Organotypic Model Research Articles

Patient-Derived Organoids Recapitulate Pathological Intrinsic and Phenotypic Features of Fibrous Dysplasia

Fibrous dysplasia (FD) is a rare bone disorder characterized by the replacement of normal bone with benign fibro-osseous tissue. Developments in our understanding of the pathophysiology and treatment options are impeded by the lack of suitable research models. In this study, we developed an in vitro organotypic model capable of recapitulating key intrinsic and phenotypic properties of FD. Initially, transcriptomic profiling of individual cells isolated from patient lesional tissues unveiled intralesional molecular and cellular heterogeneity. Leveraging these insights, we established patient-derived organoids (PDOs) using primary cells obtained from patient FD lesions. Evaluation of PDOs demonstrated preservation of fibrosis-associated constituent cell types and transcriptional signatures observed in FD lesions. Additionally, PDOs retained distinct constellations of genomic and metabolic alterations characteristic of FD. Histological evaluation further corroborated the fidelity of PDOs in recapitulating important phenotypic features of FD that underscore their pathophysiological relevance. Our findings represent meaningful progress in the field, as they open up the possibility for in vitro modeling of rare bone lesions in a three-dimensional context and may signify the first step towards creating a personalized platform for research and therapeutic studies.

Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity.

Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally "anergic", i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the "anergic" phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the "exhausted" state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies.

Exploring host-commensal-pathogen dynamics in cell line and organotypic human intestinal epithelial models

Host and microbiome intricately interact in the ecosystem of the human digestive tract, playing a crucial role in our health. These interactions can initiate immune responses in the epithelial cells, which, in turn, activate downstream responses in other immune cells. Here, we used a CaCo-2 and a human intestinal enteroid (HIE) model to explore epithelial responses to both commensal and pathogenic bacteria, individually and combined. CaCo-2 cells were co-cultured with peripheral blood mononuclear cells, revealing downstream activation of immune cells. While both systems showed comparable cytokine profiles, they differed in their responses to the different bacteria, with the organoid system being more representative of responses observed in humans. We provide evidence of the pro-inflammatory responses associated with these bacteria. These models contribute to a deeper understanding of the interactions between the microbiota, intestinal epithelium, and immune cells in the gut, promoting advances in the field of host-microbe interactions.

Modeling PAH Mixture Interactions in a Human In Vitro Organotypic Respiratory Model.

One of the most significant challenges in human health risk assessment is to evaluate hazards from exposure to environmental chemical mixtures. Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous contaminants typically found as mixtures in gaseous and particulate phases in ambient air pollution associated with petrochemicals from Superfund sites and the burning of fossil fuels. However, little is understood about how PAHs in mixtures contribute to toxicity in lung cells. To investigate mixture interactions and component additivity from environmentally relevant PAHs, two synthetic mixtures were created from PAHs identified in passive air samplers at a legacy creosote site impacted by wildfires. The primary human bronchial epithelial cells differentiated at the air-liquid interface were treated with PAH mixtures at environmentally relevant proportions and evaluated for the differential expression of transcriptional biomarkers related to xenobiotic metabolism, oxidative stress response, barrier integrity, and DNA damage response. Component additivity was evaluated across all endpoints using two independent action (IA) models with and without the scaling of components by toxic equivalence factors. Both IA models exhibited trends that were unlike the observed mixture response and generally underestimated the toxicity across dose suggesting the potential for non-additive interactions of components. Overall, this study provides an example of the usefulness of mixture toxicity assessment with the currently available methods while demonstrating the need for more complex yet interpretable mixture response evaluation methods for environmental samples.

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs. Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (≥30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

Postnatal development of rat retina: a continuous observation and comparison between the organotypic retinal explant model and in vivo development

Abstract The organotypic retinal explant culture has been established for more than a decade and offers a range of unique advantages compared with in vivo experiments and cell cultures. However, the lack of systematic and continuous comparison between in vivo retinal development and the organotypic retinal explant culture makes this model controversial in postnatal retinal development studies. Thus, we aimed to verify the feasibility of using this model for postnatal retinal development studies by comparing it with the in vivo retina. In this study, we showed that postnatal retinal explants undergo normal development, and exhibit a consistent structure and timeline with retinas in vivo. Initially, we used SOX2 and PAX6 immunostaining to identify retinal progenitor cells. We then examined cell proliferation and migration by immunostaining with Ki-67 and doublecortin, respectively. Ki-67- and doublecortin-positive cells decreased in both in vivo and explants during postnatal retinogenesis, and exhibited a high degree of similarity in abundance and distribution between groups. Additionally, we used Ceh-10 homeodomain-containing homolog, glutamate-ammonia ligase (glutamine synthetase), neuronal nuclei, and ionized calcium-binding adapter molecule 1 immunostaining to examine the emergence of bipolar cells, Müller glia, mature neurons, and microglia, respectively. The timing and spatial patterns of the emergence of these cell types were remarkably consistent between in vivo and explant retinas. Our study showed that the organotypic retinal explant culture model had a high degree of consistency with the progression of in vivo early postnatal retina development. The findings confirm the accuracy and credibility of this model and support its use for long-term, systematic, and continuous observation.

Electrical Impedance Spectroscopy Quantifies Skin Barrier Function in Organotypic In Vitro Epidermis Models

3 D human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in pre-clinical investigative dermatology and regulatory toxicology. Here, we investigated the utility of electrical impedance spectroscopy (EIS) for non-invasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for seven consecutive days did not impact epidermal morphology and readouts showed comparable trends to HEEs measured only once. We determined two frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9 engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to pro-inflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a non-invasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects and repair.

Keratinocyte-derived small extracellular vesicles supply antigens for CD1a-resticted T cells and promote their type 2 bias in the context of filaggrin insufficiency.

Exosome-enriched small extracellular vesicles (sEVs) are nanosized organelles known to participate in long distance communication between cells, including in the skin. Atopic dermatitis (AD) is a chronic inflammatory skin disease for which filaggrin (FLG) gene mutations are the strongest genetic risk factor. Filaggrin insufficiency affects multiple cellular function, but it is unclear if sEV-mediated cellular communication originating from the affected keratinocytes is also altered, and if this influences peptide and lipid antigen presentation to T cells in the skin. Available mRNA and protein expression datasets from filaggrin-insufficient keratinocytes (shFLG), organotypic models and AD skin were used for gene ontology analysis with FunRich tool. sEVs secreted by shFLG and control shC cells were isolated from conditioned media by differential centrifugation. Mass spectrometry was carried out for lipidomic and proteomic profiling of the cells and sEVs. T cell responses to protein, peptide, CD1a lipid antigens, as well as phospholipase A2-digested or intact sEVs were measured by ELISpot and ELISA. Data analysis revealed extensive remodeling of the sEV compartment in filaggrin insufficient keratinocytes, 3D models and the AD skin. Lipidomic profiles of shFLGsEV showed a reduction in the long chain (LCFAs) and polyunsaturated fatty acids (PUFAs; permissive CD1a ligands) and increased content of the bulky headgroup sphingolipids (non-permissive ligands). This resulted in a reduction of CD1a-mediated interferon-γ T cell responses to the lipids liberated from shFLG-generated sEVs in comparison to those induced by sEVs from control cells, and an increase in interleukin 13 secretion. The altered sEV lipidome reflected a generalized alteration in the cellular lipidome in filaggrin-insufficient cells and the skin of AD patients, resulting from a downregulation of key enzymes implicated in fatty acid elongation and desaturation, i.e., enzymes of the ACSL, ELOVL and FADS family. We determined that sEVs constitute a source of antigens suitable for CD1a-mediated presentation to T cells. Lipids enclosed within the sEVs secreted on the background of filaggrin insufficiency contribute to allergic inflammation by reducing type 1 responses and inducing a type 2 bias from CD1a-restricted T cells, thus likely perpetuating allergic inflammation in the skin.

Abstract 2613: Treatment of solid tumors with antibody drug conjugates targeting aberrant O-glycoproteins

Abstract The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Many solid tumors display aberrant glycosylation that results in the expression of tumor-associated carbohydrate antigens (TACAs) distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes provides the cancer selectivity needed for immunotherapy of solid tumors. However, only a few such glycopeptide epitopes have been targeted to date. We have developed a strategy to predict, design, and produce cancer-specific antibodies targeting novel O-glycoprotein epitopes with exquisite cancer specificity. Our mAbs avoid on-target off-tumor toxicity due to the specificity of aberrant glycosylation, and cover a broad range of cancer indications, including breast, lung, colon, pancreatic, and ovarian cancer. Additionally, our antibodies can be used as an IHC companion diagnostic and can be functionalized across a number of modalities, including next-generation CAR-T, T-cell bispecifics, and antibody-drug-conjugates (ADCs).Here, we present two novel antibodies, GO-08 and GO-013, selectively targeting cancer-specific Tn-glycosylated proteins in a site-specific manner with low nanomolar affinities. GO-008 and GO-013 were collectively shown to be highly cancer-selective by immunohistochemistry of sections from a large array of different squamous and adenocarcinomas, including triple-negative breast cancer, lung cancer, gastric cancer, ovarian cancer, pancreatic, and colorectal cancer. The two antibodies were functionalized utilizing ConjuAllTM technology, conjugating the microtubule disrupting payload MMAE via a site-specific beta-glucuronidase cleavable linker. ConjuAllTM functions as a plasma stable, tumor labile linkage, and due to the minimal expression pattern of beta-glucuronidase in healthy tissues, reduces both off- and on-target toxicity due to the minimization of release of free payload in non-tumor tissues. The ADCs based on GO-08 and GO-013 (LCB45A and LCB22A) demonstrated target-specific cytotoxicity invitro and in vivo with tumor elimination in breast and lung PDX models. GO-013 conjugated to MMAE (LCB22A) was well tolerated in cynomolgus monkey toxicity studies. Moreover, the ADCs selectively kill target cells in a human mixed organotypic cancer model without affecting healthy cells, indicating tolerability and safety. The combination of specificity of TACA targeting to differentiate ADC binding from healthy tissue and ConjuAll’s tumor selective payload release promises to enable an excellent anti-cancer therapy. Citation Format: Aaron C. Groen, Nisha Shrestha, Boris Klebanov, Byeongjun Yu, BeomYong Lee, Chul-Woong Chung, Changsik Park, Mikkel K. Aasted, Sally Dabelsteen, Hans H. Wandall. Treatment of solid tumors with antibody drug conjugates targeting aberrant O-glycoproteins [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 2613.

Engineered human osteoarthritic cartilage organoids

The availability of human cell-based models capturing molecular processes of cartilage degeneration can facilitate development of disease-modifying therapies for osteoarthritis [1], a currently unmet clinical need. Here, by imposing specific inflammatory challenges upon mesenchymal stromal cells at a defined stage of chondrogenesis, we engineered a human organotypic model which recapitulates main OA pathological traits such as chondrocyte hypertrophy, cartilage matrix mineralization, enhanced catabolism and mechanical stiffening. To exemplify the utility of the model, we exposed the engineered OA cartilage organoids to factors known to attenuate pathological features, including IL-1Ra, and carried out mass spectrometry-based proteomics. We identified that IL-1Ra strongly reduced production of the transcription factor CCAAT/enhancer-binding protein beta [2] and demonstrated that inhibition of the C/EBPβ-activating kinases could revert the degradative processes. Human OA cartilage organoids thus represent a relevant tool towards the discovery of new molecular drivers of cartilage degeneration and the assessment of therapeutics targeting associated pathways.

Abstract 6768: Matched XPDX™derivative models as a platform for continuity across preclinical drug development

Abstract Drug discovery is a reductive process in which druglike entities are carried through increasingly complex assay formats to tease out the compounds best suited for human studies. In vivo efficacy remains the benchmark for successful preclinical drug development. Cell-based models are a mainstay in early stages but translatability of different models across platforms remains a challenge. In oncology, patient-derived xenografts (PDX) are the gold standard for animal studies because they recapitulate key features of human tumors including the cellular heterogeneity, growth patterns, and drug response of the original patient tumor. Ex vivo, PDX derivatives have the potential to harness these same traits thereby extending the utility of PDX models to earlier stages of preclinical drug development. The KIYA-Predict™ platform is a clinically validated tool for the assessment of ex vivo drug responses across a broad range of drug classes that when applied to XPDX models shows good correlation to in vivo drug responses and excellent intra-sample reproducibility over time. Working with the well-characterized XenoSTART™ XPDX model repository, Kiyatec has derived 2D cell lines and organotypic 3D-expansion cultures to complement the ex vivo predictive value of our KIYA-PREDICT™ XPDX drug response profiling (DRP) platform. The development of syngeneic models applicable to simpler, high-throughput library screening (2D formats) and lower-throughput more complex mechanistic studies (3D organotypic models) could alleviate the burden and pitfalls of extrapolating data across unrelated models. Matched sets of 2D cell lines (2D-XPDX™) and 3D organotypic (XPDXO™) cultures were established from single cell suspensions of digested, mouse-cell depleted XPDX™ tissue samples. Drug response profiling of the XPDX™ in their various ex vivo culture formats showed relative similarities across platforms. Genomic profiling of the derivates confirmed shared tissue origin and retention of genomic and phenotypic signatures to varying degrees across models. Mechanistic evaluation of 3D organotypic culture demonstrated that disease-relevant biomarkers and processes are maintained. Together these results demonstrate the feasibility of this novel drug discovery platform that avoids ambiguity imparted by discordant models. Citation Format: Melissa Millard, Aaron Carlson, Danielle Y. Nadeau, Kimberly J. Burgess, Alyssa Simonson, Armando Diaz, Michael Wick, Teresa DesRochers. Matched XPDX™derivative models as a platform for continuity across preclinical drug development [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 6768.

3D approaches to culturing bovine skin: explant culture versus organotypic skin model.

Digital Dermatitis (DD) in cattle appears with high prevalence, nevertheless the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD. Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, read-out parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (day 7, 14 and 21), cultured skin samples were taken for (immuno)histological analysis. Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture. Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell-interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as e.g. DD.

DPM1 modulates desmosomal adhesion and epidermal differentiation through SERPINB5.

Glycosylation is essential to facilitate cell-cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell-cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as a DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in connecting desmosomal adhesion with epidermal differentiation.

Mitigation of human iris angiogenesis through uPAR/LRP-1 interaction antagonism in an organotypic exvivo model.

Rubeosis Iridis (RI) is characterized by an increase in neovascularization and inflammation factors in the iris. During angiogenesis, the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a pivotal role in extracellular matrix remodeling, where uPAR regulates endothelial cell migration and proliferation through assembly with transmembrane receptors. Here, in the context of hypoxia-induced angiogenesis, the uPA/uPAR system blockage was investigated by using UPARANT in a novel exvivo human iris organotypic angiogenesis assay. The effects of uPA/uPAR system antagonism in the humanized model of ocular pathologic angiogenesis were analyzed by sprouting angiogenesis and protein assays (western, dot blots, and co-immunoprecipitation) and correlated to vascular endothelial growth factor (VEGF) inhibition. Phosphoprotein and co-immunoprecipitation assay illustrated an unidentified antagonism of UPARANT in the interaction of uPAR with the low-density lipoprotein receptor-related protein-1 (LRP-1), resulting in inhibition of β-catenin-mediated angiogenesis in this model. The effects of uPA/uPAR system inhibition were focal to endothelial cells exvivo. Comparison between human iris endothelial cells and human retinal endothelial revealed an endothelial-specific mechanism of β-catenin-mediated angiogenesis inhibited by uPA/uPAR system blockage and not by VEGF inhibition. Collectively, these findings broaden the understanding of the effects of the uPA/uPAR system antagonism in the context of angiogenesis, revealing non-canonical β-catenin downstream effects mediated by LRP-1/uPAR interaction.

Abstract A101: A novel ovarian cancer organotypic tumor slice culture model

Abstract A101: A novel ovarian cancer organotypic tumor slice culture model

Abstract B068: FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models

Abstract The unfavorable prognosis of high-grade serous ovarian cancer, HGSOC, may be driven by a small subset of ovarian cancer stem cells (OCSC), which are endowed with tumor-initiating ability and are intrinsically resistant to cytotoxic treatments. These characteristics are sustained by signals coming from the tumor microenvironment (TME). As a common site of HGSOC presentation is the omentum, we partially recapitulated the architecture of human omentum in vitro using 3D co-cultures of primary human omental fibroblasts and mesothelial cells. Patient-derived HGSOC cells were then integrated in these organotypic models, either as bulk cancer cells or as OCSC from the same patients. After co-culture in this model, malignant cells were re-isolated by FACS sorting and subjected to whole-transcriptome RNA sequencing, comparing them to control cells that have not been in contact with the in vitro TME. Our data revealed transcriptional reprogramming induced by the in vitro TME in HGSOC cells. We found a number of pathways differentially regulated in the bulk population of OC cells vs OCSC, shedding light on the impact of the TME on OC stemness. Moreover, we identified a panel of transcription factors (TFs) specifically altered by the in vitro TME in OCSC. These TF-regulated gene networks represent OCSC vulnerabilities and, hence, potential targets in relapsing tumors. Among these, we focused on FOXM1, a TF with pleiotropic functions in cancer cells, whose activity is altered in 84% of ovarian cancer patients. The FOXM1 pathway was strongly induced by the in vitro TME specifically in OCSC. Mechanistically, the contact with the in vitro TME triggered a FAK/YAP axis which, in turn, accounted for the induction of FOXM1 and its downstream target genes. Moreover, the pharmacological inhibition of FOXM1 significantly impaired OCSC fitness, supporting, on one hand, the key role of TME in orchestrating OC stemness and suggesting, on the other, a new approach for the eradication of OCSC from the omental niche. Overall, our patient-derived organotypic platform offers the opportunity to unravel the impact of the TME on OCSC at the molecular level in experimental models with clinical relevance and can be exploited to design innovative HGSOC-eradicating treatments. Citation Format: Chiara Battistini, Hilary A. Kenny, Valentina Nieddu, Valentina Melocchi, Alessandra Decio, Beatrice Malacrida, Alessia Gatto, Fabrizio Bianchi, Raffaella Giavazzi, Nicoletta Colombo, Frances R. Balkwill, Ernst Lengyel, Ugo Cavallaro. FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B068.

Abstract A098: Microenvironment-regulated matrix Gla protein drives ovarian cancer stemness

Abstract The clinical evolution of ovarian cancer (OC), with peritoneal dissemination, tumor relapse and chemoresistance, seems to be accounted for by a small population of ovarian cancer stem cells (OCSC), thus making them an attractive target for more efficacious treatments. However, this could only be achieved through the detailed characterization of the molecular and functional traits of this elusive cell subpopulation. Therefore, the identification of novel OCSC-associated targets and therapeutic approaches may offer alternative treatment options. We established a new platform of patient-derived organotypic models of OC and found that Matrix Gla Protein (MGP) was upregulated in cancer cells upon contact with the peritoneal tumor microenvironment. MGP is classically known as a vitamin K-dependent calcium chelator, mainly associated with the inhibition of tissue calcification and cardiovascular disease. In addition, MGP is also aberrantly expressed in different cancer types, but the biological mechanisms that regulate its functional contribution to cancer progression is still unknown. Our transcriptomic profiling of primary OCSC versus their non-stem counterpart pointed to MGP as a potential driver of cancer stemness. Indeed, we obtained evidence that MGP promotes, and is required for, stemness-associated biological features in vitro and in animal models, including sphere formation and tumor initiation. MGP was also implicated in the regulation of the expression of several key genes involved in stemness and/or EMT. Finally, MGP was upregulated in OC patients following chemotherapy, suggesting that the protein may be involved in OCSC-driven chemoresistance. The molecular mechanisms underlying MGP-regulated OCSC pathophysiology were investigated by RNA sequencing experiments on MGP-manipulated OC cells, which unveiled genetic networks and pathways that are orchestrated by MGP to promote OCSC function. The biological role of MGP in OC provides novel insight into the biology of OCSC, thus defining new pathways involved in OC stemness and paving the way to innovative therapeutic strategies for OC eradication. Citation Format: Valentina Nieddu, Valentina Melocchi, Chiara Battistini, Giulia Franciosa, Michela Lupia, Jesper Olsen, Nicoletta Colombo, Fabrizio Bianchi, Ugo Cavallaro. Microenvironment-regulated matrix Gla protein drives ovarian cancer stemness [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A098.

GelMA hydrogel as a scaffold to enhance the survival and differentiation of human induced lateral ganglionic eminence precursor cells

Cell reprogramming holds enormous potential to revolutionize our understanding of neurological and neurodevelopmental disorders, as well as enhance drug discovery and regenerative medicine. We have developed a direct cell reprogramming technology that allows us to generate lineage-specific neural cells. To extend our technology, we have investigated the incorporation of directly reprogrammed human lateral ganglionic eminence precursor cells (hiLGEPs) in a 3-dimensional (3D) matrix. Hydrogels are one of the most promising bio-scaffolds for 3D cell culture, providing cells with a supportive environment to adhere, proliferate, and differentiate. In particular, gelatin methacryloyl (GelMA) hydrogels have been used for a variety of 3D biomedical applications due to their biocompatibility, enzymatic cleavage, cell adhesion and tunable physical characteristics. This study therefore investigated the effect of GelMA hydrogel encapsulation on the survival and differentiation of hiLGEPs, both in vitro and following ex vivo transplantation into a quinolinic acid (QA) lesion rat organotypic slice culture model. We demonstrate, for the first time, that the encapsulation of hiLGEPs in GelMA hydrogel significantly enhances the survival and generation of DARPP32+ striatal neurons both in vitro and following ex vivo transplant. Furthermore, GelMA-encapsulated hiLGEPs were predominantly located away from the reactive astrocyte network that forms following QA lesioning, suggesting GelMA provides a protective barrier for cells in regions of inflammatory activation. Overall, these results indicate that GelMA hydrogel has the potential to act as a 3D bio-scaffold to augment the viability and differentiation of hiLGEPs for research and translation of pharmaceutical development and regenerative medicine.

Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models.

Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction-associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.

Long-term Rapamycin Treatment Inhibit AKT Activity and Lower Intracellular Calcium Expression in Organotypic Hippocampal Slice Cultures Model of Epilepsy

Despite the development of anti-epilepsy drugs, drug-refractory epilepsy still becomes a challenging problem along with an increased incidence of epilepsy. To face that challenge and increase patients’ quality of life, treatment of epilepsy must effectively prevent epileptogenesis, not only symptomatic treatment. AKT signaling pathway was proven to have important roles in epilepsy through its function in the synaptic plasticity, neurogenesis, axon guidance, modulation of the glutamate transporter, and activation of the Ca2+ channel. AKT also activated mTOR signaling pathway as activator of mTORC1 and also effector of mTORC2. Several studies showed the ability of long-term rapamycin treatment to inhibit mTORC2. This study used organotypic hippocampal slice cultures (OHSC) and long-term rapamycin treatment was administered for 3, 5, 8, and 10 days at a dose of 20 nM after induction of epilepsy by low-Mg2+ medium administration for 40 minutes. Low-Mg2+ medium administration induced seizure activity in OHSC showed by significant increase in intracellular Ca2+expressionand also significantly increase AKT activity. After administration of long-term rapamycin treatment AKT activity and intracellular Ca2+expression were significantly reduced. The longer the treatment of rapamycin, the lower the AKT activity and intracellular Ca2+expression. Long-term rapamycin treatment has the potential to become a novel epilepsy drug through its ability to attenuate AKT activity and suppress the seizures proven by lower intracellular Ca2+expression.