3D Organotypic Culture Research Articles

Abstract P2-23-19: Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform

Abstract Tumors are multicellular ecosystems that communicate through the exchange of extracellular signaling molecules. In breast cancer, this multicellular communication can enable tumor cells to cooperate in a range of contexts, including tumor invasion and the outgrowth of distal metastases. Illuminating the mechanisms by which tumor cells communicate may reveal new therapies. To this end, three-dimensional tumor organoid models have emerged as versatile platforms for modeling multicellular behavior ex vivo. However, organoid culture typically requires the use of poorly defined, animal-derived extracellular matrices, such as Matrigel. These exogenous matrices contain thousands of proteins that dominate over and conceal cell-secreted factors in conventional proteomics approaches. Revealing low abundance cell-secreted factors from this complex exogenous background presents a formidable challenge. To surmount this challenge, we develop a new method to isolate the pericellular proteome in 3D organotypic culture models. This technology harnesses biorthogonal click chemistry to bypass exogenous factors, infiltrate intercellular spaces, and retrieve the endogenous intercellular proteome. This approach requires no genetic manipulation, requires only 1 million cells, and can be adapted to diverse organotypic models. These capabilities enable isolation of intercellular signaling factors in models that yield precious amounts of material and that can sustain only limited manipulation ex vivo, which we demonstrate using organoids established from patient-derived xenografts. To establish the generalizability of our approach, we apply this technology to a panel of breast cancer and small-cell lung cancer organoid models. Furthermore, we demonstrate that this method can be readily adapted to different extracellular matrix environments that are widely employed in organoid research, including basement membrane matrices and collagen gels. Taken together, our results establish this technology as a scalable and generalizable platform that may open opportunities for researchers investigating diverse questions in organoid models of cancer. Moving forward, we are leveraging these capabilities to investigate changes in the intercellular proteome that emerge after the acquisition of therapy resistance. We hope to uncover new mechanisms of collective signaling that may be targeted to overcome therapy resistance. Citation Format: Brad A. Krajina, Ami Yamamoto, Kevin J. Cheung, Samuel Madasu. Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-19.

Technological advances in three-dimensional skin tissue engineering

Abstract Tissue engineering is an enabling technology that can be used to repair, replace, and regenerate different types of biological tissues and holds great potential in various biomedical applications. As the first line of defense for the human body, the skin has a complex structure. When skin is injured by trauma or disease, the skin tissues may regenerate under natural conditions, though often resulting in irreversible and aesthetically unpleasant scarring. The development of skin tissue engineering strategies was reviewed. Although the traditional approaches to skin tissue engineering have made good progress, they are still unable to effectively deal with large-area injuries or produce full-thickness grafts. In vitro three-dimensional (3D) skin constructs are good skin equivalent substitutes and they have promoted many major innovative discoveries in biology and medicine. 3D skin manufacturing technology can be divided into two categories: scaffold-free and scaffold-based. The representatives of traditional scaffold-free approaches are transwell/Boyden chamber approach and organotypic 3D skin culture. Because of its low cost and high repeatability, the scaffold-free 3D skin model is currently commonly used for cytotoxicity analysis, cell biochemical analysis, and high-throughput cell function. At present, many drug experiments use artificial skin developed by traditional approaches to replace animal models. 3D bioprinting technology is a scaffold-based approach. As a novel tissue manufacturing technology, it can quickly design and build a multi-functional human skin model. This technology offers new opportunities to build tissues and organs layer by layer, and it is now used in regenerative medicine to meet the increasing need for tissues and organs suitable for transplantation. 3D bioprinting can generate skin substitutes with improved quality and high complexity for wound healing and in vitro disease modeling. In this review, we analyze different types of conventional techniques to engineer skin and compare them with 3D bioprinting. We also summarized different types of equipment, bioinks, and scaffolds used in 3D skin engineering. In these skin culture techniques, we focus on 3D skin bioprinting technology. While 3D bioprinting technology is still maturing and improvements to the techniques and protocols are required, this technology holds great promise in skin-related applications.

Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro.

Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.

Incorporating NK Cells in a Three-Dimensional Organotypic Culture System for Human Skin Stem Cells: Modeling Skin Diseases and Immune Cell Interplay.

Natural killer (NK) cells are a part of a sophisticated immune system that is necessary for the skin because it is a crucial organ that is continually exposed to environmental influences. Recent studies have shown that NK cell incorporation into three-dimensional (3D) organotypic culture systems for human skin stem cells provides a physiologically relevant environment to study the interactions between immune cells and skin cells, making it a powerful tool for simulating skin diseases and researching these interactions. It has been shown that adding NK cells to 3D organotypic culture systems can improve keratinocyte differentiation and control inflammation in a variety of skin conditions, including psoriasis. In order to increase our knowledge of skin diseases and immune cell interactions, this work intends to propose an optimum approach for adding NK cells to a 3D organotypic culturing system for human skin stem cells. By better comprehending these relationships, researchers hope to develop novel treatments for skin diseases that are more effective and cause fewer side effects than current treatments. To completely understand the mechanisms underlying these interactions and to create new treatments for skin diseases, more research is required. In conclusion, NK cell integration into 3D organotypic culture systems offers a potent tool to investigate immune cell interactions with skin cells in a physiologically appropriate setting, which may result in major improvements in the treatment of skin diseases.

Collective cell migration is spatiotemporally regulated during mammary epithelial bifurcation.

Branched epithelial networks are generated through an iterative process of elongation and bifurcation. We sought to understand bifurcation of the mammary epithelium. To visualize this process, we utilized three-dimensional (3D) organotypic culture and time-lapse confocal microscopy. We tracked cell migration during bifurcation and observed local reductions in cell speed at the nascent bifurcation cleft. This effect was proximity dependent, as individual cells approaching the cleft reduced speed, whereas cells exiting the cleft increased speed. As the cells slow down, they orient both migration and protrusions towards the nascent cleft, while cells in the adjacent branches orient towards the elongating tips. We next tested the hypothesis that TGF-β signaling controls mammary branching by regulating cell migration. We first validated that addition of TGF-β1 (TGFB1) protein increased cleft number, whereas inhibition of TGF-β signaling reduced cleft number. Then, consistent with our hypothesis, we observed that pharmacological inhibition of TGF-β1 signaling acutely decreased epithelial migration speed. Our data suggest a model for mammary epithelial bifurcation in which TGF-β signaling regulates cell migration to determine the local sites of bifurcation and the global pattern of the tubular network.

3D Oral Mucosal Models for Studying Host-Pathogen Interactions in Periodontal Disease

The host immune response to the oral biofilm plays a central role in the aetiology of periodontitis. Our understanding of hostpathogen interactions has historically been reliant on monolayer culture systems stimulated by microorganisms. However, 2D culture systems possess major shortcomings as these do not adequately mimic the complexities of the native tissue and therefore fail to provide meaningful information on the pathogenesis of periodontal disease. To mimic the in vivo condition, various types of 3D oral mucosal equivalents (OME) have been employed, including reconstituted models and 3D organotypic culture models. The rationale for OME is to serve as a relevant in vitro tool to examine the interactions of human epithelial cells with bacterial biofilms, to understand the process of epithelial layer damage, molecular mechanisms underpinning the development of periodontitis and models for testing novel therapeutics. Furthermore, to gain a deeper understanding of periodontal dysbiosis, it is important to appreciate the molecular mechanisms underlying host epithelial cell production of cytokines and chemokines in response to bacterial stimulation; and signalling pathways which underpin host-pathogen interactions such as mitogen-activated protein kinases (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFkB). Thus, the aim of this narrative review is to summarise the evidence regarding the application of 3D oral mucosal models in host-pathogen infection studies relating to periodontal disease, review the molecular mechanisms underlying host response and provide recommendations for future studies in this field.

A New Organotypic 3D Slice Culture of Mouse Meibomian Glands Reveals Impact of Melanocortins

The meibomian glands (MGs) within the eyelids produce a lipid-rich secretion that forms the superficial layer of the tear film. Meibomian gland dysfunction (MGD) results in excessive evaporation of the tear film, which is the leading cause of dry eye disease (DED). To develop a research model similar to the physiological situation of MGs, we established a new 3D organotypic slice culture (OSC) of mouse MGs (mMGs) and investigated the effects of melanocortins on exocrine secretion. Tissue viability, lipid production and morphological changes were analyzed during a 21-day cultivation period. Subsequently, the effects on lipid production and gene expression were examined after stimulation with a melanocortin receptor (MCR) agonist, α-melanocyte-stimulating hormone (α-MSH), and/or an MCR antagonist, JNJ-10229570. The cultivation of mMGs OSCs was possible without impairment for at least seven days. Stimulation with the MCR agonists induced lipid production in a dose-dependent manner, whereas this effect was tapered with the simultaneous incubation of the MCR antagonist. The new 3D OSC model is a promising approach to study the (patho-) physiological properties of MG/MGD while reducing animal studies. Therefore, it may accelerate the search for new treatments for MGD/DED and lead to new insights, such as that melanocortins likely stimulate meibum production.

A High-Throughput Platform for Culture and 3D Imaging of Organoids.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

Human Papillary and Reticular Fibroblasts Show Distinct Functions on Tumor Behavior in 3D-Organotypic Cultures Mimicking Melanoma and HNSCC.

Human dermis can be morphologically divided into the upper papillary and lower reticular dermis. Previously, we demonstrated that papillary (PFs) and reticular (RFs) fibroblasts show distinct morphology and gene expression profiles. Moreover, they differently affect tumor invasion and epithelial-to-mesenchymal transition (EMT) in in vitro 3D-organotypic cultures of cutaneous squamous cell carcinoma (cSCC). In this study, we examined if these distinct effects of PFs and RFs can be extrapolated in other epithelial/non-epithelial tumors such as melanoma and head and neck squamous cell carcinoma (HNSCC). To this end, 3D-Full-Thickness Models (FTMs) were established from melanoma (AN and M14) or HNSCC cell lines (UM-SCC19 and UM-SCC47) together with either PFs or RFs in the dermis. The interplay between tumor cells and different fibroblasts was investigated. We observed that all the tested tumor cell lines showed significantly stronger invasion in RF-FTMs compared to PF-FTMs. In addition, RF-FTMs demonstrated more tumor cell proliferation, EMT induction and basement membrane disruption. Interestingly, RFs started to express the cancer-associated fibroblast (CAF) biomarker α-SMA, indicating reciprocal interactions eventuating in the transition of RFs to CAFs. Collectively, in the melanoma and HNSCC FTMs, interaction of RFs with tumor cells promoted EMT and invasion, which was accompanied by differentiation of RFs to CAFs.

Lymphatic cells do not functionally integrate into 3D organotypic brain slice cultures, but aggregate around penetrating blood vessels.

Brain slice culture (BSC) is a well-known three-dimensional model of the brain. In this study, we use organotypic slices for studying neuro-lymphatic physiology, to directly test the longstanding assumption that the brain is not a hospitable milieu for typical lymphatic vessels. An additional objective is to model fluid egress through brain perivascular space systems and to visualize potential cellular interactions among cells in the leptomeninges including alterations of cellular geometry and number of processes. Immortalized lymphatic rat cell lines were used to seed organotypic brain slices. The brain slice model was characterized by monitoring morphologies, growth rates, degree of apoptosis, and transport properties of brain slices with or without a lymphatic component. The model was then challenged with fibroblast co-cultures, as a control cell that is not normally found in the brain. Immortalized lymphatic cells penetrated the brain slices within 2-4days. Typical cell morphology is spindly with bipolar and tripolar forms well represented. Significantly more indigo carmine marker passed through lymphatic seeded BSCs compared to arachnoid BSCs. Significantly more indigo carmine passed through brain slices co-cultured with fibroblast compared to lymphatic and arachnoid BSCs alone. We have developed an organotypic model in which lymphatic cells are able to interact with parenchymal cells in the cerebrum. Their presence appears to alter the small molecule transport ability of whole-brain slices. Lymphatic cells decreased dye transport in BSCs, possibly by altering the perivascular space. Given their direct contact with the CSF, they may affect convectional and diffusional processes. Our model shows that a decrease in lymphatic cell growth may reduce the brain slice's transport capabilities.

847 Modeling basal cell carcinoma in 3D organotypic raft cultures

Basal cell carcinoma (BCC) is the most-commonly diagnosed cancer worldwide with limited availability for non-surgical treatments. The Hedgehog signaling cascade drives tumorigenesis, but efforts to develop effective chemotherapeutics have been thwarted by chemoresistant tumor recurrence. A contributing factor to this is an overall lack of human-based preclinical models. We endeavored to develop 3D organotypic raft models that mimic characteristics of human BCC by combining primary normal human epidermal keratinocytes (NHEK) with established murine BCC cells.

Xeno-free self-assembling peptide scaffolds for building 3D organotypic skin cultures.

Organotypic skin cultures represent in vitro models of skin which can be used for disease modeling, tissue engineering, and screening applications. Non‐human collagen is currently the gold standard material used for the construction of the supporting matrix, however, its clinical applications are limited due to its xenogeneic origin. We have developed a novel peptide hydrogel‐based skin construct that shows a pluristratified epidermis, basement membrane, and dermal compartment after 3 weeks of in vitro culture. Peptide‐based constructs were compared to collagen‐based constructs and stratification marker expression was histologically higher in peptide constructs than in collagen constructs. Transepithelial electrical resistance also showed mature barrier function in peptide constructs. This study presents a novel application of the self‐assembling peptide hydrogel in a defined xeno‐free in vitro system.

Abstract 1096: Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy

Abstract Aggressive lung cancers such as non-small cell lung carcinomas (NSCLC) commonly recur following first line therapy. Recurring tumors metastasize more rapidly. We hypothesize that drug-resistant phenotypes created by the selection pressure of first line treatments such as platinum-based cytotoxic agents contribute to the increased aggressivity of recurring NSCLC. We showed previously that an engineered model of cisplatin-resistant NSCLC tissues secretes more inflammatory cytokines compared to untreated controls and induces more severe muscle tissue injury in a microphysiological model of cachexia processes. Here, we report methods for the systematic generation of cisplatin-resistant A549 lung adenocarcinoma cell cultures and demonstrate their increased aggressivity in 3D spheroid culture and in 3D organotypic culture with patient-derived lung cancer-associated fibroblasts (CAF). We treated A549 cells with cisplatin for 96 hours at doses of 15-45µM, yielding cultures of 54.8-8.9% surviving cells as quantified via flow cytometric of cell size and permeability. Here we report characterization of spheroids and tissues composed of cells selected for resistance with 25 µM cisplatin (Cis-R) compared with untreated parent A549. This dose was selected because we were unable to recover proliferating cultures with lower percentages of surviving. Cis-R spheroids exhibited a significant reduction in circularity that is indicative of increased asymmetry. We measured a Ki67 index of 6.4 +/- 1.4% for parent A549 spheroids and 49.7 +/- 9.5% for Cis-R spheroids. The marked increase in cell proliferation of CisR spheroids correlated with increased distances of invasion and more mesenchymal-like cell morphologies of invading cells in 3D cultures of Cis-R spheroids without CAF. These data suggest that cisplatin resistant A549 created using our protocol are more proliferative and invasive in 3D culture environments due to cell intrinsic changes. Organotypic 3D cultures of Cis-R spheroids and CAF exhibited an increased density and alignment of myofibroblast-like CAF with smooth muscle actin cytoskeleton when compared to cultures of parent spheroids with the same CAF. Our results demonstrate proof of principle for modeling recurrent tumors with more aggressive phenotypes using this approach. Future experiments will translate these methods to patient-derived cultures and test the hypothesis that cisplatin-resistant recurrent NSCLC stimulates more severe local and distant stromal reactions as a driver of metastasis. Engineering validated models of drug-resistant recurring cancers will help accelerate the discovery of anti-metastasis therapies in human cell-based microphysiological systems. Citation Format: Omar M. Ahmad, Elisabet A. Olsen, Delia A. Carlino, Charles E. Byrne, Mark Mondrinos. Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy [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 1096.

Abstract 5948: Modeling lung squamous cell carcinoma progression using primary human bronchial basal cells

Abstract Background: Lung squamous cell carcinoma (LUSC) arises from the accumulation of genetic driver alterations in bronchial basal cells. LUSC genomes lack obvious actionable mutations and present with a high degree of inter-tumor heterogeneity. Tractable, patient relevant models are needed to study LUSC pathogenesis and investigate the role of recurrent genomic alterations in driving cancer progression. Therefore, we are developing a tractable human model of LUSC progression to increase our understanding of this disease. Methods: To investigate the contribution and co-operation of frequently mutated genes (TP53 and CDKN2A) and commonly deregulated pathways (SOX2/P63, PI3K/AKT and KEAP1/NRF2 pathways) in driving LUSC progression, we genetically engineered primary human bronchial basal cells. This culminated in eight basal cell mutants harboring ubiquitous LOF alterations in TP53 and CDKN2A combined with additional pathway alterations. To assess the function of the genetic alterations in LUSC progression, we investigated proliferation using cell cycle analysis and colony forming assays. Air-liquid interface organotypic cultures were used to model histological changes in the bronchial epithelium driven by different combinations of mutations. The resultant cultures were analyzed for changes in histology indicative of LUSC progression using immunostaining to evaluate changes to epithelial cell proliferation, and expression of LUSC biomarkers. Results: Increases in proliferation were driven by inactivation of TP53, CDKN2A and PTEN, whereas SOX2 overexpression inhibited mutant basal cell proliferation. LUSC alterations also drove striking phenotypic changes in 3D organotypic cultures of the bronchial epithelium. All mutant cultures displayed thickening of the bronchial epithelium and significantly increased cellularity (p<0.001), indicating hyper-proliferation. SOX2 overexpression inhibited mucociliary differentiation and drove the appearance of a disordered epithelium with clear cellular and nuclear pleomorphism, indicative of high-grade premalignant lesions. Conclusions: We developed a basal cell model of recurrent LUSC genetic alterations. This approach is unique due to [1] the use of primary human cells and [2] the representation of interpatient heterogeneity and developmental stages by introducing different combinations of pathway alterations. Early results indicate that basal cells carrying cumulative alterations display increasingly complex phenotypes with features of low and high-grade premalignant lesions. Further work is required to assess the tumorigenicity of mutant cells. Citation Format: Julia Ogden, Caroline Dive, Carlos Lopez Garcia. Modeling lung squamous cell carcinoma progression using primary human bronchial basal cells [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 5948.

Automated high-speed 3D imaging of organoid cultures with multi-scale phenotypic quantification.

Current imaging approaches limit the ability to perform multi-scale characterization of three-dimensional (3D) organotypic cultures (organoids) in large numbers. Here, we present an automated multi-scale 3D imaging platform synergizing high-density organoid cultures with rapid and live 3D single-objective light-sheet imaging. It is composed of disposable microfabricated organoid culture chips, termed JeWells, with embedded optical components and a laser beam-steering unit coupled to a commercial inverted microscope. It permits streamlining organoid culture and high-content 3D imaging on a single user-friendly instrument with minimal manipulations and a throughput of 300 organoids per hour. We demonstrate that the large number of 3D stacks that can be collected via our platform allows training deep learning-based algorithms to quantify morphogenetic organizations of organoids at multi-scales, ranging from the subcellular scale to the whole organoid level. We validated the versatility and robustness of our approach on intestine, hepatic, neuroectoderm organoids and oncospheres.

Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

Collective detachment of cancer cells at the invading front could generate efficient metastatic spread. However, how cancer cell clusters shed from the leading front remains unknown. We previously reported that the dynamic expression of CD44 in breast cancers (BrCas) at collectively invading edges was associated with tumor-associated macrophages (TAMs). In this study, we first observed that the highly expressed CD44 (CD44high) cancer cell clusters were located in the BrCa circulating vessels, accompanied by CD206+ TAMs. Next, we identified that the cancer cell clusters can be converted to an invasive CD44high state which was induced by TAMs, thus giving rise to CD44-associated signaling mediated cohesive detachment. Then, we showed that disrupting CD44-signaling inhibited the TAMs triggered cohesive detaching using 3D organotypic culture and mouse models. Furthermore, our mechanistic study showed that the acquisition of CD44high state was mediated by the MDM2/p53 pathway activation which was induced by CCL8 released from TAMs. Blocking of CCL8 could inhibit the signaling cascade which decreased the CD44-mediated cohesive detachment and spread. Our findings uncover a novel mechanism underlying collective metastasis in BrCas that may be helpful to seek for potential targets.

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells.

Simple SummaryThree-dimensional (3D) cell cultures have several advantages over conventional monolayer two-dimensional (2D) cultures as they can better mimic tumor biology. This study delineates the changes in microRNAs (miRNAs) expression patterns of breast cancer cells cultured in 3D and 2D conditions. 3D organotypic cultures showed morphological changes such as cell–cell and cell–extracellular matrix interactions associated with a loss of polarity and reorganization on bulk structures in both basal Hs578T and luminal T47D breast cancer cells. Data indicate that downregulated miRNAs in Hs578T 3D cultures, relative to the 2D condition, contribute to a positive regulation of biological processes such as response to hypoxia and focal adhesion, whereas overexpressed miRNAs were related to negative regulation of the cell cycle. Remarkably, the reprogramming of miRNAs transcriptional profile was accompanied by changes in the expression of key miRNA/mRNA coregulation networks, such as miR-935/HIF-1A, which correlated with the expression found in clinical breast tumors and predicted poor patient outcomes. These data have implications in our understanding of cancer biology and impact the miRNA/mRNA regulatory axes of cells grown in 3D cultures. Our data represent a guide for novel miRNAs candidates for functional analysis, including the response to therapy and biomarkers discovery in breast cancer.The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell–cell, and cell–ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer.

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium.

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast‐derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D‐organotypic (3D‐OT) cultures were constructed. Various growth factors were added at a range of 0.1‐100 ng/ml. 3D‐OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D‐OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage‐colony stimulating factor (GM‐CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM‐CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1‐integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL‐positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM‐CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast‐derived diffusible factor(s).

Technologies to Assess Drug Response and Heterogeneity in Patient-Derived Cancer Organoids.

Patient-derived cancer organoids (PDCOs) are organotypic 3D cultures grown from patient tumor samples. PDCOs provide an exciting opportunity to study drug response and heterogeneity within and between patients. This research can guide new drug development and inform clinical treatment planning. We review technologies to assess PDCO drug response and heterogeneity, discuss best practices for clinically relevant drug screens, and assert the importance of quantifying single-cell and organoid heterogeneity to characterize response. Autofluorescence imaging of PDCO growth and metabolic activity is highlighted as a compelling method to monitor single-cell and single-organoid response robustly and reproducibly. We also speculate on the future of PDCOs in clinical practice and drug discovery.Future development will require standardization of assessment methods for both morphology and function in PDCOs, increased throughput for new drug development, prospective validation with patient outcomes, and robust classification algorithms.

In vitro modelling of the mucosa of the oesophagus and upper digestive tract: narrative review

: This review discusses the utility and limitations of model gut systems in accurately modelling the mucosa of the digestive tract from both an anatomical and functional perspective, with a particular focus on the oesophagus and the upper digestive tract, and what this means for effective in vitro modelling of oesophageal pathology. Disorders of the oesophagus include heartburn, dysphagia, eosinophilic oesophagitis, achalasia, oesophageal spasm and gastroesophageal reflux disease. 3D in vitro models of the oesophagus, such as organotypic 3D culture and spheroid culture, have been shown to be effective tools for investigating oesophageal pathology. However, these models are not integrated with modelling of the upper digestive tract—presenting an opportunity for future development. Reflux of upper gastrointestinal contents is a major contributor to oesophageal pathologies like gastroesophageal reflux disease and Barratt’s oesophagus, and in vitro models are essential for understanding their mechanisms and developing solutions. The limitations of current model gut systems in modelling the mucosa is not only limited to the oesophagus. Integration of modelling of the mucus covered epithelia of the stomach and small intestine in to upper digestive tract models is limited and often not considered at all. In this paper we discuss mucus structure and function and current approaches to modelling of the mucus layer in isolation, and in integrated systems with cell culture systems and digestive models. We identify a need for relevant modelling of the viscoelastic properties of mucus and its protective function to allow complete integration in modelling. Addressing limitations of current in vitro models and integrating upper gastrointestinal models with those of the oesophagus presents an opportunity for better understanding oesophageal physiology and pathophysiology where reflux of digestive fluids is involved.