Organotypic Co-cultures Research Articles

From Cancer to Immune Organoids: Innovative Preclinical Models to Dissect the Crosstalk between Cancer Cells and the Tumor Microenvironment

Genomic-oriented oncology has improved tumor classification, treatment options, and patient outcomes. However, genetic heterogeneity, tumor cell plasticity, and the ability of cancer cells to hijack the tumor microenvironment (TME) represent a major roadblock for cancer eradication. Recent biotechnological advances in organotypic cell cultures have revolutionized biomedical research, opening new avenues to explore the use of cancer organoids in functional precision oncology, especially when genomics alone is not a determinant. Here, we outline the potential and the limitations of tumor organoids in preclinical and translational studies with a particular focus on lung cancer pathogenesis, highlighting their relevance in predicting therapy response, evaluating treatment toxicity, and designing novel anticancer strategies. Furthermore, we describe innovative organotypic coculture systems to dissect the crosstalk with the TME and to test the efficacy of different immunotherapy approaches, including adoptive cell therapy. Finally, we discuss the potential clinical relevance of microfluidic mini-organ technology, capable of reproducing tumor vasculature and the dynamics of tumor initiation and progression, as well as immunomodulatory interactions among tumor organoids, cancer-associated fibroblasts (CAFs) and immune cells, paving the way for next-generation immune precision oncology.

Targeting the cancer cells and cancer-associated fibroblasts with next-generation FGFR inhibitors in prostate cancer co-culture models.

Inhibition of androgen receptor (AR) signaling is the main treatment strategy in advanced prostate cancer (PCa). A subset of castration resistant prostate cancer (CRPC) bypasses the AR blockade by increased fibroblast growth factor receptor (FGFR) signaling. The first- and second-generation, non-covalent FGFR inhibitors (FGFRis) have largely failed in the clinical trials against PCa. In this study, we tested the drug sensitivity of LNCaP, VCaP, and CWR-R1PCa cell lines to second-generation, covalent FGFRis (FIIN1, FIIN2) and a novel FGFR downstream molecule inhibitor (FRS2αi). 2D and 3D mono- and co-cultures of cancer cells, and cancer-associated fibroblasts (CAFs) were used to mimic tumor-stroma interactions in the extracellular matrix (ECM). The treatment responses of the FGFR signaling molecules, the viability and proliferation of cancer cells, and CAFs were determined through immunoblotting, migration assay, cell viability assay, and real-time imaging. Immunofluorescent and confocal microscopy images of control and treated cultures of cancer cells and CAFs, and their morphometric data were deduced. The FGFRis were more effective in mono-cultures of the cancer cells compared with co-cultures with CAFs. The FRS2αi was specifically effective in co-cultures with CAFs but was not cytotoxic to CAF mono-cultures as in the case of FIIN1 and FIIN2. At the molecular level, FRS2αi decreased p-FRS2α, p-ERK1/2, and activated apoptosis as monitored by cleaved caspase-3 activity in a concentration-dependent manner in the co-cultures. We observed no synergistic drug efficacy in the combination treatment of the FGFRi with ARi, enzalutamide, and darolutamide. The FRS2αi treatment led to a decrease in proliferation of cancer cell clusters in co-cultures as indicated by their reduced size and Ki67 expression. CAFs exert a protective effect on cancer cells and should be included in the invitro models to make them physiologically more relevant in screening and testing of FGFRis. The FRS2αi was the most potent agent in reducing the viability and proliferation of the 3D organotypic co-cultures, mainly by disrupting the contact between CAFs and cancer cell clusters. The next-generation FGFRi, FRS2αi, may be a better alternative treatment option for overcoming ARi treatment resistance in advanced PCa.

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Tumor microenvironment-induced FOXM1 regulates ovarian cancer stemness

In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

Recruitment of T-cells to the lung using an ex vivo co-culture paradigm

Precision cut lung slices (PCLS) bridge a gap between in vivo and in vitro studies by maintaining anatomical organization with structural integrity and intercellular signaling pathways. Applications of PCLS have included the modeling of inflammatory lung diseases, metabolism studies, and drug development. In the lungs, immune responses are carried out by a network of T- and B- cells, the latter of which are resident. The limited resident T-cell population of the lung diminishes accurate representations of pathogen response capacity in PCLS. Addressing this, we set out to increase pulmonary T-cell populations ex vivo. We hypothesized that thymus and bone marrow-derived T-cells would work synergistically to populate the lung in co-culture experiments. A murine organotypic lung co-culture model was developed and characterized for tissue health and T-cell recruitment over 3 days ex vivo using adult neurobasal media with 4 mM glucose + 2% B27 supplement. Lung slices were cultured independently, with bone marrow, thymus, or both. Immune colonization of the lung was assessed using immunohistochemistry for CD3+ T-cells and ACK2+ cells. Cells were counted in alveolar and airway spaces after 3 days of culture. Co-culture of lung slices with bone marrow did not increase CD3+ immunoreactive T-cells while thymus co-culture increased CD3+ T-cells by 76% in the alveolar space and by 39% in the airway, relative to lung alone. When lung slices were cultured with bone marrow plus thymus, CD3+ T-cells increased by 206% in the alveolar space and by 251% in the airway, relative to the lung alone. Co-culture with thymus increased ACK2+ cells by 48% in the airway, while lung culture with both thymus and bone marrow, ACK2+ cells increased by 35% in the alveolar space and 85% in the airway, relative to the lung alone. These results suggest that the increased T-cell population corresponding with thymus and bone marrow co-culture could be a result of cell-cell interaction or the secretion of growth factors. Cell secretions or growth factor release could stimulate thymic secretion of T-cells or could stimulate T-cell proliferation in the lung, suggesting that co-culture with thymus and bone marrow can elicit a T-cell response ex vivo. Future studies will center around differentiating what drives T-cell population to be increased in lung co-cultures and what T-cell subsets are being recruited in PCLS co-cultures while analyzing their capacity for response to pathogens ex vivo. Anschutz Foundation Pandemic Preparedness Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1261: In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability

Abstract Introduction: How tumor cells colonize an inherently distinct environment and survive the pressures of therapeutic treatment during metastasis is incompletely understood. In patients with medulloblastoma, the most common malignant pediatric brain tumor, mortality is almost exclusively dictated by metastasis to the leptomeninges, the inner covering of the brain and spinal cord. We aim to mechanistically understand the molecular drivers of leptomeningeal colonization, including how leptomeningeal adhesion mediates therapy resistance. Experimental approach: Progress in leptomeningeal metastasis research has been hindered by the scarcity of matched primary and metastatic tissue from patients given the lack of clinical benefit to obtaining metastatic tissue. Furthermore, end points from pre-clinical human xenograft mouse models are often driven by primary cerebellar tumors, not leptomeningeal dissemination. These barriers underscore the necessity for novel in vitro leptomeningeal metastasis models that allow for rigorous experimental analysis. These models must simultaneously recapitulate tumor cell-host and tumor cell-environment interactions driving colonization to produce translatable findings. Accordingly, our lab has developed two novel in vitro leptomeningeal metastasis models that allow for cell-based experimental analysis: a primary meningeal cell-medulloblastoma organotypic co-culture and an acellular leptomeningeal matrix mimetic monoculture. Results: Proteomic analysis identified that leptomeningeal cells promote medulloblastoma chemotactic honing to the leptomeninges through CXCL12-CXCR4 signaling. Additionally, loss of CXCL12/CXCR4 signaling impaired medulloblastoma adhesion to the leptomeninges. CXCL12/CXCR4 are known to mediate adhesion of stem cells in stromal niches via activation of integrin receptor ITGA4/ITGB1 (VLA4) binding to stromal VCAM1. Indeed, the VLA4-VCAM1 axis is required for medulloblastoma leptomeningeal adhesion and requires activation by CXCL12/CXCR4 signaling. From a translational perspective, we found that leptomeningeal adhesion promotes therapy resistance and that inhibition of adhesion signaling sensitized adherent medulloblastoma cells to therapy. Conclusions: These findings provide proof-of-concept that appropriate in vitro models of metastasis can be developed and utilized to define the biology driving adhesion and chemotherapy resistance in medulloblastoma metastasis. Furthermore, we show that medulloblastoma leptomeningeal adhesion is mediated by a CXCL12/CXCR4-VLA4/VCAM1 axis. Finally, we demonstrate the therapeutic potential of targeting this axis in sensitizing medulloblastoma metastases to current therapies. Citation Format: Leyre Jimenez Garcia, Amy C. Gross, Ryan D. Roberts, James B. Reinecke. In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1261.

The dopamine receptor D1 inhibitor, SKF83566, suppresses GBM stemness and invasion through the DRD1-c-Myc-UHRF1 interactions.

Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.

The mechanistic study of cancer-associated adipocytes promoting colorectal cancer peritoneal metastasis via FKBP5/UCP1-mediated lipid metabolism reprogramming.

178 Background: Colorectal cancer (CRC) is a prevalent malignant tumor globally, with peritoneal metastasis as a significant late-stage dissemination pathway, severely impacting patient prognosis. Recent research increasingly suggests the critical role of lipid metabolism within the tumor microenvironment in tumor progression. Cancer associated adipocytes (CAAs), a vital component, contribute to tumor progression, yet the exact molecular mechanisms remain unclear. We aim to explore the role of CAAs in CRC peritoneal metastasis and their specific regulatory mechanisms. Methods: In this study, we analyzed clinical samples from 312 CRC patients to assess the expression levels of FKBP5 and UCP1 proteins in tumor-adjacent tissues. Purified exosomes from co-cultured adipocytes and tumor cells, along with exosomes from ordinary tumor cells, underwent miRNA profiling via sequencing to evaluate exosomal miR-200a-3p expression. We established FKBP5 stable knockdown (FKBP5-/-) transgenic mouse models, organotypic co-culture models, and patient-derived xenograft (PDX) models. Through experiments involving metabolomics, mass spectrometry, immunoprecipitation, western blotting, and qPCR, we investigated the role of the miR-200a-3p/FKBP5/PPARγ/UCP1 signaling pathway in CAAs in CRC peritoneal metastasis. Results: After co-culturing adipocytes and tumor cells, we observed brown adipocyte differentiation, resulting in significant downregulation of FKBP5 protein expression. FKBP5 exhibited a strong association with UCP1 expression. Tumor cells highly expressed miR-200a-3p, released via exosomes into the tumor microenvironment and internalized by adipocytes. Within adipocytes, miR-200a-3p orchestrated metabolic reprogramming via the FKBP5/PPARγ/UCP1 pathway, inducing brown adipocyte differentiation, generating CAAs. CAAs released FFAs, promoting tumor peritoneal metastasis by activating tumor cell surface G-protein coupled receptors (GPCRs). Conversely, miR-200a-3p inhibition in tumor cells yielded opposing effects. In a spontaneous mouse model of colorectal cancer using azoxymethane (AOM)/dextran sulfate sodium salt (DSS), FKBP5-/- mice exhibited increased brown adipocyte content in the tumor microenvironment, along with notably higher spontaneous colorectal tumor numbers and sizes compared to wild-type mice. Conclusions: This study elucidates the pivotal role of CAAs in the peritoneal metastasis of CRC, confirming the regulatory role of the FKBP5/UCP1 pathway-mediated lipid metabolism reprogramming in this process. These findings offer a fresh perspective for further exploration of the molecular mechanisms underlying CRC peritoneal metastasis, and provide a theoretical foundation for the identification of novel therapeutic targets.

CXCR2-Blocking Has Context-Sensitive Effects on Rat Glioblastoma Cell Line Outgrowth (S635) in an Organotypic Rat Brain Slice Culture Depending on Microglia-Depletion (PLX5622) and Dexamethasone Treatment

In glioblastoma (GBM), the interplay of different immune cell subtypes, cytokines, and/or drugs shows high context-dependencies. Interrelations between the routinely applied dexamethasone (Dex) and microglia remain elusive. Here, we exploited rat organotypic brain slice co-cultures (OBSC) to examine the effects on a rat GBM cell line (S635) outgrowth resulting from the presence of Dex and pretreatment with the colony-stimulating factor receptor 1 (CSF1-R) inhibitor PLX5622: in native OBSC (without PLX5622-pretreatment), a diminished S635 spheroid outgrowth was observable, whereas Dex-treatment enhanced outgrowth in this condition compared to PLX5622-pretreated OBSC. Screening the supernatants of our model with a proteome profiler, we found that CXCL2 was differentially secreted in a Dex- and PLX5622-dependent fashion. To analyze causal interrelations, we interrupted the CXCL2/CXCR2-axis: in the native OBSC condition, CXCR2-blocking resulted in increased outgrowth, in combination with Dex, we found potentiated outgrowth. No effect was found in the PLX5622-pretreated. Our method allowed us to study the influence of three different factors—dexamethasone, PLX5622, and CXCL2—in a well-controlled, simplified, and straight-forward mechanistic manner, and at the same time in a more realistic ex vivo scenario compared to in vitro studies. In our model, we showed a GBM outgrowth enhancing synergism between CXCR2-blocking and Dex-treatment in the native condition, which was levelled by PLX5622-pretreatment.

338.9: Human organotypic co-culture model of pleura

338.9: Human organotypic co-culture model of pleura

Selective ROCK Inhibitor Enhances Blood Flow Recovery after Hindlimb Ischemia.

The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.

Organotypic 3D Co-Culture of Human Pleura as a Novel In Vitro Model of Staphylococcus aureus Infection and Biofilm Development.

Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen is Staphylococcus aureus (S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects of S. aureus infection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model with S. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1β) proved host-pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected with S. aureus resulting in the formation of biofilm, including host-pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema.

Decreased RNA polymerase III subunit expression leads to defects in oligodendrocyte development.

RNA polymerase III (Pol III) is a critical enzymatic complex tasked with the transcription of ubiquitous non-coding RNAs including 5S rRNA and all tRNA genes. Despite the constitutive nature of this enzyme, hypomorphic biallelic pathogenic variants in genes encoding subunits of Pol III lead to tissue-specific features and cause a hypomyelinating leukodystrophy, characterized by a severe and permanent deficit in myelin. The pathophysiological mechanisms in POLR3- related leukodystrophy and specifically, how reduced Pol III function impacts oligodendrocyte development to account for the devastating hypomyelination seen in the disease, remain poorly understood. In this study, we characterize how reducing endogenous transcript levels of leukodystrophy-associated Pol III subunits affects oligodendrocyte maturation at the level of their migration, proliferation, differentiation, and myelination. Our results show that decreasing Pol III expression altered the proliferation rate of oligodendrocyte precursor cells but had no impact on migration. Additionally, reducing Pol III activity impaired the differentiation of these precursor cells into mature oligodendrocytes, evident at both the level of OL-lineage marker expression and on morphological assessment, with Pol III knockdown cells displaying a drastically more immature branching complexity. Myelination was hindered in the Pol III knockdown cells, as determined in both organotypic shiverer slice cultures and co-cultures with nanofibers. Analysis of Pol III transcriptional activity revealed a decrease in the expression of distinct tRNAs, which was significant in the siPolr3a condition. In turn, our findings provide insight into the role of Pol III in oligodendrocyte development and shed light on the pathophysiological mechanisms of hypomyelination in POLR3-related leukodystrophy.

Abstract 4386: Comparison of cytotoxicity of anthracycline based antineoplastic drugs in breast cancer

Abstract Mitoxantrone (MTX) is used for the chemotherapeutic treatment of breast cancer, but it has a dose-limiting cardiotoxicity. One of the mechanisms of MTX cytotoxicity is by inhibiting the topoisomerase II enzyme, which is crucial for maintaining cellular processes like replication. The aim of the study is to investigate if an alternative analog of MTX, named KP71 would be a suitable antineoplastic drug with reduced cardiovascular side effects. Cytotoxicity of both drugs in breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF7, the non-neoplastic breast cell line MCF10, cardiac human fibroblasts (HFB), and human umbilical vein endothelial cells (HUVEC) was assessed by MTT at 96h. The IC50 values (nM) for MTX/KP71 were as shown in table below. DNA decatenation assay demonstrated topoisomerase II inhibition with values of IC50 = 2.9/12.3 µM for topoisomerase IIα, and 2.1/7.0 µM for topoisomerase IIβ for MTX/KP71 respectively. Western blotting showed DNA damage by the gradation in γH2AX expression at different concentrations of MTX and KP71 at 6 h and 24 h treatment. KP71 was found to retain the ability to inhibit the proliferation of neoplastic cell lines in vitro with a slower profile of cytotoxicity on non-neoplastic cell lines as compared to MTX. KP71 partly exerted cytotoxicity by damaging DNA and inhibiting topoisomerase II enzyme activity. Organotypic HFB-HUVEC co-cultures were used to investigate the effects of anthracycline drugs in angiogenesis. Although both drugs reduced the number of tubules (and branches) compared to control (absence of drug), there were not significant differences between MTX and KP71. MTT IC50 (nM) MTX KP71 MDA-MB-468 27 50 MDA-MB231 38 169 MCF7 63 150 MCF10 35 90 HFB 160 1177 HUVEC 56 93 Citation Format: Mitali Singhal, Jacobo Elies Gomez, Sanjit Nayak, Kirsten Riches Suman, Amalia Ruiz Estrada. Comparison of cytotoxicity of anthracycline based antineoplastic drugs in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4386.

Organotypic 3D co-culture of human pleura as a novel in vitro model of Staphylococcus aureus infection and biofilm development

Organotypic 3D co-culture of human pleura as a novel in vitro model of Staphylococcus aureus infection and biofilm development

Cortactin expression in a Norwegian cohort of human papilloma virus negative oral squamous cell carcinomas of the mobile tongue.

Oral squamous cell carcinoma of the tongue (OTSCC) is the most common malignancy among oral squamous cell carcinomas and is frequently associated with an unfavorable prognosis. Local spread and distant metastasis are important causes of poor prognosis in OTSCC. Cortactin amplification and overexpression, a common molecular alteration in oral squamous cell carcinomas, have been linked to invasion and metastasis of tumor cells. However, the intra-tumor expression pattern and prognostic significance of cortactin in human papillomavirus (HPV) negative OTSCC is not fully investigated. Immunohistochemical analysis using tissue microarray consisting of formalin-fixed and paraffin-embedded HPV negative OTSCC (n=123) specimens showed overexpression of cortactin at tissue cores from invading fronts as compared to the corresponding center cores. High overall cortactin expression was found to be associated with advanced (larger) tumor size and the occurrence of distance metastasis. Kaplan-Meier survival analysis showed that patients with high overall cortactin expression were associated with reduced 5-year survival. Multivariate Cox regression analysis identified high cortactin expression to be an independent prognostic factor in OTSCC. Additionally, siRNA-mediated silencing of cortactin was found to suppress the proliferative and invasive abilities of OTSCC cells in an organotypic co-culture model. Overexpression of cortactin is a promising prognostic marker in HPV-negative OTSCC.

Dendritic cell phenotype and function in a 3D co-culture model of patient-derived metastatic colorectal cancer organoids.

Colorectal cancer (CRC) remains one of the most aggressive and lethal cancers, with metastasis accounting for most deaths. As such, there is an unmet need for improved therapies for metastatic CRC (mCRC). Currently, the research focus is shifting towards the reciprocal interactions within the tumor microenvironment (TME), which prevent tumor clearance by the immune system. Dendritic cells (DCs) play a key role in the initiation and amplification of anti-tumor immune responses and in driving the clinical success of immunotherapies. Dissecting the interactions between DCs and CRC cells may open doors to identifying key mediators in tumor progression, and possible therapeutic targets. This requires representative, robust and versatile models and tools. Currently, there is a shortage of such in vitro systems to model the CRC TME and its tumor-immune cell interactions. Here we develop and establish a dynamic organotypic 3D co-culture system to recapitulate and untangle the interactions between DCs and patient-derived mCRC tumor organoids. To our knowledge, this is the first study investigating human DCs in co-culture with tumor organoids in a 3D, organotypic setting. This system reveals how mCRC organoids modulate and shape monocyte-derived DCs (MoDCs) behavior, phenotype, and function, within a collagen matrix, using techniques such as brightfield and fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting. Our 3D co-culture model shows high viability and extensive interaction between DCs and tumor organoids, and its structure resembles patient tissue sections. Furthermore, it is possible to retrieve DCs from the co-cultures and characterize their phenotypic and functional profile. In our study, the expression of activation markers in both mature and immature DCs and their ability to activate T cells were impacted by co-culture with tumor organoids. In the future, this direct co-culture platform can be adapted and exploited to study the CRC-DC interplay in more detail, enabling novel and broader insights into CRC-driven DC (dys)function.

Highly concentrated trehalose induces prohealing senescence-like state in fibroblasts via CDKN1A/p21

Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates. The living skin equivalent (LSE) is an organotypic coculture containing keratinocytes cultivated on fibroblast-populated dermal substitutes. We demonstrated that human primary fibroblasts treated with highly concentrated trehalose promote significantly extensive spread of the epidermal layer of LSE without any deleterious effects. The RNA-seq analysis of trehalose-treated 2D and 3D fibroblasts at early time points revealed the involvement of the CDKN1A pathway, the knockdown of which significantly suppressed the upregulation of DPT, ANGPT2, VEGFA, EREG, and FGF2. The trehalose-treated fibroblasts were positive for senescence-associated β-galactosidase. Finally, transplantation of the dermal substitute with trehalose-treated fibroblasts accelerated wound closure and increased capillary formation significantly in the experimental mouse wounds in vivo, which was canceled by the CDKN1A knockdown. These data indicate that high-concentration trehalose can induce the senescence-like state in fibroblasts via CDKN1A/p21, which may be therapeutically useful for optimal wound repair.

Revisiting the involvement of tau in complex neural network remodeling: analysis of the extracellular neuronal activity in organotypic brain slice co-cultures

Objective. Tau ablation has a protective effect in epilepsy due to inhibition of the hyperexcitability/hypersynchrony. Protection may also occur in transgenic models of Alzheimer’s disease by reducing the epileptic activity and normalizing the excitation/inhibition imbalance. However, it is difficult to determine the exact functions of tau, because tau knockout (tauKO ) brain networks exhibit elusive phenotypes. In this study, we aimed to further explore the physiological role of tau using brain network remodeling. Approach. The effect of tau ablation was investigated in hippocampal-entorhinal slice co-cultures during network remodeling. We recorded the spontaneous extracellular neuronal activity over 2 weeks in single-slice cultures and co-cultures from control and tauKO mice. We compared the burst activity and applied concepts and analytical tools intended for the analysis of the network synchrony and connectivity. Main results. Comparison of the control and tauKO co-cultures revealed that tau ablation had an anti-synchrony effect on the hippocampal-entorhinal two-slice networks at late stages of culture, in line with the literature. Differences were also found between the single-slice and co-culture conditions, which indicated that tau ablation had differential effects at the sub-network scale. For instance, tau ablation was found to have an anti-synchrony effect on the co-cultured hippocampal slices throughout the culture, possibly due to a reduction in the excitation/inhibition ratio. Conversely, tau ablation led to increased synchrony in the entorhinal slices at early stages of the co-culture, possibly due to homogenization of the connectivity distribution. Significance. The new methodology presented here proved useful for investigating the role of tau in the remodeling of complex brain-derived neural networks. The results confirm previous findings and hypotheses concerning the effects of tau ablation on neural networks. Moreover, the results suggest, for the first time, that tau has multifaceted roles that vary in different brain sub-networks.

Novel 3D organotypic co-culture model of pleura.

Pleural mesothelial cells are the predominant cell type in the pleural cavity, but their role in the pathogenesis of pleural diseases needs to be further elucidated. 3D organotypic models are an encouraging approach for an in vivo understanding of molecular disease development. The aim of the present study was to develop a 3D organotypic model of the pleural mesothelium. Specimens of human pleura parietalis were obtained from patients undergoing surgery at the University Hospital Leipzig, Germany. 3D co-culture model of pleura was established from human pleural mesothelial cells and fibroblasts. The model was compared to human pleura tissue by phase-contrast and light microscopy, immunochemistry and -fluorescence as well as solute permeation test. Histological assessment of the 3D co-culture model displayed the presence of both cell types mimicking the morphology of the human pleura. Vimentin and Cytokeratin, PHD1 showed a similar expression pattern in pleural biopsies and 3D model. Expression of Ki-67 indicates the presence of proliferating cells. Tight junctional marker ZO-1 was found localized at contact zones between mesothelial cells. Each of these markers were expressed in both the 3D co-culture model and human biopsies. Permeability of 3D organotypic co-culture model of pleura was found to be higher for 70 kDa-Dextran and no significant difference was seen in the permeability for small dextran (4 kDa). In summary, the presented 3D organoid of pleura functions as a robust assay for pleural research serving as a precise reproduction of the in vivo morphology and microenvironment.