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Cancer cells require nutrients to grow and proliferate. The availability of the amino acid cysteine impacts whether cancer cells die by an iron-dependent, non-apoptotic form of cell death executed by lipid peroxidation called ferroptosis. Ferroptosis can be induced if the availability of the amino acid cysteine is limiting or by inhibiting the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). Extracellular metabolites such as monounsaturated fatty acids (MUFAs) and cysteine can protect from ferroptosis. However, it is unclear how cancer cells acquire cysteine to evade ferroptosis or what MUFAs they can use to evade ferroptosis. Using human-derived cancer cell lines in both adherent (2D) and suspension (3D) culture, this study demonstrates that the cysteine-rich extracellular protein albumin protects from ferroptosis caused by cysteine deprivation. Albumin in combination with inhibition of the nutrient and energy sensing kinase, mammalian target of rapamycin complex 1 (mTORC1), is required to robustly protect from cysteine deprivation-induced ferroptosis in 2D. This combination treatment protects from ferroptosis by increasing intracellular levels of cysteine. Furthermore, this process is dependent on the catabolism of albumin into its amino acid constituents. Additionally, albumin protects from cystine deprivation-induced ferroptosis in 3D culture. This study reveals that a variety of exogenous MUFAs that have not previously been characterized as ferroptosis-protective metabolites protect HT-1080 cells in monolayer and HT-1080 and Panc-1 spheroids from GPX4 inhibition-induced ferroptosis. These MUFAs exhibit different protective abilities in adherent compared to spheroid culture and this suggests growth-context dependent differences in MUFA protection. These results describe novel metabolite-mediated strategies of ferroptosis evasion in 2D and 3D culture. Understanding how cells utilize exogenous protective metabolites such as albumin and MUFAs in death-inducing conditions can reveal novel ways to induce more complete cancer cell death by curtailing alternative avenues of protection. Targeting these mechanisms to induce more effective cell death in cancer cells reveals potential therapeutic relevance of this work.

Tumors are three-dimensional (3D) entities characterized by complex structural architecture which is necessary for adequate intercellular, intracellular and cell-to-matrix interactions among the aberrant cells in cancer. In the field of cancer research, 2D cell cultures are traditionally used for decades in the majority of experiments. The reasons for this are the vast benefits these models provide, including simplicity and cost effectiveness. However, it is now known that these models are exposed to much higher stiffness, they lose physiological extracellular matrix (ECM) on artificial plastic surfaces as well as differentiation, polarization and cell-cell communication. This leads to the loss of crucial cellular signaling pathways and changes in cell responses to stimuli when compared to in vivo conditions. Moreover, they cannot adequately mimic the complexity and dynamic interactions of the tumor microenvironment (TME) which is of great importance in anticancer drug treatments. 3D models seem more biomimetic compared to 2D cell monolayers because they offer the opportunity to model the cancer mass together with its environment which seems the key factor in promoting and directing cancer invasion. 3D cell culture with its additional dimensionality makes the difference in cellular responses because it influences the spatial and physical aspects of the cells in 3D culture. This affects the signal transduction and makes the behavior of 3D-cultured cells more physiologically relevant and reflective of in vivo cellular responses. This review focuses on major differences between 2D and 3D cell cultures, highlighting the importance of considering bioengineering humanized 3D cancer models as the future in cancer research. Additionally, it presents diverse 3D models currently used in cancer research, outlining their benefits and limitations. Precisely, this review highlights the differences between the 3D models with the focus on tumor stroma interactions, cell population and extracellular matrix composition providing methods and examples for each model from the studies done so far.

Spheroids based on chondrocytes are gaining interest in the treatment of injured cartilage tissue due to their regenerative potential. In this study, chondrocyte-based aggregates were formed by culturing cells on a polymeric coating of a lactose-modified chitosan (CTL) and their evolution was followed for 14 days of culture. Morphological analyses through scanning electron microscopy indicated that these cell aggregates formed by a process of cell aggregation rather than proliferation, resulting in structures with an irregular morphology that were up to 1 mm in size. Chondrocytes interact very closely with CTL. In the early days of cellular aggregation, CTL is distributed among the cells, while in later stages, it is localized in the inner part of the aggregates, forming an amorphous matrix. The initial outer position of the polymer is replaced over time by an increased matrix deposition whose main component is type II collagen. Transmission electron microscopy pointed out that the chondrocytes internalized the polymer during the first days of culture. The presence of CTL does not affect the ability of cells to migrate from chondro-aggregates when transferred to uncoated wells, mimicking the ability of cells to colonize a cartilage defect. Overall, these results suggest that CTL plays a role as a temporary matrix for chondrocytes aggregation during chondro-aggregates formation. This opens up innovative therapeutic approaches for cartilage regeneration without the need to use a scaffold to deliver cells to the damaged site.KEYWORDS: chondrocytes, spheroids, lactose-modified chitosan, cartilage regeneration, bioactive polysaccharide

Breast cancer (BC) is a heterogenous disease classified into four molecular subtypes (Luminal A, Luminal B, HER2 and triple-negative (TNBC)) depending on the expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal receptor 2 (HER2). The development of effective treatments for BC, especially TNBC, remains a challenge. Aminosteroid derivative RM-581 has previously shown an antiproliferative effect in multiple cancers in vitro and in vivo. In this study, we evaluated its effect in BC cell lines representative of BC molecular subtypes, including metastatic TNBC. We found that RM-581 has an antiproliferative effect on all BC molecular subtypes, especially on Luminal A and TNBC, in 2D and 3D cultures. The combination of RM-581 and trastuzumab or trastuzumab-emtansine enhanced the anticancer effect of each drug for HER2-positive BC cell lines, and the combination of RM-581 and taxanes (docetaxel or paclitaxel) improved the antiproliferative effect of RM-581 in TNBC and metastatic TNBC cell lines. We also confirmed that RM-581 is an endoplasmic reticulum (EnR)-stress aggravator by inducing an increase in EnR-stress-induced apoptosis markers such as BIP/GRP78 and CHOP and disrupting lipid homeostasis. This study demonstrates that RM-581 could be effective for the treatment of BC, especially TNBC.

Brain metastases (BM) occur when cancer cells from the primary location spread to the brain through the bloodstream. BM affects approximately 20-40 % of cancer patients, with the majority originating from lung, breast, and skin cancers. If left untreated, the median survival time is usually only one month, and aggressive treatment only extends survival to around four months. Despite the advances in modern medicine, the blood-brain barrier (BBB) poses a significant challenge in treating BM, restricting the entry of approximately 98 % of chemotherapeutic drugs into the brain. Thus, better model systems are needed to understand the complex interplay between cancer cells and normal brain cells. In order to generate novel models, a range of cell lines derived from lung, breast and skin BM were characterized to understand their behaviour in vitro. Subsequently, their distinguishing features were analyzed to identify the most suitable candidates for generating three-dimensional models, which can later be used for drug testing. We found that LBM1, derived from a lung cancer BM, and H16, developed from a melanoma BM, were the best candidates for spheroid formation. The two cell lines underwent treatment with thioridazine, an antipsychotic drug that has been previously tested by our group on various melanoma BM cell lines and demonstrated a significant reduction in their viability. Comparable results were seen with LBM1 and H16 cells. The potential of LBM1- and H16-derived exosomes to serve as imaging agents was also investigated, with focus on iron oxide labelling using carboxyl-coated superparamagnetic iron oxide nanoparticles (SPIONs), through electroporation. To verify the electroporation, further investigation is necessary to confirm the presence of the iron oxide particles within the exosomes. However, dextran-coated SPIONs were successfully internalised by LBM1 cells following 24 h incubation, suggesting its potential labelling attributes and potential uptake by LBM1-derived exosomes. Lastly, LBM1 and H16 cells were injected intracardially into four non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice each for evaluation of their tumourigenic potential. Despite a small sample size, H16-injected mice demonstrated a 23 % increased survival time compared to LBM1-injected mice, but no tumours were observed at week 4. In contrast, 3 out of 4 LBM1-injected mice displayed brain tumours four weeks following injection, thus demonstrating its potential as a model system for future BM research. Masteroppgave i biomedisin BMED395 MAMD-MEDBI

Induced pluripotent stem cells (iPSCs) may develop into any form of cell and are being intensively investigated. The influence on iPSCs of nanostructures generated using two-dimensional colloidal arrays was examined in this study. Colloidal arrays were formed using the following procedure. First, core–shell colloids were adsorbed onto a glass substrate using a layer-by-layer method. Second, the colloids were immobilized via thermal fusion. Third, the surface of the colloids was modified by plasma treatment. By adjusting the number density of colloids, cultured iPSCs were easily detached from the substrate without manual cell scraping. In addition to planar culture, cell aggregation of iPSCs attached to the substrate was achieved by combining hydrophilic surface patterning on the colloidal array. Multilayered cell aggregates with approximately four layers were able be cultured. These findings imply that colloidal arrays might be an effective tool for controlling the strength of cell adhesion.

My technical work and my STS research are connected primarily through the process of obtaining a patent and understanding what implications it has for future engineers who want to develop a novel invention. A patent is a type of intellectual property that gives its owner the legal right to exclude others from creating, utilizing, or selling a novel invention for a limited period of time in exchange for publishing a sufficiently detailed invention. While my technical project focuses on the development of a novel biomedical device, it will require intellectual property for various components and applications. My technical project focuses on the development and testing of an automatic organoid/microsphere movement device. My STS research explores two large pharmaceutical companies that had overlaps in a chemical compound leading to an infringed patent. For my technical project, it is important to understand my STS research as the commercialization of my device would require the protection of my intellectual property. My technical work focuses on designing a device that precisely withdraws and places organoids into a designated location within a permissive biomaterial using an automated set-it-and-forget-it approach. My capstone team created a prototype of an automated device and completed preliminary testing to verify the concept. Our team built the device from an initial design drawing utilizing linear guide rails, stepper motors, a high-definition camera, a nanoliter injector, and CAD-printed components. The device is designed to be used for the application of research on organoids, which are three-dimensional cell cultures constructed from stem cells that function to mimic human organs in-vitro. The machine will be utilized in Dr. Christopher Highley’s lab for continued development and research applications. Our device aims to improve a manual process and improve the precision of current research methods. We also hope to patent the components of the device and commercialize it to allow other researchers and engineers globally to advance research in drug development, the study of diseases, and transplantable organs. My STS research explores an infringed patent between Novartis and Plexxikon Inc. over a pharmaceutical drug sold to Novartis by GlaxoSmithKline. My research focuses on Novartis’s acquisition of the drug Tafinlar and explores the groups involved in the process. My claim is that Novartis did not willingly infringe on Plexxikon’s chemical compound. Instead, GSK willingly sold an infringed drug to Novartis for monetary benefits and to satisfy its shareholders. My paper explores the idea that the United States Patent Office and GlaxoSmithKline did not adequately perform their roles during Novartis’s acquisition of GSK’s oncology portfolio. The goal of my research is to ensure that future engineers develop novel technologies and drugs that do not overlap with proprietary technologies. Working on these projects simultaneously has greatly impacted my overall work. My technical work allowed me to understand the applications of my team's novel device and the proprietary components that could be patented in the future. Similarly, the research I conducted for my STS paper allowed me to understand the complexities of patents and how it is important that I ensure the patents I receive do not overlap with others. This increased my motivation to have a completed prototype capable of picking up and placing organoids to allow for discussions involving the intellectual property of the device. In summary, working on both my STS research paper and my technical project together this past year has allowed me to learn the stages involved in developing technologies and ensuring that the rights of my work are not stolen.

Organoids are 3D tissue constructs that have the ability to replicate structural and functional properties of human organs and tissues, giving them great potential in the field of medicine with applications in the study of disease progression, drug testing and development, and even organ transplantation. Despite this potential, the development of organoids is hindered by the fact that organoids must be seeded into biomaterials for growth and differentiation, a process that is currently done by hand, making it imprecise and time-consuming. These issues caused by manual seeding lead to slower progression in organoid research and technological development. To address these issues our team has created an automated organoid seeding device that utilizes image detection software to precisely and efficiently seed organoids into biomaterials. The current technical limitations are not the only factor holding back organoids as a medical technology, there are also important social factors to be considered, particularly ethics. Organoids are derived from embryonic stem cells (ESCs), which carry significant ethical concerns in the fact that harvesting ESCs can be seen as the ending of a human life. This is an important factor to consider as these concerns potentially impact the amount of investment that organoid research receives and thus its technological development. In order to analyze how a social factor such as ethics can impact the development and evolution of a technology, my research will be done in the context of the theory of social construction of technology (SCOT). This research will be done in the form of semi-structured interviews with both stem-cell researchers and investors in the space along with literature review in order to determine if organoid and ESC research receives lower investment as a result of ethical concerns and if so, how this lack of investment has affected the development of organoids as a technology. Through this research, I expect to discover the extent to which a social factor like ethics can actually impact how a technology like organoids is able to evolve, particularly in relation to the theory of SCOT. The overall goal of the combined results from both the STS research and capstone project is to allow for organoids to reach their full potential as a medical technology so that they can be used in their extensive applications to save lives and create treatments for diseases that currently cannot be effectively treated. With the capstone project addressing one of the major technical issues involved with organoids and the STS research addressing a major social problem, the combination of these results will aim to remove some of the barriers currently holding organoids from reaching their potential.

The interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-β) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-β, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-β, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-β and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach.

Liver is the foremost organ of human being for drug metabolism, and it played a significant role in toxicity evaluation of drugs. Establishing a liver model in vitro can accelerate the process of the drug screening and new drug research and development. We provide a 3D printing based hepatic sinusoid-on-a-chip microdevice that reconstitutes organ-level liver functions to create a drug screening model of toxicity evaluation on chip. The microfluidic device, which recapitulates the hepatic sinusoid microenvironment, consists of PET polyporous membranes which mimic the perisinusoidal space, and experience fluid flow to mimic the hepatic arterial capillaries. The PET membrane was used to separate the hepatocyte and endotheliocyte. The endotheliocyte was cultured on the downside of the membrane and the hepatocyte were 3D seeded on the membrane via the 3D printer. This device was used to reproduce the in vitro liver model for drug toxicity assays. The expression of several biomarkers of liver was compared with the monoculture and 2D cultured conditions, and the results reveal that this organ-on-a-chip microdevice mimics the drug hepatoxicity that has not been possible by 2D cell-based and animal models, providing a useful platform for screening the drugs and developing an effective therapy in hepatopathy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by increased levels of desmoplasia that contributes to reduced drug delivery and poor treatment outcomes. In PDAC, the stromal content can account for up to 90% of the total tumor volume. The complex interplay between stromal components, including pancreatic cancer associated fibroblasts (PCAFs), and PDAC cells in the tumor microenvironment (TME) have a significant impact on prognoses and thus needs to be recapitulated in vitro when evaluating various treatment strategies. This study is a systematic evaluation of photodynamic therapy (PDT) in 3D heterotypic coculture models of PDAC with varying ratios of patient derived PCAFs that simulate heterogenous PDAC tumors with increasing stromal content. The efficacy of antibody-targeted PDT (photoimmunotherapy; PIT) using cetuximab photoimmunoconjugates (PICs) of benzoporphyrin derivative (BPD) is contrasted with that of liposomal BPD (Visudyne®), which is currently in PDT clinical trials for PDAC. We demonstrate that both Visudyne®-PDT and PIT were effective in heterotypic PDAC 3D spheroids with a low stromal content. However, as the stromal content increases above 50% in the 3D spheroids, the efficacy of Visudyne®-PDT is reduced by up to 10-fold, while PIT retains its efficacy. PIT was found to be 10-fold, 19-fold and 14-fold more phototoxic in spheroids with 50%, 75% and 90% PCAFs, respectively, as compared to Visudyne®-PDT. This marked difference in efficacy is attributed to the ability of PICs to penetrate and distribute within spheroids with a higher stromal content, whereas Visudyne® is restricted to the spheroid periphery. This study thus demonstrates how the stromal content in PDAC spheroids directly impacts their responsiveness to PDT and proposes PIT to be a highly suited treatment option for desmoplastic tumors with particularly high degrees of stromal content.

BACKGROUND: Lesions of articular cartilage represent a crucial risk factor for the early development of osteoarthritis. Autologous chondrocyte implantation (ACI) is a well-established procedure in therapy of those lesions in the knee. The aim of the presented study is to detect differences in short-term radiological outcome depending on defect localization (femoral condyle vs. retropatellar) after spheroid-based ACI. OBJECTIVE: This study aimed to demonstrate that radiological outcome after spheroid-based ACI in the knee is independent of defect localization. METHODS: MRI-scans after retropatellar ACI and ACI of the medial/lateral femoral condyle, with a preoperative Outerbridge grade of III or IV were evaluated regarding MOCART 2.0. RESULTS: The mean defect-size was 5.0 ± 1.8 cm2, with a minimum size of 2 cm2 and a maximum size of 9 cm2. Scans were performed 7.7 months (± 3.1 months) postoperatively. The mean MOCART 2.0 score was 78.5 ± 15.6. No statistically significant influence neither of the localization (p= 0.159), the gender (p= 0.124) nor defect size (< 5 cm2 vs. ⩾ 5 cm2; p= 0.201) could be observed. CONCLUSIONS: The presented data demonstrate good to excellent radiological short-term results after spheroid-based ACI. Data indicates, that at least radiological results are independent of gender, defect-size and defect-localization.

Introduction Regenerative medicine is a promising approach for hair loss; however, its primary challenge is the inductivity of human dermal papilla cells (DPCs), which rapidly lose hair growth-inducing properties in 2D culture. Despite extensive research efforts to construct DPCs, current 3D microenvironments fabricated to restore hair inductivity remain insufficient. Objectives Here, we aimed to fabricate ECM-enriched controllable vascularized dermal papilla (DP) spheroids that highly mimic in vivo DPCs microenvironments to restore their hair inductivity. Methods We employed layer-by-layer (LbL) self-assembly using gelatin and alginate to construct nanoscale biomimetic ECM for DPCs, with Ca2+ as a cross-linking agent to create controllable DP spheroids. DPCs were also co-cultured with human umbilical vein endothelial cells to construct vascularized DP spheroids. Immunofluorescence staining and angiography was used to detect angiogenesis in vitro and in vivo. RNA sequencing and in vivo implantation were employed to investigate DPCs signature. Results LbL technology enabled DPCs to aggregate into controllable DP spheroids of size and cell numbers similar to those of primary DP. Vascularization prevented hypoxia-induced necrosis and functioned in association with host vessels post-transplantation. Compared with traditional 3D culture, nanoscale ECM and vascularization were found to restore the transcriptional signature of DPCs and triple hair induction efficiency following engraftment. Conclusion Our novel biomimetic developmental tissue engineering strategy is a crucial step toward the recovery of human DPC hair inductivity, which would enable the rapid clinical application of large-scale hair regeneration platforms.

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments. ispartof: Cell Mol Life Sci vol:80 issue:4 pages:93- ispartof: location:Switzerland status: Published online

Le cancer du poumon non-à-petites-cellules (CPNPC) représente 85% des cas de cancer du poumon. Cependant, les modèles utilisés de culture cellulaire en deux dimensions (2D) représentent partiellement les caractéristiques physiopathologiques du CPNPC. Notre objectif est de réaliser un modèle in vitro plus représentatif des caractéristiques de ce type de cancer. La culture tridimensionnelle (3D) dans laquelle les cellules forment un sphéroïde, est considérée comme un modèle plus fidèle aux tumeurs retrouvées chez les patients grâce à la structure et aux interactions intercellulaires du modèle. Pour mieux représenter le microenvironnement tumoral, nous intégrons une lignée cellulaire de fibroblastes dans le sphéroïde de CPNPC, afin d’imiter l’interaction entre cellules cancéreuses et cellules stromales. En effet, bien que les fibroblastes représentent un faible pourcentage des cellules au sein des tumeurs de CPNPC, elles jouent un rôle prépondérant dans la biologie tumorale et la réponse aux médicaments. Nous avons testé différentes lignées de fibroblastes et différents ratios de co-culture afin de déterminer les conditions optimales de notre modèle de co-culture 3D. Après 7 jours de co-culture, les cellules cancéreuses démontrent un potentiel migratoire plus élevé lorsque mesuré dans des chambres de Boydens. Cet effet n’est dépendant ni de la prolifération, ni d’un changement de phase dans le cycle cellulaire. Nous avons caractérisé la localisation des fibroblastes au sein du sphéroïde par des expériences de microscopie à fluorescence. L’expression de la protéine α-SMA du cytosquelette a aussi été déterminée par immunofluorescence. Nous avons par la suite établi un modèle de co-culture sur 24 jours afin de maximiser la communication entre les cellules cancéreuses et les fibroblastes. Ce modèle de co-culture long terme a par la suite été analysé selon son contenu en cytokines, chimiokines et métabolites. Enfin, nous avons réalisé un criblage de médicaments au jour 24 de notre co-culture long terme afin d’évaluer une réponse thérapeutique des cellules cancéreuses, dans des conditions plus semblables au microenvironnement tumoral., Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases. However, the two-dimensional (2D) cell culture models used represent partially the pathophysiological characteristics of NSCLC. Our goal is to develop an in vitro model that is more representative of the characteristics of this type of cancer. The three-dimensional (3D) culture, in which the cells form a spheroid, is considered to be a model more faithful to the tumors found in patients due to the structure and intercellular interactions of the model. In order to better represent the tumor microenvironment, we are integrating a fibroblast cell line into the spheroid of NSCLC to mimic the interaction between cancer cells and stromal cells. Although fibroblasts represent a small percentage of cells in NSCLC tumors, they play a major role in tumor biology and drug response. We tested different fibroblast cell lines and co-culture ratios to determine the optimal conditions of our 3D co-culture model. After 7 days of co-culture, the cancer cells show a higher migratory potential when measured in Boydens chambers. This effect is not dependent on proliferation or change of phase in the cell cycle. We characterized the localization of fibroblasts within the spheroid by fluorescence microscopy experiments. The expression and localization of the cytoskeletal protein α-SMA was also determined by immunofluorescence. We then established a 24-day co-culture model to maximize communication between cancer cells and fibroblasts. This long-term co-culture model was subsequently analyzed for cytokine, chemokine and metabolite content. Finally, we performed drug screening on day 24 of our long-term co-culture to evaluate a therapeutic response of cancer cells under conditions more similar to the tumor microenvironment.

Here we aimed to establish a simple detection method for detecting circulating tumor cells (CTCs) in the blood sample of colorectal cancer (CRC) patients using poly(2-methoxyethyl acrylate) (PMEA)-coated plates. Adhesion test and spike test using CRC cell lines assured efficacy of PMEA coating. A total of 41 patients with pathological stage II–IV CRC were enrolled between January 2018 and September 2022. Blood samples were concentrated by centrifugation by the OncoQuick tube, and then incubated overnight on PMEA-coated chamber slides. The next day, cell culture and immunocytochemistry with anti-EpCAM antibody were performed. Adhesion tests revealed good attachment of CRCs to PMEA-coated plates. Spike tests indicated that ~75% of CRCs from a 10-mL blood sample were recovered on the slides. By cytological examination, CTCs were identified in 18/41 CRC cases (43.9%). In cell cultures, spheroid-like structures or tumor-cell clusters were found in 18/33 tested cases (54.5%). Overall, CTCs and/or growing circulating tumor cells were found in 23/41 CRC cases (56.0%). History of chemotherapy or radiation was significantly negatively correlated with CTC detection (p = 0.02). In summary, we successfully captured CTCs from CRC patients using the unique biomaterial PMEA. Cultured tumor cells will provide important and timely information regarding the molecular basis of CTCs.

BackgroundOvarian cancer cells aggregate during or after exfoliation from the primary tumor to form threedimensional spheroids. Spheroid formation provides a survival advantage during peritoneal dissemination in nutrient and oxygen-depleted conditions which is accompanied by a suppressed metabolic phenotype and fragmented mitochondria. Upon arrival to their metastatic sites, spheroids adhere to peritoneal organs and transition to a more epithelial phenotype to support outgrowth and invasion. In this study, we investigated the plasticity of mitochondrial morphology, dynamics, and function upon adhesion.MethodsUsing our slow-developing (MOSE-L) and fast-developing (MOSE-LTICv) ovarian cancer models, we mimicked adhesion and reoxygenation conditions by plating the spheroids onto tissue culture dishes and changing culture conditions from hypoxia and low glucose to normoxia with high glucose levels after adhesion. We used Western Blot, microscopy and Seahorse analyses to determine the plasticity of mitochondrial morphology and functions upon adhesion, and the impact on proliferation and invasion capacities.ResultsIndependent of culture conditions, all spheroids adhered to and began to grow onto the culture plates. While the bulk of the spheroid was unresponsive, the mitochondrial morphology in the outgrowing cells was indistinguishable from cells growing in monolayers, indicating that mitochondrial fragmentation in spheroids was indeed reversible. This was accompanied by an increase in regulators of mitobiogenesis, PGC1a, mitochondrial mass, and respiration. Reoxygenation increased migration and invasion in both cell types but only the MOSE-L responded with increased proliferation to reoxygenation. The highly aggressive phenotype of the MOSE-LTICv was characterized by a relative independence of oxygen and the preservation of higher levels of proliferation, migration and invasion even in limiting culture conditions but a higher reliance on mitophagy. Further, the outgrowth in these aggressive cells relies mostly on proliferation while the MOSE-L cells both utilize proliferation and migration to achieve outgrowth. Suppression of proliferation with cycloheximide impeded aggregation, reduced outgrowth and invasion via repression of MMP2 expression and the flattening of the spheroids.DiscussionOur studies indicate that the fragmentation of the mitochondria is reversible upon adhesion. The identification of regulatory signaling molecules and pathways of these key phenotypic alterations that occur during primary adhesion and invasion is critical for the identification of druggable targets for therapeutic intervention to prevent aggressive metastatic disease.

Redan innan födseln fastställs kvinnors fertilitet, som karakteriseras av ett begränsat antal äggblåsor, så kallade folliklar. Beståndet av folliklar minskar naturligt med åldern, samtidigt som kvinnors fertilitet beror på deras steroid- och äggproduktion. Patologiska tillstånd som polycystiskt ovarialsyndrom (PCOS), prematur ovariell insufficiens (POI) och cancer, samt ett flertal livsstils- och miljöfaktorer, kan ha en negativ inverkan på kvinnors fertilitet. Eftersom alla dessa faktorer kan leda till minskad fertilitet, har fertilitetsbevarande tekniker samt assisterade fortplantningstekniker blivit allt mer viktiga. Aktuell forskning gällande regeneration av äggstockar innefattar bildandet av sfäroider och organoider, en ny typ av in vitro 3D-odling, som kan användas för att etablera modell system av olika organ. I denna masteruppsats jämfördes två olika system för 3D-odling, three-layer gradient system (3-LGS) samt Biosilk™, för att bilda äggstockssfäroider baserade på celler från cortex och medulla. I 3-LGS påträffades icke-organiserade cellaggregat, endast från medulla, fram till 25 dagar av odling. Dock resulterade 3-LGS inte i några sfäroider, för varken cortex eller medulla, efter 6 veckor av odling. Därför krävs det fortsatta försök för att kunna dra slutsatser om 3-LGS kan användas för att bilda 3D-kulturer från vuxna äggstocksceller. Däremot lyckades äggstockssfäroider framställas, från både cortex och medulla, med hjälp av Biosilk™ efter sammanlagt 6 veckor av odling. Dessa sfäroider antingen fixerades eller förvarades i -80°C för framtida bulk RNA-sekvensiering eller RNA fluorescerande in situ-hybridiserings (RNA-FISH). Ett RNA-FISH protokoll (RNAscope®) förfinades, för att bättre kunna visualisera äggstocksvävnad samt äggstockssfäroider. Vävnaden snittades antingen på behandlat täckglas eller på mikroskopglas. Då både äggstocksvävnad och Biosilk™ är autofluorescerande, blektes vävnadssnitten olika länge. Dessutom testades olika fluoroforer för att få en så bra signal som möjligt. Slutsatsen var att, vävnad snittad på behandlat täckglas inte genererade i en klarare signal från fluoroforerna som först förväntat. Istället hade vävnaden hög autofluorescens samt antingen vek sig eller föll av de behandlade täckglasen. Möjligtvis skulle RNAscope® protokollet kunna förfinas ytterligare, för att vidare undersöka om användandet av vävnad snittad på behandlade täckglas skulle vara fördelaktigt. Vävnad som blektes längre än vad det ursprungliga RNAscope® protokollet angav hade en lägre autofluorescens. Därav ledde förfiningen av protokollet till att det blev mer anpassat för äggstockssfäroider framställda med Biosilk™. Slutligen, uppvisade fluoroforerna TSA 520, TSA 570 och TSA 650 en skarpare signal än fluoroforerna Fluorescein Tyramide, Cy 3 och Cy 5. In humans, female fertility is established before birth and is characterized by a limited follicular reserve, which decreases naturally with age. Women’s fertility depends on the steroid and oocyte production, which can be negatively influenced by pathological conditions, such as polycystic ovary syndrome (PCOS), premature ovarian insufficiency (POI) and cancer, as well as several lifestyle and environmental factors. Since all of these factors could lead to a decrease in fertility, fertility preservation techniques and assisted reproductive technologies (ARTs) have become increasingly important. Current research for gonad regeneration include organoid and spheroid formation, as a novel type of in vitro 3D culturing, which enables the establishment of an organ model systems. During this thesis, two different 3D-culture systems: three-layer gradient system (3-LGS) and Biosilk™, were compared for spheroid formation from adult human ovarian cells, stemming from both cortex and medulla. In the 3-LGS, some unorganized aggregates, based on medulla, could be found up to 25 days of culture, but did not yield in any final 3D-cultures after 6 weeks of culture. Consequently, further investigations are needed to conclude whether 3-LGS can be used for human adult ovarian cells. In contrast to this, ovarian 3D-cultures could be achieved with Biosilk™, using cells from both cortex and medulla. These were either fixed or stored in -80°C for later bulk RNA-sequencing and RNA fluorescence in situ hybridization (RNA-FISH). An RNA-FISH protocol (RNAscope®) was refined for this purpose. Tested adjustments included varying glass-type for sectioning, and tissue bleaching time. Moreover, different fluorophores were tested for clearer visualization of ovarian tissue material. Tissue sectioned on coated coverslips did not result in a sharper signal as first anticipated. Instead, the tissue was both autofluorescent, and folding or falling off of the coated coverslips. Therefore, sectioning on coverslips was not continued. There is, however, a possibility that it could be used, if the RNAscope® protocol was adjusted accordingly. Since both ovarian tissue and Biosilk™ are autofluorescent, it was concluded that a longer bleaching time than in the original RNAscope® protocol, resulted in less autofluorescense and a more clear signal. Thereby, making the protocol more suitable for application on ovary 3D-cultures based on Biosilk™, than it was before. Lastly, the tested fluorophores TSA 520, TSA 570 and TSA 650 resulted in a sharper signal than Fluorescein Tyramide, Cy 3 and Cy 5.

The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.

Oxygen concentration plays a crucial role in (3D) cell culture. However, the oxygen content in vitro is usually not comparable to the in vivo situation, which is partly due to the fact that most experiments are performed under ambient atmosphere supplemented with 5% CO2, which can lead to hyperoxia. Cultivation under physiological conditions is necessary, but also fails to have suitable measurement methods, especially in 3D cell culture. Current oxygen measurement methods rely on global oxygen measurements (dish or well) and can only be performed in 2D cultures. In this paper, we describe a system that allows the determination of oxygen in 3D cell culture, especially in the microenvironment of single spheroids/organoids. For this purpose, microthermoforming was used to generate microcavity arrays from oxygensensitive polymer films. In these oxygen-sensitive microcavity arrays (sensor arrays), spheroids cannot only be generated but also cultivated further. In initial experiments we could show that the system is able to perform mitochondrial stress tests in spheroid cultures to characterize mitochondrial respiration in 3D. Thus, with the help of sensor arrays, it is possible to determine oxygen label-free and in real-time in the immediate microenvironment of spheroid cultures for the first time.

Mit mehr als eine Million neuer Fälle im Jahr 2020 ist Magenkrebs weltweit die fünfthäufigste Krebsart mit einer hohen Mortalitätsrate. Während die meisten Krankheitsfälle sporadisch auftreten, unterliegen 10 % der betroffenen Patienten einer familiären Häufung, von denen nur bis zu 3 % der Fälle mit einer bereits bekannten genetischen Ursache in Verbindung gebracht werden. Das hereditäre diffuse Magenkarzinom ist ein häufig vererbtes Magenkrebssyndrom, das durch Keimbahnmutationen in CDH1 ausgelöst wird. Es wurden jedoch bisher keine weiteren Gene mit dieser Krankheit in Verbindung gebracht. Daten der Whole-exome Sequenzierung aus einer Kohorte von Magenkrebspatienten ergaben, dass CTNND1 einen interessanten Beitrag zum hereditären Magenkrebs leistet. Ziel der vorliegenden Arbeit war es, die identifizierten CTNND1-Mutationen funktionell zu charakterisieren. Aus diesem Grund wurden zwei verschiedene Techniken, die Hanging-Drop-Methode und Ultra-Low-Attachment-Platten, für die Sphäroid-Modellierung an NCI-N87-Zellen getestet, um drei genetische CTNND1-Varianten zu bewerten: c.28_29delinsCT (p.Ala10Leu), c.1105C>T (p.Pro369Ser) und c.1537A>G (p.Asn513Asp). Mit Hilfe statistischer Methoden konnte bestätigt werden, dass die Ultra-Low-Attachment-Platten die effektivste Technik für die Sphäroid-Modellierung sind, da sie skalierbare und reproduzierbare Sphäroide liefern. Anschließend wurde die Pathogenität von drei CTNND1-Varianten, die bei Magenkrebspatienten gefunden wurden, durch Sphäroid-Modellierung getestet. Die CTNND1-Varianten wirkten sich negativ auf die Funktion des Proteinprodukts (p120) aus, indem sie vor allem die Zell-Zell-Bindung störten. Da p120 zur Struktur der Zelle beiträgt, führte es dazu, dass die produzierten Sphäroide anders aggregierten als die mit dem CTNND1-Wildtyp. Außerdem konnte beobachtet werden, dass die c.1105C>T-Mutation die Bindung von E-Cadherin und p120 beeinträchtigte, was zu einer Internalisierung von E-Cadherin führte. Daraus lässt sich schließen, dass die CTNND1-Varianten die Funktion des Proteins beeinträchtigen, indem sie die Zell-Zell-Adhäsionseigenschaften verändern und die Position von E-Cadherin beeinflussen With more than one million cases in 2020, gastric cancer is the 5th most common cancer worldwide linked to a high mortality rate. Whilst most of the disease cases occur sporadically, 10% of the affected patients show familial aggregation, from which only up to 3% of the cases are associated with an already known genetic cause. Hereditary diffuse gastric cancer is a frequent inherited gastric cancer syndrome triggered by germline mutations in CDH1, but no additional genes have been linked with this disease. Whole exome sequencing data from a cohort of gastric cancer patients revealed CTNND1 as an interesting contributor to hereditary gastric cancer. The present work aimed to functionally characterize the identified CTNND1 mutations. To do so, two different techniques, the Hanging Drop method, and Ultra-Low Attachment plates, were tested for spheroid modeling on NCI-N87 cells to evaluate three CTNND1 genetic variants: c.28_29delinsCT (p.Ala10Leu), c.1105C>T (p.Pro369Ser) and c.1537A>G (p.Asn513Asp). With the help of statistical methods, it could be confirmed that the Ultra- Low Attachment plates were the most effective technique for spheroid modeling, as it provided scalable and reproducible spheroids. Afterwards, the pathogenicity of three CTNND1 variants found in gastric cancer patients was tested by spheroid modeling. CTNND1 variants negatively impacted its protein product (p120) function by mainly disturbing cell-to-cell attachment. As p120 contributes to the cell's structure, it caused the produced spheroids to aggregate differently than those with the CTNND1 wild type. Moreover, it could also be observed that the c.1105C>T mutation impaired the E-cadherin and p120 binding, resulting in E-cadherin internalization. It could be concluded that CTNND1 variants are impairing its protein function by altering cell-cell adhesion properties and by influencing the location of E-cadherin.

Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) are valuable for the understanding/treatment of the deadly heart diseases and their drug screening. However, the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging. Here, it is discovered surprisingly that Rock inhibitor (RI), used ubiquitously to improve the survival/yield of human iPSCs, induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 μM). This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 μM for 3D culture, the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95 % in ~7 days (compared to less than ~50 % in 14 days for the 10 μM RI condition). Furthermore, the outset beating time (OBT) of all resultant cardiac spheroids (CSs) is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol (GelMA) hydrogel, showing high homogeneity (in terms of the OBT) in functional maturity of the CSs. Moreover, the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs. These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases., Graphical abstract Rock inhibitor (RI) at 10 μM induces early gastrulation-like heterogeneous differentiation of human iPSCs and compromises their cardiac differentiation in 3D. Importantly, reducing RI to 1 μM retains human iPSCs in solid inner cell mass-like spheroids with high pluripotency for efficient and homogeneous cardiac differentiation in 3D. This discovery may greatly facilitate human iPSCs-based therapy and modeling of heart diseases.Image 1, Highlights • Rock inhibitor (RI) at the widely used 10 μM induces early gastrulation-like differentiation of human iPSCs (hiPSCs) in 3D. • Reducing RI to 1 μM retains the high pluripotency of hiPSCs in inner cell mass-like solid spheroids in 3D. • Reducing RI to 1 μM shortens the time needed for 3D cardiac differentiation of hiPSCs by ~1 week. • Reducing RI to 1 μM doubles the percentage of beating cardiac spheroids after 3D cardiac differentiation of hiPSCs.

Triple-negative breast cancer constitutes 15 – 20% of all breast cancer and is considered one of the most aggressive forms of breast cancer. Clinical studies have suggested that the high numbers of infiltrating tumor-associated macrophage (TAM) act as a critical contributor behind the aggression of TNBC. Therefore, this study was focused on the preparation of a TNBC spheroid model with M2-like macrophages (M2-M) since it closely simulated the tumor microenvironment observed in clinical TNBC tumors. High EGFR expression in TNBC highlights the importance of cetuximab (Cmab)-assisted cellular internalization into the EGFR overexpressing TNBC cell line. The Cmab-anchored gold nanorod (GNR)-mediated photothermal approach was successfully developed to treat the TAM-infiltrated TNBC spheroid model. MDA-MB-231 spheroids with a diameter of 260 ± 10 μm were successfully prepared. An increase in the IC50 of doxorubicin in MDA-MB-231 spheroids with M2-M compared to the without M2-M group suggested that TAM-mediated development of resistance. The in vitro cytotoxicity assay demonstrated that there was elevated apoptosis expression (PARP and caspase-9), cell cycle arrest (G2/M phase), and cytotoxic effects following treatment with Cmab-GNR plus NIR irradiation. Moreover, there was no significant difference between the cytotoxic effect of Cmab-GNR plus NIR with M2-M and without M2-M group. Thus, our study highlighted that Cmab-GNR with NIR were able to overcome TAM-acquired resistance in the tumor model, which might be due to the polarization of the protumoral phenotype to the antitumoral phenotype.

Drug-resistant cancer spheroids were fabricated by three-dimensional (3D) bioprinting for the quantitative evaluation of drug resistance of cancer cells, which is a very important issue in cancer treatment. Cancer spheroids have received great attention as a powerful in vitro model to replace animal experiments because of their ability to mimic the tumor microenvironment. In this work, the extrusion printing of gelatin-alginate hydrogel containing MCF-7 breast cancer stem cells successfully provided 3D growth of many single drug-resistant breast cancer spheroids in a cost-effective 3D-printed mini-well dish. The drug-resistant MCF-7 breast cancer spheroids were able to maintain their drug-resistant phenotype of CD44high/CD24low/ALDH1high in the gelatin-alginate media during 3D culture and exhibited higher expression levels of drug resistance markers, such as GRP78 chaperon and ABCG2 transporter, than bulk MCF-7 breast cancer spheroids. Furthermore, the effective concentration 50 (EC50) values for apoptotic and necrotic spheroid death could be directly determined from the 3D printed-gelatin-alginate gel matrix based on in situ 3D fluorescence imaging of cancer spheroids located out of the focal point and on the focal point. The EC50 values of anti-tumor agents (camptothecin and paclitaxel) for apoptotic and necrotic drug-resistant cancer spheroid death were higher than those for bulk cancer spheroid death, indicating a greater drug resistance. Statement of significance This study proposed a novel 3D bioprinting-based drug screening model, to quantitatively evaluate the efficacy of anticancer drugs using drug-resistant MCF-7 breast cancer spheroids formed within a 3D-printed hydrogel. Quantitative determination of anticancer drug efficacy using EC50, which is extremely important in drug discovery, was achieved by 3D printing that enables concurrent growth of many single spheroids efficiently. This study verified whether drug-resistant cancer spheroids grown within 3D-printed gelatin-alginate hydrogel could maintain and present drug resistance. Also, the EC50 values of the apoptotic and necrotic cell deaths were directly acquired in 3D-embedded spheroids based on in situ fluorescence imaging. This platform provides a single-step straightforward strategy to cultivate and characterize drug-resistant spheroids to facilitate anticancer drug screening.

Tumour spheroid experiments are routinely used to study cancer progression and treatment. Various and inconsistent experimental designs are used, leading to challenges in interpretation and reproducibility. Using multiple experimental designs, live-dead cell staining, and real-time cell cycle imaging, we measure necrotic and proliferation-inhibited regions in over 1000 4D tumour spheroids (3D space plus cell cycle status). By intentionally varying the initial spheroid size and temporal sampling frequencies across multiple cell lines, we collect an abundance of measurements of internal spheroid structure. These data are difficult to compare and interpret. However, using an objective mathematical modelling framework and statistical identifiability analysis we quantitatively compare experimental designs and identify design choices that produce reliable biological insight. Measurements of internal spheroid structure provide the most insight, whereas varying initial spheroid size and temporal measurement frequency is less important. Our general framework applies to spheroids grown in different conditions and with different cell types., Tumour spheroid experiments with real-time fluorescent ubiquitination-based cell cycle imaging are interpreted using mathematical modelling and statistical identifiability analysis. A framework is provided to compare data obtained across different experimental designs and the experimental design choices that provide reliable biological insight are revealed.

Loss of ARID1A, a tumor suppressor gene, is associated with the higher grade of colorectal cancer (CRC). However, molecular and cellular mechanisms underlying the progression and aggressiveness of CRC induced by the loss of ARID1A remain poorly understood. Herein, we evaluated cellular mechanisms underlying the effects of ARID1A knockdown on the carcinogenesis features and aggressiveness of CRC cells. A human CRC cell line (Caco-2) was transfected with small interfering RNA (siRNA) specific to ARID1A (siARID1A) or scrambled (non-specific) siRNA (siControl). Cell death, proliferation, senescence, chemoresistance and invasion were then evaluated. In addition, formation of polyploid giant cancer cells (PGCCs), self-aggregation (multicellular spheroid) and secretion of an angiogenic factor, vascular endothelial growth factor (VEGF), were examined. The results showed that ARID1A knockdown led to significant decreases in cell death and senescence. On the other hand, ARID1A knockdown enhanced cell proliferation, chemoresistance and invasion. The siARID1A-transfected cells also had greater number of PGCCs and larger spheroid size and secreted greater level of VEGF compared with the siControl-transfected cells. These data, at least in part, explain the cellular mechanisms of ARID1A deficiency in carcinogenesis and aggressiveness features of CRC.

Islet transplantation is an effective replacement therapy for type 1 diabetes (T1D) patients. However, shortage of donor organ for allograft is obstacle for further development of the treatment. Subcutaneous transplantation with stem cell-derived β-cells might overcome this, but poor vascularity in the site is burden for success in the transplantation. We investigated the effect of subcutaneous transplantation of vascularized β-cell spheroid tissue constructed 3-dimensionally using a layer-by-layer (LbL) cell-coating technique in a T1D model mouse.We used MIN6 cells to determine optimal conditions for the coculture of β-cell spheroids, normal human dermal fibroblasts, and human umbilical vein endothelial cells, and then, under those conditions, we constructed vascularized spheroid tissue using human induced pluripotent stem cell-derived β-cells (hiPS β cells). The function of insulin secretion of the vascularized hiPS β-cell spheroid tissue was evaluated in vitro. Furthermore, the function was investigated in T1D model NOD/SCID mice subcutaneously transplanted with the tissue.In vitro, the vascularized hiPS β-cell spheroid tissue exhibited enhanced insulin secretion. The vascularized hiPS β-cell spheroid tissue also significantly decreased blood glucose levels in diabetic immunodeficient mice when transplanted subcutaneously. Furthermore, host mouse vessels were observed in the explanted vascularized hiPS β-cell spheroid tissue.Vascularized hiPS β-cell spheroid tissue decreased blood glucose levels in the diabetic mice. This therapeutic effect was suggested due to host angiogenesis in the graft. This method could lead to a promising regenerative treatment for T1D patients.

Introduction: Mesenchymal stromal/stem cells (MSCs) are multipotent, self-renewing cells that are extensively used in tissue engineering. Dedifferentiated fat (DFAT) cells are derived from adipose tissues and are similar to MSCs. Three-dimensional (3D) spheroid cultures comprising MSCs mimic the biological microenvironment more accurately than two-dimensional cultures; however, it remains unclear whether DFAT cells in 3D spheroids possess high osteogenerative ability. Furthermore, it is unclear whether DFAT cells from 3D spheroids transplanted into calvarial bone defects are as effective as those from two-dimensional (2D) monolayers in promoting bone regeneration. Methods: We compared the in vitro osteogenic potential of rat DFAT cells cultured under osteogenic conditions in 3D spheroids with that in 2D monolayers. Furthermore, to elucidate the ability of 3D spheroid DFAT cells to promote bone healing, we examined the in vivo osteogenic potential of transplanting DFAT cells from 3D spheroids or 2D monolayers into a rat calvarial defect model. Results: Osteoblast differentiation stimulated by bone morphogenetic protein-2 (BMP-2) or osteogenesis-inducing medium upregulated osteogenesis-related molecules in 3D spheroid DFAT cells compared with 2D monolayer DFAT cells. BMP-2 activated phosphorylation in the canonical Smad 1/5 pathways in 3D spheroid DFAT cells but phosphorylated ERK1/2 and Smad2 in 2D monolayer DFAT cells. Regardless of osteogenic stimulation, the transplantation of 3D DFAT spheroid cells into rat calvarial defects promoted new bone formation at a greater extent than that of 2D DFAT cells. Conclusions: Compared with 2D DFAT cells, 3D DFAT spheroid cells promote osteoblast differentiation and new bone formation via canonical Smad 1/5 signaling pathways. These results indicate that transplantation of DFAT cells from 3D spheroids, but not 2D monolayers, accelerates bone healing.

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

Lung regeneration is dependent on the availability of progenitor lung cells. Large numbers of self-renewing, patient-specific induced pluripotent stem cell-derived alveolar epithelial cells (iPSC-AECs) are needed to adequately recellularize whole-organ constructs. Prior methods to generate functional iPSC-AECs are not feasible for large-scale cell production. We present a novel protocol to produce iPSC-AECs, which is scalable for whole-organ regeneration. Differentiation of iPSCs was performed with genetically modified iPSCs with fluorescent reporters, which underwent differentiation in a stepwise protocol mimicking lung development. Cells were purified, sorted, and embedded in a liquid Matrigel precursor either to form adherent droplets or to form cell-laden Matrigel spheroids, which were subsequently transferred to spinner flasks with media as floating droplets. After culture, monolayer spheres of iPSC-AECs were isolated to form single cell suspensions. Equal numbers of iPSC-AECs from the two culture conditions were seeded into decellularized lung scaffolds. IPSC-AECs cultured in floating droplets were significantly more proliferative than those in adherent droplets, with significantly higher total cell counts and Ki67 expression. Equivalent expression of the distal lung markers was observed for both culture conditions. Lungs recellularized from both culture groups had similar histological appearance. Media changes took significantly less time with the floating droplet method and was more cost effective. The floating droplet culture method demonstrated enhanced proliferative capacity, stable distal lung epithelial phenotype, and reduced resources compared with prior culture methods. In this study, we provide a means for iPSC-AEC production for regeneration of whole-lung constructs. IMPACT STATEMENT: We describe a novel culture method for induced pluripotent stem cell-derived alveolar epithelial cell (AEC) expansion with enhanced proliferative capacity and reduced resource requirements compared with previously described methods. This method is scalable for human whole-lung regeneration bioengineering or could be automated for commercial cell production. This culture method may have implications for the differentiation of type I AECs from type II AECs.

Osteocytes play an important role to modulate the bone remodeling and are also known as terminally differentiated cells originated from the osteoblast precursor cells, but its differentiation mechanism remains unclear. Since an efficient in vitro method to evoke the osteocyte differentiation from the osteoblast precursor cells has not been established, we conducted the comparative gene expression analysis for mouse pre-osteoblast MC3T3-E1 cells in order to elucidate the key factors to induce the osteocyte differentiation from the pre-osteoblast cells. In this study, we prepared four different culture environments by modulating their cell-substrate interaction and cell-cell interaction; (i) low and (ii) high cell density on the adhesive culture models, and (iii) low and (iv) high cell density on the non-adhesive floating culture models. By comparing these conditions in terms of cell-substrate and cell-cell interaction, we showed that the elimination of cell-substrate interaction under non-adhesive floating culture greatly up-regulated the gene expression of osteocyte markers in the pre-osteoblast cells. Moreover, the presence of moderate cell-cell interaction in the non-adhesive spheroid culture further enhanced the up-regulation of osteocyte markers for the pre-osteoblast cells. The results altogether suggest the most appropriate culture environment to induce the in vitro osteocyte differentiation of pre-osteoblast cells.

Three-dimensional cell cultures, or spheroids, are important model systems for cancer research because they recapitulate chemical and phenotypic aspects of in vivo tumors. Spheroids develop radially symmetric chemical gradients, resulting in distinct cellular populations. Stable isotopic labeling by amino acids in cell culture (SILAC) is a well-established approach to quantify protein expression and has previously been used in a pulse-chase format to evaluate temporal changes. In this article, we demonstrate that distinct isotopic signatures can be introduced into discrete spatial cellular populations, effectively tracking proteins to original locations in the spheroid, using a platform that we refer to as spatial SILAC. Spheroid populations were grown with light, medium, and heavy isotopic media, and the concentric shells of cells were harvested by serial trypsinization. Proteins were quantitatively analyzed by ultraperformance liquid chromatography–tandem mass spectrometry. The isotopic signatures correlated with the spatial location and the isotope position do not significantly impact the proteome of each individual layer. Spatial SILAC can be used to examine the proteomic changes in the different layers of the spheroid and to identify protein biomarkers throughout the structure. We show that SILAC labels can be discretely pulsed to discrete positions, without altering the spheroid’s proteome, promising future combined pharmacodynamic and pharmacokinetic studies.

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.

Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRASG12V exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRASG12V-expressing cells. This represents an applicable approach to explore protein–protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells.

Objective The dopaminergic nigrostriatal neurons (DA cells) in healthy people present a slow degeneration with aging, which produces cellular debris throughout life. About 2%–5% of people present rapid cell degeneration of more than 50% of DA cells, which produces Parkinson’s disease (PD). Neuroinflammation accelerates the cell degeneration and may be critical for the transition between the slow physiological and the rapid pathological degeneration of DA cells, particularly when it activates microglial cells of the medial forebrain bundle near dopaminergic axons. As synaptic debris produced by DA cell degeneration may trigger the parkinsonian neuroinflammation, this study investigated the removal of axonal debris produced by retrograde degeneration of DA cells, paying particular attention to the relative roles of astrocytes and microglia. Methods Rats and mice were injected in the lateral ventricles with 6-hydroxydopamine, inducing a degeneration of dopaminergic synapses in the striatum which was not accompanied by non-selective tissue damage, microgliosis or neuroinflammation. The possible retrograde degeneration of dopaminergic axons, and the production and metabolization of DA-cell debris were studied with immunohistochemical methods and analyzed in confocal and electron microscopy images. Results The selective degeneration of dopaminergic synapses in the striatum was followed by a retrograde degeneration of dopaminergic axons whose debris was found within spheroids of the medial forebrain bundle. These spheroids retained mitochondria and most (e.g., tyrosine hydroxylase, the dopamine transporter protein, and amyloid precursor protein) but not all (e.g., α-synuclein) proteins of the degenerating dopaminergic axons. Spheroids showed initial (autophagosomes) but not late (lysosomes) components of autophagy (incomplete autophagy). These spheroids were penetrated by astrocytic processes of the medial forebrain bundle, which provided the lysosomes needed to continue the degradation of dopaminergic debris. Finally, dopaminergic proteins were observed in the cell somata of astrocytes. No microgliosis or microglial phagocytosis of debris was observed in the medial forebrain bundle during the retrograde degeneration of dopaminergic axons. Conclusions The present data suggest a physiological role of astrocytic phagocytosis of axonal debris for the medial forebrain bundle astrocytes, which may prevent the activation of microglia and the spread of retrograde axonal degeneration in PD.

Patient-derived cells and xenografts retain the biological characteristics of clinical cancers and are instrumental in gaining a better understanding of the chemoresistance of cancer cells. Here, we have established a panel of patient-derived spheroids from clinical materials of ovarian cancer. Systematic evaluation using therapeutic agents indicated that sensitivity to platinum-based compounds significantly varied among the spheroids. To understand the molecular basis of drug sensitivity, we performed integrative analyses combining chemoresistance data and gene expression profiling of the ovarian cancer patient-derived spheroids. Correlation analyses revealed that cisplatin resistance was significantly associated with elevated levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione-producing redox enzymes. Accordingly, cisplatin-resistant spheroids established in vitro showed elevated levels of G6PD and active glutathione. Moreover, treatment with a G6PD inhibitor in combination with cisplatin suppressed spheroid proliferation in vitro and largely eradicated peritoneal metastasis in mouse xenograft models. Furthermore, G6PD expression was elevated during carcinogenesis and associated with poor prognosis. Thus, the combination of gene expression data and chemosensitivity revealed the essential roles of G6PD-driven redox metabolism in cisplatin resistance, underscoring the significance of an integrative approach using patient-derived cells.

Chemotherapy for glioblastoma multiforme (GBM) remains ineffective due to insufficient penetration of therapeutic agents across the blood–brain barrier (BBB) and into the GBM tumor. Herein, is described, the optimization of the lipid composition and fabrication conditions for a BBB- and tumor penetrating terpolymer-lipid-hybrid nanoparticle (TPLN) for delivering doxorubicin (DOX) to GBM. The composition of TPLNs was first screened using different lipids based on nanoparticle properties and in vitro cytotoxicity by using 23 full factorial experimental design. The leading DOX loaded TPLNs (DOX-TPLN) were prepared by further optimization of conditions and used to study cellular uptake mechanisms, in vitro cytotoxicity, three-dimensional (3D) glioma spheroid penetration, and in vivo biodistribution in a murine orthotopic GBM model. Among various lipids studied, ethyl arachidate (EA) was found to provide excellent nanoparticle properties e.g., size, polydispersity index (PDI), zeta potential, encapsulation efficiency, drug loading, and colloidal stability, and highest anticancer efficacy for DOX-TPLN. Further optimized EA-based TPLNs were prepared with an optimal particle size (103.8 ± 33.4 nm) and PDI (0.208 ± 0.02). The resultant DOX-TPLNs showed ~ sevenfold higher efficacy than free DOX against human GBM U87-MG-RED-FLuc cells in vitro. The interaction between the TPLNs and the low-density lipoprotein receptors also facilitated cellular uptake, deep penetration into 3D glioma spheroids, and accumulation into the in vivo brain tumor regions of DOX-TPLNs. This work demonstrated that the TPLN system can be optimized by rational selection of lipid type, lipid content, and preparation conditions to obtain DOX-TPLN with enhanced anticancer efficacy and GBM penetration and accumulation.

Three-dimensional (3D) spheroid culture provides opportunities to model tumor growth closer to its natural context. The collagen network in the extracellular matrix supports autonomic tumor cell proliferation, but its presence and role in tumor spheroids remain unclear. In this research, we developed an in vitro 3D co-culture model in a microwell 3D (μ-well 3D) cell-culture array platform to mimic the tumor microenvironment (TME). The modular setup is used to characterize the paracrine signaling molecules and the role of the intraspheroidal collagen network in cancer drug resistance. The μ-well 3D platform is made up of poly(dimethylsiloxane) that contains 630 round wells for individual spheroid growth. Inside each well, the growth surface measured 500 μm in diameter and was functionalized with the amphiphilic copolymer. HCT-8 colon cancer cells and/or NIH3T3 fibroblasts were seeded in each well and incubated for up to 9 days for TME studies. It was observed that NIH3T3 cells promoted the kinetics of tumor organoid formation. The two types of cells self-organized into core-shell chimeric tumor spheroids (CTSs) with fibroblasts confined to the shell and cancer cells localized to the core. Confocal microscopy analysis indicated that a type-I collagen network developed inside the CTS along with increased TGF-β1 and α-SMA proteins. The results were correlated with a significantly increased stiffness in 3D co-cultured CTS up to 52 kPa as compared to two-dimensional (2D) co-culture. CTS was more resistant to 5-FU (IC50 = 14.0 ± 3.9 μM) and Regorafenib (IC50 = 49.8 ± 9.9 μM) compared to cells grown under the 2D condition 5-FU (IC50 = 12.2 ± 3.7 μM) and Regorafenib (IC50 = 5.9 ± 1.9 μM). Targeted collagen homeostasis with Sclerotiorin led to damaged collagen structure and disrupted the type-I collagen network within CTS. Such a treatment significantly sensitized collagen-supported CTS to 5-FU (IC50 = 4.4 ± 1.3 μM) and to Regorafenib (IC50 = 0.5 ± 0.2 μM). In summary, the efficient formation of colon cancer CTSs in a μ-well 3D culture platform allows exploration of the desmoplastic TME. The novel role of intratumor collagen quality as a drug sensitization target warrants further investigation.

In recent years, the successful construction of tissues derived from established iPSCs has been disclosed, but it has been reported that the constructed tissues encounter problems of internal necrosis when their size increases. To solve this problem, a simulated microgravity device is used. However, the culture of early developing kidney cells using this device has not yet been reported. This study investigated whether developing kidney cells cultured in a simulated microgravity environment can differentiate into glomerular cells and renal epithelial cells. The results showed that both mouse developing kidney cells cultured in simulated microgravity and static environment formed kidney spheroids. In static culture, ureteric bud and glomerular structures were not found. While ureteric buds, podocytes, PECAM-1 positive cell aggregates, and primordial vascular plexus were formed in the kidney spheroids in simulated microgravity culture. Moreover, the expression level of the PECAM-1 gene was significant in simulated microgravity culture as compared to that of static culture. These results indicate that simulated microgravity is effective for the differentiation of developing kidney cells.

Solid phase microextraction (SPME) in combination with high-resolution mass spectrometry was employed for the determination of metabolomic profile of mouse melanoma growth within in vitro 2D, in vitro 3D, and in vivo models. Such multi-model approach had never been investigated before. Due to the low-invasiveness of SPME, it was possible to perform time-course analysis, which allowed building time profile of biochemical reactions in the studied material. Such approach does not require the multiplication of samples as subsequent analyses are performed from the very same cell culture or from the same individual. SPME already reduces the number of animals required for experiment; therefore, it is with good concordance with the 3Rs rule (replacement, reduction, and refinement). Among tested models, the largest number of compounds was found within the in vitro 2D cell culture model, while in vivo and in vitro 3D models had the lowest number of detected compounds. These results may be connected with a higher metabolic rate, as well as lower integrity of the in vitro 2D model compared to the in vitro 3D model resulting in a lower number of compounds released into medium in the latter model. In terms of in vitro-in vivo extrapolation, the in vitro 2D model performed more similar to in vivo model compared to in vitro 3D model; however, it might have been due to the fact that only compounds secreted to medium were investigated. Thus, in further experiments to obtain full metabolome information, the intraspheroidal assessment or spheroid dissociation would be necessary.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

The expression of pluripotency factors, and their associations with clinicopathological parameters and drug response have been described in various cancers, including gastric cancer. This study investigated the association of pluripotency factor expression with the clinicopathological characteristics of gastric cancer patients, as well as changes in the expression of these factors upon the stem cell-enriching spheroid culture of gastric cancer cells, regulation of sphere-forming capacity, and response to cisplatin and TRAIL treatments by Nanog and KLF4. Nanog expression was significantly associated with the emergence of a new tumor and a worse prognosis in gastric cancer patients. The expression of the pluripotency factors varied among six gastric cancer cells. KLF4 and Nanog were expressed high in SNU-601, whereas SOX2 was expressed high in SNU-484. The expression of KLF4 and SOX2 was increased upon the spheroid culture of SNU-601 (KLF4/Nanog-high) and SNU-638 (KLF4/Nanog-low). The spheroid culture of them enhanced TRAIL-induced viability reduction, which was accompanied by the upregulation of death receptors, DR4 and DR5. Knockdown and overexpression of Nanog in SNU-601 and SNU-638, respectively, did not affect spheroid-forming capacity, however, its expression was inversely correlated with DR4/DR5 expression and TRAIL sensitivity. In contrast, KLF4 overexpression in SNU-638 increased spheroid formation, susceptibility to cisplatin and TRAIL treatments, and DR4/DR5 expression, while the opposite was found in KLF4-silenced SNU-601. KLF4 is supposed to play a critical role in DR4/DR5 expression and responses to TRAIL and cisplatin, whereas Nanog is only implicated in the former events only. Direct regulation of death receptor expression and TRAIL response by KLF4 and Nanog have not been well documented previously, and the regulatory mechanism behind the process remains to be elucidated.

Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes. In vitro 3D culture systems simulate the in vivo tumor growth and preserve the biological characteristics of tumors more accurately than conventional monolayer cell cultures. We describe a protocol for the development of 3D tumor organoids using conjunctival melanoma (CM) and uveal melanoma (UM) cell lines as well as the use of hand-held customized electrodes, suitable for in vitro ECT in the culture well without destruction of the tumor environment. This protocol analyzes the culture and growth of 3D CM and UM spheroids and their reaction to bleomycin (2.5 µg/mL) alone, electroporation (EP) (750 Volts/cm, 8 pulses, 100 µs, 5 Hz) alone, and ECT as a combination of EP and bleomycin. The drug concentration and the EP settings used in this protocol were established as preferred ECT conditions according to previous experiments. The assay used to determine the spheroid viability was conducted 3-7 days following treatment. The effect on viability and growth of the 3D tumor spheroids was significant only after ECT. The customized electrodes are described in detail in order to facilitate the application of pulses in the culture well. This novel treatment of 3D UM and CM spheroids sets a steppingstone for future clinical application.

Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

International audience; An increasing number of studies concerning solid cancers, including prostate cancer, are tending to demonstrate the predominant role of the interactions of tumor cells with their microenvironment, and underlining the relevance of therapeutic approaches co-targeting these two components. Artificial in vitro 3D culture models, such as spheroids, are therefore being designed to allow intercellular interactions between tumor cells and the matrix, under hypoxic conditions mimicking a microtumor. This project aims to develop and characterize a multicellular tumor spheroid (MCTS) model of human prostate cancer cells expressing PSMA, for in vitro drug screening. To this end, 1,000 cells/well were seeded in 100 µl of culture medium with 0.5% of methylcellulose in 96-well, non-adherent, V-shaped bottom plates. Bioluminescent imaging of the spheroids enabled the measurement of spheroid growth. From Day 7 of growth, immunofluorescence studies showed cellular proliferation (Ki-67), mainly located in the periphery of the spheroid section, associated with the formation of an apoptotic core (TUNEL). Scanning electron microscopy and fluorescent imaging (Lox-1 probe) showed the presence of an extracellular matrix and the installation of an oxygen gradient leading to the formation of a hypoxic area during growth. This hypoxia was correlated with increased VEGF excretion. Drug sensitivity was assessed on 2D and 3D cultures. The LNCaP-Luc spheroids are more resistant to docetaxel and TH-302, a hypoxia-activated prodrug, compared with cells grown in a monolayer. For docetaxel, this resistance increased with the spheroid growth stage, whereas the activity of TH-302 was potentiated by the hypoxic environment. In conclusion, the development of LNCaP-Luc cell MCTS provides a simple model mimicking a microtumor; it appears to be particularly well-suited to the validation of new therapeutic approaches targeting proliferation and the microenvironment.

International audience; An increasing number of studies concerning solid cancers, including prostate cancer, are tending to demonstrate the predominant role of the interactions of tumor cells with their microenvironment, and underlining the relevance of therapeutic approaches co-targeting these two components. Artificial in vitro 3D culture models, such as spheroids, are therefore being designed to allow intercellular interactions between tumor cells and the matrix, under hypoxic conditions mimicking a microtumor. This project aims to develop and characterize a multicellular tumor spheroid (MCTS) model of human prostate cancer cells expressing PSMA, for in vitro drug screening. To this end, 1,000 cells/well were seeded in 100 µl of culture medium with 0.5% of methylcellulose in 96-well, non-adherent, V-shaped bottom plates. Bioluminescent imaging of the spheroids enabled the measurement of spheroid growth. From Day 7 of growth, immunofluorescence studies showed cellular proliferation (Ki-67), mainly located in the periphery of the spheroid section, associated with the formation of an apoptotic core (TUNEL). Scanning electron microscopy and fluorescent imaging (Lox-1 probe) showed the presence of an extracellular matrix and the installation of an oxygen gradient leading to the formation of a hypoxic area during growth. This hypoxia was correlated with increased VEGF excretion. Drug sensitivity was assessed on 2D and 3D cultures. The LNCaP-Luc spheroids are more resistant to docetaxel and TH-302, a hypoxia-activated prodrug, compared with cells grown in a monolayer. For docetaxel, this resistance increased with the spheroid growth stage, whereas the activity of TH-302 was potentiated by the hypoxic environment. In conclusion, the development of LNCaP-Luc cell MCTS provides a simple model mimicking a microtumor; it appears to be particularly well-suited to the validation of new therapeutic approaches targeting proliferation and the microenvironment.

Nanoparticles with a strong absorption of incident radio frequency (rf) or microwave irradiation are desirable for remote hyperthermia treatments. While controversy has surrounded the absorption properties of spherical metallic nanoparticles, other geometries, such as prolate and oblate spheroids, have not received sufficient attention for application in hyperthermia therapies. Here, we use the electrostatic approximation to calculate the relative-absorption ratio of metallic nanoparticles in various biological tissues. We consider a broad parameter space, sweeping across frequencies from 1 MHz to 10 GHz, while also tuning the nanoparticle dimensions from spheres to high-aspect-ratio spheroids approximating nanowires and nanodisks. We find that, while spherical metallic nanoparticles do not offer differential heating in tissue, large absorption cross sections can be obtained from long prolate spheroids, while thin oblate spheroids offer minor potential for absorption. Our results suggest that metallic nanowires should be considered for rf- and microwave-based wireless hyperthermia treatments in many tissues going forward.

Mathematical modeling for 3D bioprinting allows us to avoid widespread errors and also time and financial losses. It is necessary for such critical processes as tissue spheroids fusion and diffusion of nutrients in them. The reason is that tissue spheroids fusion is the base of the 3D bioprinting technology. In this work, we propose an approach for tissue spheroids fusion modeling considering a need to compromise between fidelity of the geometric form and viability of the whole bioconstruct.