3D Culture Research Articles

Three-dimensional tissue engineered skeletal muscle modelling facioscapulohumeral muscular dystrophy

Abstract Facioscapulohumeral muscular dystrophy (FSHD) is caused by sporadic misexpression of the transcription factor double homeobox 4 (DUX4) in skeletal muscles. So far, monolayer cultures and animal models have been used to study the FSHD disease mechanism and for FSHD therapy development, but these models do not fully recapitulate the disease and there is a lack of knowledge on how DUX4 misexpression leads to skeletal muscle dysfunction. To overcome these barriers, we have developed a three-dimensional tissue engineered skeletal muscle (3D-TESM) model by generating genetically matched myogenic progenitors (MPs) from human induced pluripotent stem cells of three mosaic FSHD patients. 3D-TESMs derived from genetically affected MPs recapitulate pathological features including DUX4 and DUX4 target gene expression, smaller myofiber diameters, and reduced absolute forces upon electrical stimulation. RNA sequencing data illustrates increased expression of DUX4 target genes in 3D-TESMs compared to two-dimensional (2D) myotubes, and cellular differentiation was improved by 3D culture conditions. Treatment of 3D-TESMs with three different small molecules identified in drug development screens in 2D muscle cultures showed no improvements, and sometimes even declines, in contractile force and sarcomere organization. These results suggest that these compounds either have a detrimental effect on the formation of 3D-TESMs, an effect that might have been overlooked or was challenging to detect in 2D cultures and in vivo models, and/or that further development of the 3D-TESM model is needed. In conclusion, we have developed a 3D skeletal muscle model for FSHD that can be employed for preclinical research focusing on DUX4 expression and downstream pathways of FSHD in relation to contractile properties. In the future, we expect that this model can also be used for preclinical drug screening.

ONC206, an imipridone derivative, demonstrates anti-colorectal cancer activity against stem/progenitor cells in 3D cell cultures and in patient-derived organoids

ONC206, an imipridone derivative, demonstrates anti-colorectal cancer activity against stem/progenitor cells in 3D cell cultures and in patient-derived organoids

Physical, biochemical, and biological characterization of olive-derived lipid nanovesicles for drug delivery applications

AbstractExtracellular vesicles (EVs) have shown great promise as drug delivery system (DDS). However, their complex and costly production limit their development for clinical use. Interestingly, the plant kingdom can also produce EV-like nanovesicles that can easily be isolated and purified from a large quantity of raw material at a high yield. In this study, olive-derived nanovesicles (ODNVs) were isolated from raw fruits using serial centrifugations and their physical and biological features characterized to demonstrate their promising potential to be used as a DDS. Nanotracking particle analysis indicated an average size of 109.5 ± 3.0 nm and yield of 1012 ODNVs/mL for the purest fraction. Microscopy imaging, membrane fluidity assay and lipidomics analysis showed the presence of a rich lipid bilayer that significantly varied between different sources of ODNVs but showed a distinct signature compared to human EVs. Moreover, ODNVs were enriched in PEN1 and TET8 compared to raw fruits, suggesting an extracellular origin. Interestingly, ODNVs size and yield stayed unchanged after exposure to high temperature (70 °C for 1 h), wide pH range (5–10), and 50–100 nm extrusion, demonstrating high resistance to physical and chemical stresses. This high resistance allowed ODNVs to stay stable in water at 4 °C for a month, or with the addition of 25 mM trehalose for long-term freezing storage. Finally, ODNVs were internalized by both 2D and 3D cell culture without triggering significant cytotoxicity and immunogenicity. Importantly, the anticancer drug doxorubicin (dox) could be loaded by passive incubation within ODNVs and dox-loaded ODNVs decreased cell viability by 90% compared to only 70% for free dox at the same concentration, indicating a higher efficiency of drug delivery by ODNVs. In addition, this high cytotoxicity effect of dox-loaded ODNVs was shown to be stable after a 2-week storage at 4 °C. Together, these findings suggested that ODNVs represent a promising candidate as drug nanocarrier for various DDS clinical applications, as demonstrated by their biocompatibility, high resistance to stress, good stability in harsh environment, and improvement of anticancer drug efficacy.

Abstract C049: Implementing human bone marrow organoids to interrogate microenvironmental influences on the efficacy of blood cancer immunotherapies

Abstract Cancers perpetuate immunosuppressive niches that fuel progression and hinder the clinical utility of immunotherapies. Pre-clinical validation of immunotherapies often relies on 2D models that lack spatial complexity, or mouse models that do not fully recapitulate the human tumor microenvironment (TME). We recently developed a human induced pluripotent stem cell (iPSC)-derived bone marrow organoid platform, creating a robust system to interrogate cancer-stroma cross talk. Here, we further develop the model to create the first in vitro system for evaluation of blood cancer immunotherapies in a human tissue environment. We focused on targeting myeloproliferative neoplasms (MPNs) – chronic blood cancers affecting ∼400,000 individuals in the US that are currently largely incurable. 1/3 of MPNs are driven by a mutation in calreticulin (mutCALR), and the mutCALR oncoprotein is displayed on the cell surface of disease-initiating stem cells and fibrosis-driving megakaryocytes. MPNs are characterized by pronounced inflammation and fibrosis (“myelofibrosis”). Therapeutic antibodies have entered first-in-human trials, and we recently presented pre-clinical data for the first mutCALR-directed CAR-T cell therapy. However, it is unclear how the TME may limit the efficiency of these agents. To address this, we first tested the utility of the organoid platform to evaluate target killing of leukemic cell lines and cells from patients using the anti-CD33 antibody-drug conjugate (ADC) gemtuzumab. The ADC showed good penetration and potent target cell killing in organoids, with only minimal reduction in efficacy in TGFβ-rich/fibrotic microenvironments. We then developed more sophisticated ‘chimeroids’ comprising the iPSC organoid scaffold engrafted with malignant stem cells plus CAR-Ts. Addition of mutCALR-directed CAR-Ts induced highly selective killing of mutCALR+ stem/progenitor cells with minimal toxicity to non-malignant cells including niche cells (n=5 donors), replicating the highly specific and robust killing seen in 2D cultures. To evaluate the impact of TME perturbations on CAR-T phenotype and function, we generated a scRNAseq dataset of 161,970 cells from ‘healthy’ and ‘myelofibrotic’ organoids engrafted with patient-derived mutCALR+ cells plus CAR-Ts, pre-stimulating organoids with two immunoregulatory cytokines highly abundant in myelofibrosis -TGFβ and Galectin-1. CAR- Ts exposed to mutCALR+ cells showed a 2x expansion of CD8+ effector memory cells, with significant enrichment of IFNγ, TNFα and IL-2 signaling gene-sets. The proportion of immunosuppressive Tregs increased in a high TGFβ TME, but not with Galectin-1. Ongoing analyses are further interrogating crosstalk between CAR-T, target cells, and niche components, and exploring the pro-inflammatory impact of CAR-T killing on the marrow niche – a potential mediator of delayed hematopoietic recovery. These studies demonstrate the utility of human organoids to evaluate blood cancer-targeting immunotherapies within a relevant human TME, with broad relevance for mechanistic and translational studies. Citation Format: Zoë C. Wong, Yuqi Shen, Alex Rampotas, Isaac Gannon, Charlotte Brierley, Camelia Benlabiod, Nawshad Hayder, Eleanor Murphy, Aude-Anaïs Olijnik, Claire Roddie, Martin Pulé, Adam J. Mead, Abdullah O. Khan, Bethan Psaila. Implementing human bone marrow organoids to interrogate microenvironmental influences on the efficacy of blood cancer immunotherapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C049.

Abstract B041: Investigating the impact of extracellular matrix stiffness on macrophage function and organoid growth in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 11%. Current treatments mostly target the cancer cells themselves and have not been able to significantly improve survival rates, meaning there is an urgent need for novel therapies. PDAC is unique among solid tumors because it exhibits extensive desmoplasia that is characterized by an abundance of extracellular matrix (ECM) components. In recent years, an increasing body of work has shown that cancer tissue is stiffer than normal tissue by a factor of 3 or more. This increase in ECM stiffness has been shown to play an important role in affecting cell-to-cell communication as well as cancer progression and chemoresistance . However, how ECM stiffness affects tumor-associated macrophages, an important component of the pancreatic tumor microenvironment (TME), is still understudied. Previous studies have demonstrated that macrophages are highly responsive to mechanical cues, with the mechanical environment influencing macrophage polarization and function in a context- and disease-dependent manner. Elucidating these potential changes in PDAC can provide us with insights on how stiffness affects therapeutic outcome. Healthy pancreatic tissue typically exhibits a storage modulus of approximately 0.5-1 kPa, whereas PDAC tissue can range from 4-10 kPa. To study macrophage responses to varying stiffness, we cultured RAW264.7 mouse macrophage cell lines on 2D substrates with stiffness levels of 0.2, 2, and 8 kPa. We observed that macrophages cultured on stiffer matrices exhibited a trend toward a pro-inflammatory phenotype, with increased expression of M1 markers such as iNOS, IL-1β, and IL-6, compared to those cultured on softer matrices. However, conditioned media from macrophages cultured on both soft and stiff substrates similarly reduced the size of PDAC organoids, indicating a lack of functional difference between these groups. These findings suggest the need for more sophisticated models that better replicate the PDAC TME to fully capture the complex cell-to-cell interactions that are not evident in 2D cultures. To address this limitation, we developed a 3D tri-culture tunable biomimetic mouse model comprising PDAC organoids, host-matched cancer-associated fibroblasts (CAFs), and macrophages. This novel model allows us to modulate the stiffness of the 3D co-culture system through the addition of collagen I and CAFs. Rheometry data confirmed that the stiff tri-culture model had a significantly higher storage modulus than the soft tri-culture model. Using qPCR, we investigated the role of mechanosensitive ion channels, specifically Transient Receptor Potential Vanilloid-type 4 (TRPV4) and Piezo1, in mediating macrophage mechanosensing in response to varying levels of matrix stiffness. This study highlights the importance of 3D models that can recapitulate biophysical cues of cancer and will allow us to better understand how macrophages respond to stiffness, which may give rise to new therapeutic targets and development of new treatments in the future. Citation Format: Bayan Mahgoub, Weikun Xiao, Eileen Fung, Shannon Mumenthaler, Reginald Hill. Investigating the impact of extracellular matrix stiffness on macrophage function and organoid growth in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B041.

Abstract C050: Leveraging ECM deposition by CAF-like Ewing sarcoma tumor cells to target micrometastases with matrix-binding VHHs

Abstract Ewing sarcoma (EwS) is an aggressive bone and soft tissue tumor and EwS patients often succumb to their disease years after initial treatment due to relapses at metastatic sites. Novel therapeutic strategies are needed that can successfully eliminate microscopic residual disease foci that persist at the end of primary therapy. Extracellular matrix (ECM) proteins in the tumor microenvironment (TME) provide critical pro-survival and pro-invasion signals to tumor cells. Our published and unpublished data show that the glycoprotein tenascin-C (TNC) is enriched in EwS metastases and that TNC and other pro-tumorigenic ECM proteins are deposited by subpopulations of CAF-like tumor cells that are activated in response to tumor and TME-derived TGF-beta ligands. TNC is abundantly produced by multiple human tumors of epithelial and non-epithelial lineage but is otherwise rarely expressed outside of development and wound healing. Importantly, TNC is specifically enriched in metastatic lesions where it has been implicated as a mediator of metastatic competence and treatment resistance. In the current work we have tested whether the TNC-rich TME of disseminated EwS tumor foci could be leveraged to direct therapies to sites of residual micrometastatic disease. To achieve this, we generated monovalent and bivalent anti-human TNC VHHs (hTNC-VHH) using a mammalian expression platform. Camelid-derived hTNC-VHH sequences were sourced from published literature. VHHs are single domain heavy chain only antibody fragments with higher stability and tissue penetration than conventional monoclonal antibodies. Using EwS tumor spheroids in collagen-rich 3D culture, hTNC-VHH penetrated dense ECM matrices and bound through multiple cell layers in under 10 minutes. hTNC-VHH accumulated in TNC-positive but not TNC-knockout EwS spheroids and were retained beyond 96 hours. To assess their potential to drive protein and immune therapies to TNC-rich TMEs, we fused hTNC-VHH to immune modulating CD3- and CD28-activating VHHs. These hTNC-VHH-CD3/CD28-conjugates promoted immune cell activation and tumor cell death in PBMC-tumor co-culture assays as determined by CD25 flow cytometry and fluorescence microscopy, respectively. To assess in vivo localization, fluorescently-tagged hTNC-VHH were intravenously injected into mice that had been xenografted with EwS cells. Tissue microscopy confirmed selective binding of hTNC-VHH to small lung and liver EwS micrometastases in less than 4 hours. VHHs were not retained in non-tumor bearing organs including the brain and heart or in tumor-free lung and liver regions. Experiments are ongoing to assess anti-tumor efficacy and pharmacodynamics of hTNC-VHH conjugates in EwS xenografts. Together these findings suggest that the ECM-remodeling properties of CAF-like EwS cells can be exploited to recruit novel ECM-targeting protein therapeutics to micrometastases. If successful, this innovative approach could eradicate microscopic residual disease and prevent metastatic EwS recurrence. Citation Format: Emma D. Wrenn, Jason P. Price, Raymond O. Ruff, Nicolas M. Garcia, James M. Olson, Elizabeth R. Lawlor. Leveraging ECM deposition by CAF-like Ewing sarcoma tumor cells to target micrometastases with matrix-binding VHHs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C050.

PLLA Porous Scaffold as a 3D Breast Cancer Model to Investigate Drug Resistance.

Multidrug resistance remains one of the major challenges in breast cancer research, often leading to treatment failure. To better understand this mechanism, sophisticated three-dimensional (3D) tumor models are necessary, as they offer several advantages over traditional bidimensional (2D) cultures. In this study, poly-l-lactic-acid porous scaffolds were produced using a thermally induced phase separation technique and employed as 3D models for breast cancer cell lines: MDA-MB-231, MCF-7, and its multidrug-resistant variant, MCF-7R. The MTS assay was used to compare growth inhibition following doxorubicin treatment in 2D and 3D. Remarkably, the IC50 values increased in 3D cultures compared to 2D: MDA-MB-231 (445 vs. 54.5 ng/mL), MCF-7 (7.45 vs. 0.75 μg/mL), and MCF-7R (165 vs. 39 μg/mL). MCF-7R, which usually shows greater resistance in 2D, demonstrated even higher resistance in 3D. In fact, IC50 was not reached within 3 days as with the other models, but only after 6 days. Cellular morphology also played a crucial role. When treated with concentrations higher than the IC50, MDA-MB-231 cells lost their physiological 3D clustered structure, while MCF-7 and its resistant variant exhibited disrupted layers. All cell lines in 3D showed higher chemoresistance, suggesting a more biomimetic spatial architecture. Our work bridges the gap between monolayer and animal models, highlighting the potential of polymeric 3D scaffolds in breast cancer research.

Controlling spheroid attachment improves pancreatic beta cell differentiation from human iPS cells.

Regenerative medicine using human induced pluripotent stem cells (hiPSCs) is available for treating type 1 diabetes; however, the efficiency and maturation of hiPSC differentiation into pancreatic beta cells requires improvement. Various protocols, including three-dimensional (3D) culture, have been developed to improve differentiation efficiency and maturation. Several methods for 3D culture have been reported; however, they require costly and complicated equipment, special materials, and complicated operations. To solve these problems, we developed a simple 3D culture method under static conditions using a cyclo-olefin polymer (COP) characterized by high moisture barrier properties, low surface energy, and hydrophobicity. Using this 3D method and our simple and low-cost protocol, we found that differentiation into the definitive endoderm (DE) was better when the spheroids were attached. Therefore, upon the addition of Y-27632, attached spheroids with unique shapes and cavities were formed, and the differentiation efficiency into DE increased. During DE differentiation, the attachment of spheroids to the substrate and their subsequent floating improved differentiation efficiency. We found that the amount of C-peptide in spheroids differentiated using COP dishes was greater than that in rotary culture. Furthermore, INSULIN was highly expressed in areas with low cell density, suggesting that the unique shape of the spheroids made from COP dishes improved differentiation efficiency. Our study suggests that a device-free, simple 3D culture method that controls spheroid attachment improves the efficiency of hiPSC differentiation into pancreatic beta cells.

Synthesis and effect of 4-acetylphenylamine-based imidazole derivatives on migration and growth of 3D cultures of breast, prostate and brain cancer cells.

In this study, we have synthesized novel 4-acetophenone moiety-bearing functionalized imidazole derivatives containing S-, and N-ethyl substituents and evaluated their anticancer activity. Their anticancer activity was studied against human breast carcinoma (MDA-MB-231), human prostate carcinoma (PPC-1), and human glioblastoma (U-87). Compounds 4, 9, 14, and 22 were identified as the most promising anticancer agents from a series of imidazole derivatives. They showed the highest cytotoxicity by MTT assay against MDA-MB-231, PPC-1 and U-87 cell lines. Compounds 14 and 22 were most selective against PPC-1 and U-87 cell lines, and their EC50 values against these cell lines ranged from 3.1 to 47.2 µM. Most tested compounds showed lower activity against the triple-negative breast cancer MDA-MB-231 cell line. None of the imidazole derivatives possessed an inhibiting effect on the migration of PPC-1 and U-87 cells by 'wound' healing assay. In spheroid assay, the most promising were compounds 14 and 22, especially in PPC-1 3D cultures. They efficiently reduced both the size and the viability of PPC-1 spheroid cells.

Abstract A025: RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer with a high frequency of p53 mutation and hyperactivation of the AKT pathway. Even though roughly 85% of patients achieve remission after initial surgical debulking and chemotherapy, four out of five HGSOC patients will experience disease recurrence, resulting in a 5-year survival rate of 43%. We have identified and characterized two TAp63 Regulated Oncogenic Long non-coding RNAs (TROLL-2 and TROLL-3) whose transcription is induced by mutant p53 and high expression level correlate with a more aggressive ovarian cancer grade and phosphorylation of AKT. In-depth analysis demonstrated that TROLL-2 and TROLL-3 directly activate AKT in tumor cells, thus linking p53 alterations to AKT activity. The use of AKT inhibitors in the clinic is limited because of frequent adverse effects, likely due to the constitutive expression of AKT in all cells. TROLL-2 and TROLL-3 expression is limited to tumor cells and provides a more specific target for decreasing AKT activation, potentially minimizing off-target effects. We have optimized reduction of TROLL-2 and TROLL-3 expression using antisense oligonucleotides (ASOs) in an ex vivo perfusion platform for 3D culture of microtumors from primary patient derived tumors. Samples show maximum reduction of proliferation (EdU) and phosphorylation of AKT when TROLL-2 and TROLL-3 ASOs are used concurrently. We also performed phospho-proteomics and single cell RNAseq to compare individual ASO-mediated knockdown of each lncRNA to combined targeted ASO treatments. We have developed primary patient derived xenograft models using ovarian tumors orthotopically implanted into NSG mice generating a relevant preclinical model to determine the efficacy of ASO treatment. To reduce the rapid degradation and clearance of ASOs in circulation, ASOs were encapsulated into lipid nanoparticles and metal-organic frameworks (MOFs) and we are currently testing these in vivo. Since AKT activation is associated with increased staging and resistance to platinum and targeted therapies in ovarian cancer patients, our data provides preclinical rationale for the implementation of ASOs and the use of new delivery methods as a relevant translational therapy and novel method to exploit the specificity of TROLL-2 and TROLL-3 expression in HGSOC. Citation Format: Ashley Lui, Marco Napoli, Jaden R Baldwin, Christina L Carr, Angie Rivera, Duy Nguyen, W. Gregory Sawyer, Michael R Dunne, Erin George, Omar K Farha, Michelle Teplensky, Elsa R Flores. RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A025.

Hydrogel Elastic Energy: A Stressor Triggering an Adaptive Stress-Mediated Cell Response.

The crosstalk between the cells and the extracellular matrix (ECM) is bidirectional and consists of a pushing/pulling stretch exerted by the cells and a mechanical resistance counteracted by the surrounding microenvironment. It is widely recognized that the stiffness of the ECM, its viscoelasticity, and its overall deformation are the most important traits influencing the response of the cells. Here these three parameters are combined into a concept of elastic energy, which in biological terms represents the mechanical feedback that cells perceive when the ECM is deformed. It is shown that elastic energy is a stress factor that influences the response of cells in three-dimensional (3D) cultures. Strikingly, the higher the elastic energy of the matrix and thus the mechanical feedback, the higher the stress state of the cells, which correlates with the formation of G3BP-mediated stress granules. This condition is associated with an increase in alkaline phosphatase (ALP) activity but a decrease in gene expression and is mediated by the nuclear translocation of Yes-associated protein (YAP). This work supports the importance of considering the elastic energy as mechano-controller in regulating cellular stress state in 3D cultures.

Optimisation of cell fate determination for cultivated muscle differentiation.

Production of cultivated meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Although these formulations can drive myogenic differentiation levels comparable to serum-starvation-based protocols, the resulting cultures are often heterogeneous, with a significant proportion of cells not participating in myofusion, limiting maturation of the muscle. To address this problem, we employed RNA sequencing to analyse heterogeneity in differentiating bovine satellite cells at single-nucleus resolution, identifying distinct cellular subpopulations including proliferative cells that fail to exit the cell cycle and quiescent 'reserve cells' that do not commit to myogenic differentiation. Our findings indicate that the MEK/ERK, NOTCH, and RXR pathways are active during the initial stages of myogenic cell fate determination, and by targeting these pathways, we can promote cell cycle exit while reducing reserve cell formation. This optimised medium formulation consistently yields fusion indices close to 100% in 2D culture. Furthermore, we demonstrate that these conditions enhance myotube formation and actomyosin accumulation in 3D bovine skeletal muscle constructs, providing proof of principle for the generation of highly differentiated cultivated muscle with excellent mimicry to traditional muscle.

A guide for blood-brain barrier models.

Understanding the intricate mechanisms underlying brain-related diseases hinges on unraveling the pivotal role of the blood-brain barrier (BBB), an essential dynamic interface crucial for maintaining brain equilibrium. This review offers a comprehensive analysis of BBB physiology, delving into its cellular and molecular components while exploring a wide range of in vivo and in vitro BBB models. Notably, recent advancements in 3D cell culture techniques are explicitly discussed, as they have significantly improved the fidelity of BBB modeling by enabling the replication of physiologically relevant environments under flow conditions. Special attention is given to the cellular aspects of in vitro BBB models, alongside discussions on advances in stem cell technologies, providing valuable insights into generating robust cellular systems for BBB modeling. The diverse array of cell types used in BBB modeling, depending on their sources, is meticulously examined in this comprehensive review, scrutinizing their respective derivation protocols and implications. By synthesizing diverse approaches, this review sheds light on the improvements of BBB models to capture physiological conditions, aiding in understanding BBB interactions in health and disease conditions to foster clinical developments.

EHD1 promotes breast cancer metastasis through upregulating HIF2a expression via activating mTOR pathway.

The multistep dynamic process of metastasis is the primary cause of breast cancer deaths. C-terminal Eps15-homology domain-containing protein 1 (EHD1), a translocator associated with endocytic recycling, has been implicated in various oncogenic processes. However, the precise molecular mechanisms of EHD1-induced breast cancer metastases remain largely unexplored. Here we found that the upregulation of EHD1 in breast cancer was positively associated with distant lymph node metastasis in patients. Meanwhile, EHD1 promoted epithelial-mesenchymal transition (EMT), invasion, and metastasis of breast cancer cells in both two-dimensional (2D) and three-dimensional (3D) culture models invitro, as well as invivo. Remarkably, EHD1 can activate the AKT-mTOR pathway to upregulate the protein expression of hypoxia-inducible factor 2α (HIF2α) under normoxic conditions and subsequently enhance the invasive and metastatic breast cancer. Our findings indicated EHD1 as a new regulator of HIF2α and a potential therapeutic target for inhibiting breast cancer metastasis.

Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis.

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.

Differential Replication and Oncolytic Effects of Zika Virus in Aggressive CNS Tumor Cells: Insights from Organoid and Tumoroid Models

Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus’ (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus’ oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors, enabling the translation of this approach to clinical trials, are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM, MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect, replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells, but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly, ZIKV rapidly replicated in GBM, MED, and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover, ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether, our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However, such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects, suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV.

3D-environment and muscle contraction regulate the heterogeneity of myonuclei.

Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation.

EXTH-58. BLOCKING IMMUNE CHECKPOINT— LAIR1: DISRUPTING THE LAIR1—FXIII-A—COLLAGEN IMMUNOSUPPRESSIVE CIRCUIT FOR ENHANCED ANTITUMOR THERAPEUTICS

Abstract BACKGROUND Recent evidence indicates that tumor-associated myeloid cells (TAMCs), including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), play a crucial role in glioblastoma (GBM) immunosuppression and progression. These cells can constitute up to 50% of the total mass in GBM tumors and are pivotal in dampening the immune response, which negatively impacts patient survival. Targeting TAMCs represents a promising strategy to overcome the limitations of current cancer treatments. However, drug development in this area remains limited. Our study focuses on Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR1), a novel immune checkpoint overexpressed on TAMCs within GBM. We comprehensively analyze LAIR1’s inhibitory role in cancer progression, highlighting its potential as a therapeutic target. METHODS Both in vitro and in vivo experiments were conducted. LAIR1 wild-type (LAIR1+/+) and knock-out (LAIR1-/-) tumor models were tested for antitumor response and intratumoral immune landscape. We also utilized 2D and 3D cultures to evaluate the effects of antibody blockade (aLAIR1) alone and in combination with CAR-T therapy (8R-70CAR) in both human and mouse GBM models. RESULTS We have uncovered a previously unrecognized immunosuppressive LAIR1→ Factor XIII A (FXIII-A)→ Collagen IV circuit across cancer types. Inhibiting LAIR1, either through genetic LAIR1-/- or aLAIR1, effectively disrupts this immunosuppressive pathway and results in enhanced peripheral and tumor-infiltrating memory CD8 T cell populations, a phenotypic shift of TAMs towards an M1 profile, and a decrease in tumor collagen deposition. These collective effects contribute to the normalization of the TME and facilitate improved interactions between T cells and tumor cells, leading to a more effective antitumor response. Notably, using aLAIR1 as a standalone intervention or combined with CAR T cell therapy demonstrates enhanced antitumor efficacy in preclinical models resistant to chemo-radiation and anti-PD-1 treatments. CONCLUSIONS We discovered a novel mechanism by which LAIR1 contributes to GBM progression. LAIR1 blockade holds promise in reversing suppressive TME and serving as a therapeutic strategy.

Comparative evaluation of the modulatory role of 1,25-dihydroxy-vitamin D3 on endoplasmic reticulum stress-induced effects in 2D and 3D cultures of the intestinal porcine epithelial cell line IPEC-J2

BackgroundThe use of conventional two-dimensional (2D) culture of the porcine intestinal epithelial cell (IEC) line IPEC-J2 in animal nutrition research has the disadvantage that IEC function is studied under unphysiological conditions, which limits the ability of transferring knowledge to the in vivo-situation. Thus, the aim of the present study was to establish a more convincing and meaningful three-dimensional (3D) culture of IPEC-J2 cells, which allows to study cell function in a more tissue-like environment, and to compare the effect of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) on ER stress indicators and the expression of tight junction proteins (TJP), inflammatory and apoptosis-related genes and the modulatory role of 1,25-dihydroxy-vitamin D3 (1,25D3) on these parameters in 2D and 3D cultures of IPEC-J2 cells.ResultsA published protocol for 3D culture of Caco-2 cells was successfully adopted to IPEC-J2 cells as evident from fully differentiated 3D IPEC-J2 spheroids showing the characteristic spherical architecture with a single layer of IPEC-J2 cells surrounding a central lumen. Treatment of 2D IPEC-J2 cells and 3D IPEC-J2 spheroids with TM for 24 h markedly increased mRNA and/or protein levels of the ER stress target genes, heat shock protein family A (Hsp70) member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3), whereas co-treatment with TM and 1,25D3 did not mitigate TM-induced ER stress in IPEC-J2 cells in the 2D and the 3D cell culture. In contrast, TM-induced expression of pro-inflammatory [interleukin-6 (IL6), IL8] and pro-apoptotic genes [BCL2 associated X, apoptosis regulator (BAX), caspase 3 (CASP3), CASP8] and genes encoding TJP [TJP1, claudin 1 (CLDN1), CLDN3, occludin (OCLN), cadherin 1 (CDH1), junctional adhesion molecule 1 (JAM1)] was reduced by co-treatment with TM and 1,25D3 in 3D IPEC-J2 spheroids but not in the 2D cell culture.ConclusionsThe effect of 1,25D3 in the IPEC-J2 cell culture is dependent on the culture model applied. While 1,25D3 does not inhibit TM-induced expression of genes involved in inflammation, apoptosis and TJP in conventional 2D cultures of IPEC-J2 cells, TM-induced expression of these genes is abrogated by 1,25D3 in the more meaningful 3D IPEC-J2 cell culture model.

Characteristics of stem sings of EpCAM-negative and EpCAM-positive tumor cells in primary tumor and 2D and 3D cultures in breast cancer

One of the predictors of adverse prognosis in breast cancer is the overexpression of the EpCAM protein. However, a signifcant part of tumor cells have low or no EpCAM expression. The molecular features and the ability to grow in 2D and 3D cultures, indirectly refecting metastatic potential of tumor cells without EpCAM expression, have not been sufficiently studied.The aim of the study was to compare phenotypic variants of stem cells and EMT depending on the EpCAM expression in the primary tumor of breast cancer and 2D and 3D cultures.Material and Methods. The study included 7 patients with invasive breast carcinoma of no special type. Luminal A subtype was found in 2/7 patients (29 %), and luminal B HER2-negative molecular subtype was found in 5/7 patients (71 %). The patients didn’t have neoadjuvant chemotherapy. Cells from the primary tumor were cultured in 2D and 3D. The primary tumor cells, 2D and 3D cultures were phenotyped using fow cytometry with antibodies targeting: CD45, EpCAM (CD326), CD44, CD24, N-cadherin (CD325), and CD133, CK7/8, EpCAM (CD326).Results. No significant differences were found in the frequency of detection and the number of cells exhibiting stem cell features among EpCAM- and EpCAM+ tumor cells, 2D and 3D cultures. All phenotypic variants of co-expression of stemness markers CD44, CD24, CD133, and ALDH were present both in the primary tumor and 2D/3D cultures. Stem cells in mammospheres had features of both epithelial and hybrid EMT phenotypes. In primary tumors and 2D/3D cultures, the smallest proportion consisted of stem cells with the CD44+CD24- phenotype and cells co-expressing CD44+CD24- with other stemness markers, while the largest proportion comprised stem cells with ALDH+ and ALDH+CD133+ phenotypes. Marked intra- and inter-tumor heterogeneity in the phenotypic composition of primary tumors, 2D, and 3D cultures was noted.Conclusion. The results indicate that EpCAM-negative and EpCAM-positive tumor cells of luminal molecular subtype of breast cancer are capable of exhibiting various phenotypic variants of stemness and EMT in the primary tumor and 2D and 3D cultures. In most cases, individual tumor cells show co-expression of several stemness markers. The phenotypic composition of primary tumors, 2D and 3D cultures is characterized by pronounced intra- and inter-tumor heterogeneity.