxifen (TAM) in the T47D human breast cancer cell line depending on the cell culture model (three-dimensional (3D, spheroid) or two-dimensional (2D, monolayer)) and to estimate the antiproliferative activity of vitamin D in balanced TAM/β-estradiol conditions. The study was also designed to investigate whether vitamin D might influence interleukin-6 (IL-6) and metalloproteinase-2 (MMP-2) production in a co-culture of T47D cell spheroids with an endothelial cell monolayer in the presence of β-estradiol and TAM. Spectrophotometric analysis with MTT revealed that the cytotoxic and antiproliferative activity of TAM was dependent on the culture model, the density of cell culture, and culture medium supplements. In balanced TAM/β-estradiol medium, vitamin D only slightly inhibited T47D cell proliferation in both 2D and 3D cultures. Direct contact of tumor cell spheroids with the endothelium induced production of MMP-2 and IL-6, which was significantly inhibited in TAM/β-estradiol balanced medium. Addition of vitamin D further inhibited MMP-2 production, but enhanced the production of IL-6 as was shown by ELISA assay. Our co-culture model in TAM/β-estradiol balanced medium proved to be useful for examining direct and paracrine interactions of tumor cells with the endothelium in conditions that were closer to in vivo conditions than in the standard 2D model.     

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model

BackgroundMesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment.AimIn light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device.MethodsThe conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium.ResultsThe A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups.ConclusionsHyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.     

Modeling mechanical inhomogeneities in small populations of proliferating monolayers and spheroids

Understanding the mechanical behavior of multicellular monolayers and spheroids is fundamental to tissue culture, organism development, and the early stages of tumor growth. Proliferating cells in monolayers and spheroids experience mechanical forces as they grow and divide and local inhomogeneities in the mechanical microenvironment can cause individual cells within the multicellular system to grow and divide at different rates. This differential growth, combined with cell division and reorganization, leads to residual stress. Multiple different modeling approaches have been taken to understand and predict the residual stresses that arise in growing multicellular systems, particularly tumor spheroids. Here, we show that by using a mechanically robust agent-based model constructed with the peridynamic framework, we gain a better understanding of residual stresses in multicellular systems as they grow from a single cell. In particular, we focus on small populations of cells (1–100 s) where population behavior is highly stochastic and prior investigation has been limited. We compare the average strain energy density of cells in monolayers and spheroids using different growth and division rules and find that, on average, cells in spheroids have a higher strain energy density than cells in monolayers. We also find that cells in the interior of a growing spheroid are, on average, in compression. Finally, we demonstrate the importance of accounting for stochastic fluctuations in the mechanical environment, particularly when the cellular response to mechanical cues is nonlinear. The results presented here serve as a starting point for both further investigation with agent-based models, and for the incorporation of major findings from agent-based models into continuum scale models when explicit representation of individual cells is not computationally feasible.     

Transforming growth factor-β1 (TGF-β1) and acetylcholine (ACh) alter nitric oxide (NO) and interleukin-1β (IL-1β) secretion in human colon adenocarcinoma cells

Colon adenocarcinoma is one of the most common fatal malignancies in Western countries. Progression of this cancer is dependent on tumor microenvironmental signaling molecules such as transforming growth factor-β (TGF-β) or acetylcholine (ACh). The present study was conducted to assess the influence of recombinant human transforming growth factor (rhTGF)-β1 or ACh on nitric oxide (NO) and interleukin-1β (IL-1β) secretion by three human colon adenocarcinoma cell lines: HT29, LS180, and SW948, derived from different grade tumors (Duke’s stage). The cells were cultured in 2D and 3D (spheroids) conditions. Colon carcinoma cells exhibited different sensitivities to rhTGF-β1 or ACh dependent on the tumor grade and the culture model. ACh exhibited significant inhibitory effects towards NO, endothelial nitric oxide synthase (eNOS), and IL-1β secretion especially by tumor cells derived form Duke’s C stage of colon carcinoma. rhTGF-β1 also decreased NO, IL-1β, and eNOS expression, but its effect was lower than that observed after the administration of ACh. The inhibition of NO and IL-1β production was more striking in 3D tumor spheroids than in 2D culture monolayers. Taken together, the TGF-β1–ACh axis may regulate colon carcinoma progression and metastasis by altering NO secretion and influence inflammatory responses by modulating IL-1β production.     

Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents

In this study, we asked the question of whether cells in 3D culture cope more effectively with cytotoxic agents than cells in 2D. The sensitivities of human skin cells (keratinocytes, dermal fibroblasts and endothelial cells) to oxidative stress (hydrogen peroxide) and to a potentially toxic heavy metal (silver) when cultured under 2D and 3D conditions were investigated. The results show a marked resistance of cells to a given dose of hydrogen peroxide or silver nitrate causing a 50% loss of viability in 3D cultures, when compared to the same cells grown in 2D. There was also an improvement in the ability of cells to withstand both stresses when cells were in co-culture rather than in mono-culture. Foetal calf serum was found to have a mild protective effect in 2D culture but this was not extended to findings in 3D culture. This study suggests that dermatotoxicity testing using 3D co-cultures might be more likely to reflect true physiological responses to xenobiotic materials than existing models that rely on 2D mono-cultures.