Ex-vivo Culture Research Articles

Explorative Study of Modulatory Effects of Notochordal Cell-Derived Extracellular Vesicles on the IL-1β-Induced Catabolic Cascade in Nucleus Pulposus Cell Pellets and Explants.

Cell-free regenerative strategies, such as notochordal cell (NC)-derived extracellular vesicles (EVs), are an attractive alternative in developing new therapies for intervertebral disc (IVD) degeneration. NC-EVs have been reported to elicit matrix anabolic effects on nucleus pulposus cells from degenerated IVDs cultured under basal conditions. However, the degenerative process is exacerbated by pro-inflammatory cytokines contributing to the vicious degenerative cycle. Therefore, this study explores whether NC-EVs modulate interleukin (IL)-1β-mediated pro-inflammatory responses in the degenerating disc. This study utilized two IL-1β induced pro-catabolic culture models; a dog 3D nucleus pulposus (NP) cell pellet culture and a human patient-derived, exvivo NP tissue culture system. Porcine NC-EVs were generated from NC-conditioned medium by differential centrifugation followed by size exclusion chromatography. Donor matched EV-depleted media were generated by overnight ultracentrifugation, whereafter the EV-depleted NCCM supernatant was subjected to size exclusion chromatography. To investigate whether observed effects were EV-associated, NC-EVs conditions were compared to EV-depleted controls in the absence and presence of IL-1β. The size and concentration of NC-EVs were quantified by nanoparticle tracking analysis, which showed minimal donor variation and confirmed depletion of EVs in the EV-depleted media. In the IL-1β-induced catabolic cascade, the NC-EVs did not elicit anabolic effects at the matrix level nor did they rescue the pro-catabolic phenotype within dog pellets. Modification of the CCL2 secretion seemed to be context dependent in the human explants: where EVs treatment stimulated CCL2 secretion but in the presence of IL-1β this effect was counteracted. Secretion of IL-6 and C-X-C motif chemokine ligand 1 was significantly decreased in NC-EV + IL-1β vs. control+IL-1β but not compared to EV-depleted human explant controls. Altogether, this data provides evidence for a protective modulatory role of NC-EVs. Considering the homeostatic function EVs exert, inherently encompassing subtle biologic modifications, the current study may have lacked sufficient power to demonstrate statistical significance in a sample set with evident donor variation. NC-EVs may modulate the production of specific cytokines and chemokines in human degenerate explants when the key pro-inflammatory cytokine IL-1β is present. Implementation of the technical EV-depleted controls in further studies is essential to robustly demonstrate that these effects are EV-mediated and not associated with other secreted factors co-isolated during EV-isolation.

IFNγ regulates ferroptosis in KFs by inhibiting the expression of SPOCD1 through DNMT3A

Keloid is benign skin tumor, and their curing is relatively difficult due to the unclear mechanism of formation. Inducing ferroptosis of keloid fibroblasts (KFs) may become a new method for treating keloid. Here, we discover interferon (IFN)γ could induce KFs ferroptosis through inhibiting SPOC domain-containing protein 1 (SPOCD1), serving as a mode of action for CD8+T cell (CTL)-mediated keloid killing. Mechanistically, keloid IFNγ deficiency in combination with reduced DNMT3A increase the expression of SPOCD1, thereby promoting KFs’ proliferation and inhibiting its ferroptosis. Moreover, keloid SPOCD1 deficiency attenuates KFs progression and extracellular matrix (ECM) deposition. Reducing IFNγ and SPOCD1 simultaneously can increase the positive rate of reactive oxygen species (ROS) and promote mitochondrial shrinkage. Ex-vivo explant keloid culture has also confirmed that the reduction of SPOCD1 helps to reduce the proliferation rate of KFs, inhibit the angiogenesis of keloid scars, and thus inhibit keloid formation. Thus, IFNγ signaling paired with SPOCD1 is a natural keloid ferroptosis promoting mechanism and a mode of action of CTLs. Targeting SPOCD1 pathway is a potential anti-keloid approach.

IL-21 shapes the B cell response in a context-dependent manner.

The T-cell-derived cytokine IL-21 is crucial for germinal center (GC) responses, but its precise role in B cell function has remained elusive. Using IL-21 receptor (Il21r) conditional knockout mice and exvivo culture systems, we demonstrate that IL-21 has dual effects on B cells. While IL-21 induced apoptosis in a STAT3-dependent manner in naive B cells, it promoted the robust proliferation of pre-activated B cells, particularly IgG1+ B cells. Invivo, B-cell-specific Il21r deletion impaired IgG1 responses post-immunization and disrupted progression from pre-GC to GC states. Although Il21r deficiency did not affect the proportion of IgG1+ cells among GC B cells, it greatly diminished the proportion of IgG1+ cells among the plasmablast/plasma cell population. Collectively, our findings suggest that IL-21 serves as a critical regulator of B cell fates, influencing B cell apoptosis and proliferation in a context-dependent manner.

Optimizing Stem Cell Expansion: The Role of Substrate Stiffness in Enhancing Dental Pulp Stem Cell Quiescence and Regeneration.

Quiescent stem cells exhibit unique self-renewal and engraftment abilities vital for regenerative therapies, but these diminish during exvivo culture. This study investigates how substrate stiffness regulates the balance between dental pulp stem cell (DPSC) quiescence, activation, and senescence and explores the role of extracellular matrix stiffness in modulating DPSC fate via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Polydimethylsiloxane substrates with varying stiffness in 2D (2 kPa, 50 kPa) and 3D (50 kPa) were fabricated. Mechanical properties and porosity were characterized. Human DPSCs were cultured for 7 and 14 days. Senescence was assessed by senescence β-galactosidase activity, nuclear changes by immunofluorescence staining, and gene expression of quiescence, self-renewal, and senescence markers by reverse transcription quantitative polymerase chain reaction. NF-κB activation was analyzed through p65 nuclear translocation. Statistical analysis employed one-way analysis of variance with post-Tukey tests (P < .05). The porous (310 ± 63 μm) 3D substrate had 50 kPa stiffness. DPSCs on 50 kPa substrates exhibited increased nuclear size and senescence in both 2D and 3D contexts. Softer 2 kPa substrates promoted quiescence, evidenced by reduced chromatin condensation and senescence, alongside upregulation of quiescence associated genes (BMI-1) and pluripotency markers (NANOG, OCT4, SOX2). NF-κB activation was observed on soft substrates, marked by nuclear translocation of p65 and upregulated NF-κB pathway genes, correlating with enhanced stemness and reduced senescence. This study highlights the pivotal role of substrate stiffness in modulating stem cell fate. Softer substrates preserve DPSC quiescence, reduce senescence, and enhance stemness through NF-κB pathway activation, offering insights into optimizing exvivo DPSC expansion for therapeutic applications.

Accelerating CAR T cell manufacturing with an automated next-day process.

The traditional method of CAR T cell production involves lengthy ex-vivo culture times which can result in the reduction of crucial naïve T cell subsets. Moreover, traditional CAR T cell therapy manufacturing processes can prolong time-to-patient, potentially delaying patient treatment, and contribute to disease progression. In this study, we describe an innovative and semi-automated 24-hour CAR T manufacturing process that yields a higher percentage of naïve/stem-cell like T cells which showed high cytotoxic activity and cytokine release in vitro. The data supports the feasibility of implementing this streamlined manufacturing process in clinics. This approach also has the potential to enhance CAR T therapy efficacy and improve patient access to therapy.

Gene editing without exvivo culture evades genotoxicity in human hematopoietic stem cells.

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A+58 and+55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A+58 and+55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by exvivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.

Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations

Background:Mesenchymal stem cells (MSCs) derived from gestational tissues offer a promising avenue for prenatal intervention in congenital malformations although their application is hampered by concerns related to cellular plasticity and the need for invasive, high-risk surgical procedures. Here, we present naturally occurring exosomes (EXOs) isolated from amniotic fluid-derived MSCs (AF-MSCs) and their mimetic analogs (MIMs) as viable, reproducible, and stable alternatives. These nanovesicles present a minimally invasive therapeutic option, addressing the limitations of MSC-based treatments while retaining therapeutic efficacy.Methods:MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. A physicochemical, structural, and molecular comparison was conducted with exosomes (EXOs) released from the same batch of cells. Additionally, their distribution patterns in female mice were evaluated following in vivo administration, along with an assessment of their safety profile throughout gestation in a mouse strain predisposed to neural tube defects (NTDs). The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake in two different cell types(fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams.Results:Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a threefold greater yield. Biodistribution patterns following intraperitoneal administration were comparable between the two particle types, with the uterus being among targeted organs. No toxic effects were observed in the dams during gestation, nor were there any malformations or significant differences in the number of viable versus dead fetuses detected. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo.Conclusions:The present data confirms the potential application of EXOs and MIMs as suitable tools for prevention and treatment of NTDs and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.

Advancements in Dental Bioreactor Design: A Comprehensive Approach for Application in Dentistry.

The protocol introduces a novel multi-chamber bioreactor tailored for ex-vivo cell culture in dentistry research, emulating the 3D dental environment to propel research in dental applications. Constructed primarily from a polymeric material with a sophisticated 3D design, the bioreactor securely holds teeth structures within sealed chambers, enabling controlled perfusion of culture medium crucial for cell growth through a singular entry and exit point. An integrated electronic system manages flow and pressure, ensuring precise control over environmental conditions. This technology facilitates cell cultivation under conditions closely resembling natural tooth microenvironments, offering opportunities for varied studies from understanding cellular behavior in dental contexts to targeted therapy development. The bioreactor's amalgamation of polymeric components, 3D design, and electronic controls enhances adaptability and accuracy, rendering it a valuable asset in dental research. The report comprehensively delineates the bioreactor's design, operations, and potential applications, showcasing its significant contributions to dental research and regenerative medicine. By amalgamating advanced technologies, this bioreactor emerges as a pivotal tool for investigating cellular processes within dental structures, paving the way for scientific exploration and therapeutic advancements.

CSIG-25. MAPK/ERK SIGNALING IN GLIOMAS MODULATES INTERFERON RESPONSES, PROMOTES TUMOR-MICROGLIA CROSSTALK, AND CONFERS SUSCEPTIBILITY TO ANTI-PD-1 AND ANTI-CTLA-4 IMMUNOTHERAPY

Abstract While immune checkpoint blockade (ICB) is not effective in unselected glioblastoma (GBM) patients, we previously established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GBM. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain to be determined. We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T-cell response and performed survival studies to validate the most relevant hits. Additionally, paired scRNA-seq with p-ERK staining, spatial transcriptomics, and ex-vivo slice culture of BRAFV600E mutant GBM tumors treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Our CRISPR/Cas9 screening results identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to anti-tumoral immunity across all kinases. Survival studies showed that mice bearing high p-ERK gliomas exhibited prolonged survival with anti-PD-1 treatment, which was not seen when ERK phosphorylation diminished. MAPK activation in murine gliomas led to durable anti-tumoral immunity upon implant re-challenge, with memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells was associated with interferon responses and antigen presentation. Moreover, in BRAFV600E human GBM cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GBM tissue analyzed by spatial transcriptomics, we observed modulation of interferon responses by the MAPK/ERK pathway. Notably, in slice cultures from BRAFV600E mutant GBM, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia. In conclusion, the MAPK/ERK pathway is a critical driver of GBM susceptibility to anti-tumoral immunity, modulating the interferon responses and antigen-presenting machinery in glioma cells, as well as tumor cell interaction with the microenvironment.

Metalloproteinases are involved in the regulation of prenatal tooth morphogenesis.

During development, tooth germs undergo various morphological changes resulting from interactions between the oral epithelium and ectomesenchyme. These processes are influenced by the extracellular matrix, the composition of which, along with cell adhesion and signalling, is regulated by metalloproteinases. Notably, these include matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs). Our analysis of previously published scRNAseq datasets highlights that these metalloproteinases show dynamic expression patterns during tooth development, with expression in a wide range of cell types, suggesting multiple roles in tooth morphogenesis. To investigate this, Marimastat, a broad-spectrum inhibitor of MMPs, ADAMs, and ADAMTSs, was applied to ex-vivo cultures of mouse molar tooth germs. The treated samples exhibited significant changes in tooth germ size and morphology, including an overall reduction in size and an inversion of the typical bell shape. The cervical loop failed to extend, and the central area of the inner enamel epithelium protruded. Marimastat treatment also disrupted proliferation, cell polarization and organization compared to control tooth germs. Additionally, a decrease in laminin expression was observed, leading to a disruption in continuity of the basement membrane at the epithelial-mesenchymal junction. Elevated Hif-1α expression correlated with a disruption to blood vessel development around the tooth germs. These results reveal the crucial role of metalloproteinases in tooth growth, shape, cervical loop elongation, and the regulation of blood vessel formation during prenatal tooth development.

A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

Exvivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering exvivo culture necessary yet detrimental. Mechanistically, exvivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic reactive oxygen species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications.

BMP3b regulates bone mass by inhibiting BMP signaling

Bone morphogenetic protein 3b (BMP3b), also known as growth differentiation factor 10 (GDF10), is a non-osteogenic BMP highly expressed in the skeleton. Although in vitro studies have shown that BMP3b suppresses osteoblast differentiation, the physiological role of BMP3b in regulating bone mass in vivo remains unknown. Here, we show that BMP3b deletion in mice leads to a high bone mass phenotype via an unexpected novel mechanism involving de-repression of canonical BMP/Smad signaling. BMP3b null mice were viable, and exhibited no significant difference in body size compared to wildtype control. Trabecular bone parameters assessed by histomorphometry and μCT, revealed a significant increase in bone volume and bone mineral density. Expression of osteoblast-differentiation genes were elevated in bone tissue of BMP3b null mice, whereas expression of osteoclast-related genes remained unchanged. Consistent with this, Bmp3b was highly expressed in osteoblasts relative to osteoclast cells. Ex-vivo culture of primary bone marrow mesenchymal stem cells (BMSCs) and primary bone marrow-derived osteoclasts revealed that inactivation of BMP3b enhances osteogenesis without affecting osteoclastogenesis. Mechanistically, we found that BMP3b suppressed BMP4-induced Smad1/5 phosphorylation and inhibited the activity of a BMP4-driven Id-1 luciferase reporter. Protein-protein interaction assays revealed that BMP3b competitively interfered with the association of BMP4 and BMP type I receptors. These findings suggest that BMP3b regulates bone mass by acting as a BMP receptor antagonist. Thus, maintenance of bone mass involves antagonism of canonical BMP/Smad signaling by a member of the BMP family.

Spheroid culture to select theoretical therapeutic drugs in intravascular large B-cell lymphoma.

Intravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal large B-cell lymphoma that is characterized by the proliferation of lymphoma cells in the lumina of small vessels. Recent progress uncovering the genetic characteristics associated with MYD88/CD79B mutations has stimulated interest in the use of drugs targeting B-cell receptor signaling, including Bruton's tyrosine kinase. However, difficulties in culturing exvivo IVLBCL cells has hampered research on the development of novel therapies. In the present study, we demonstrated the establishment of an exvivo culture system of IVLBCL cells obtained from patient-derived xenograft (PDX) models. The spheroid culture enabled us to culture IVLBCL PDX cells for more than 10 days and to explore the efficacy of drug treatments acting on these cells. We found that carfilzomib and ibrutinib were effective for treating IVLBCL in exvivo experiments and conducted invivo analyses to assess the efficacy of these drugs. Although the efficacy of carfilzomib was difficult to confirm due to its toxicity in our models, ibrutinib showed comparable efficacy to a standard combination of chemotherapy drugs. Together, our data provide a new culture method for IVLBCL PDX cells and a rationale for translating ibrutinib to clinical use in IVLBCL patients.

Denosumab stimulates spermatogenesis in infertile men with preserved Sertoli cell capacity

Sperm production depends on proper Sertoli-germ cell interaction, and we hypothesized that receptor activator of nuclear factor κB ligand (RANKL) activity in Sertoli cells may influence spermatogenesis. Treatment with the RANKL inhibitor denosumab, normally used to treat osteoporosis, increased testicular weight, inhibin B, and germ cell proliferation in exvivo testis cultures and invivo in a humanized RANKL mouse. The effect on germ cell proliferation was positively associated with baseline serum concentrations of anti-müllerian hormone (AMH). In accordance, denosumab increased germ cell proliferation in exvivo human testis cultures with low/moderate but not severe impairment of Sertoli cell function. In a placebo-controlled randomized clinical trial, denosumab had no effect on semen quality but increased sperm concentration in a subgroup of infertile men with serum AMH ≥38 pmol/L at baseline. In conclusion, high serum AMH may increase the probability of a beneficial response to denosumab treatment in infertile men, thus suggesting a possible venue for precision medicine in male infertility.

GDF10 is a negative regulator of vascular calcification

Cardiovascular mortality is particularly high and increasing in patients with chronic kidney disease, with vascular calcification (VC) as a major pathophysiologic feature. VC is a highly regulated biological process similar to bone formation involving osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). We have previously demonstrated that loss of T-cell death-associated gene 51 (TDAG51) expression leads to an attenuation of medial VC. We now show a significant induction of circulating levels of growth differentiation factor 10 (GDF10) in TDAG51-/- mice, which was of interest due to its established role as an inhibitor of osteoblast differentiation. The objective of this study was to examine the role of GDF10 in the osteogenic transdifferentiation of VSMCs. Using primary mouse and human VSMCs, as well as exvivo aortic ring cultures, we demonstrated that treatment with recombinant human (rh) GDF10 mitigated phosphate-mediated hydroxyapatite (HA) mineral deposition. Furthermore, exvivo aortic rings from GDF10-/- mice exhibited increased HA deposition compared to C57BL/6J controls. To explain our observations, we identified that rhGDF10 treatment reduced protein expression of runt-related transcription factor 2, a key driver of osteogenic transdifferentiation of VSMCs and VC. In support of these findings, invivo treatment with rhGDF10 attenuated VD3-induced VC. Furthermore, we demonstrated an increase in circulating GDF10 in patients with chronic kidney disease with clinically defined severe VC, as assessed by coronary artery calcium score. Thus, our studies identify GDF10 as a novel inhibitor of mineral deposition and as such, may represent a potential novel biomarker and therapeutic target for the detection and management of VC.

Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

Bone morphogenetic proteins (BMPs) are involved in several cellular responsive actions, such as development, cell differentiation, and apoptosis, via their specific transmembrane receptors. In particular, BMPs promote the differentiation and maturation of bone and cartilage from mesenchymal stem cells. Based on comprehensive analyses performed with a large number of antibodies, mitogen- and stress-activated protein kinase (MSK)1 was found to be immediately phosphorylated in the mouse chondrocyte precursor cell line, ATDC5, upon BMP-6 stimulation. The overexpression and knockdown of MSK1 in ATDC5 cells also enhanced and suppressed BMP-6-induced chondrocyte differentiation, respectively. Similar to ATDC5 cells, an exvivo organ culture system using mouse embryonic metatarsal bones also demonstrated that BMP-6-mediated MSK1 activation might play a role in chondrocyte differentiation. Using several inhibitors, the p38 kinase pathway was confirmed to be implicated in BMP-6-induced phosphorylation of MSK1. Furthermore, MSK1 mutants lacking kinase activities and those lacking serine/threonine residues targeted by p38 kinase severely impaired their ability to potentiate BMP-6-induced chondrogenic differentiation of ATDC5 cells. Interestingly, a loss-of-function study for Smad4 perturbed BMP-6-induced phosphorylation of p38 kinase to inhibit BMP-6-mediated chondrocyte differentiation via MSK1 activation. Overall, both Smad-dependent and independent pathways require BMP-6-induced chondrocyte differentiation via MSK1 activation in ATDC5 cells.

MAPK/ERK signaling in gliomas modulates interferon responses, T cell recruitment, microglia phenotype, and immune checkpoint blockade efficacy.

Glioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single cell RNA-sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. CRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. Our data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.

Glycogen Phosphorylase Inhibitor Promotes Hair Growth via Protecting from Oxidative-Stress and Regulating Glycogen Breakdown in Human Hair follicles.

Hair growth cycles are mainly regulated by human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs). Protecting hDPCs from excessive oxidative stress and hORSCs from glycogen phosphorylase (PYGL) is crucial to maintaining the hair growth phase, anagen. In this study, we developed a new PYGL inhibitor, Hydroxytrimethylpyridinyl Methylindolecarboxamide (HTPI) and assessed its potential to prevent hair loss. HTPI reduced oxidative damage, preventing cell death and restored decreased level of anagen marker ALP and its related genes induced by hydrogen peroxide in hDPCs. Moreover, HTPI inhibited glycogen degradation and induced cell survival under glucose starvation in hORSCs. In ex-vivo culture, HTPI significantly enhanced hair growth compared to the control with minoxidil showing comparable results. Overall, these findings suggest that HTPI has significant potential as a therapeutic agent for the prevention and treatment of hair loss.

AP collagen peptides (APCPs) promote hair growth by activating the GSK-3β/β-catenin pathway and improve hair condition.

AP collagen peptides (APCPs) are enzymatically decomposed collagen peptides that contain tri-peptides such as glycine-proline-hydroxyproline. We found that APCPs increased the proliferation of both human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs). APCPs also stimulated the secretion of several growth factors, including IGFBP-6, PDGF-AB, PIGF and VEGF in hDPCs. Moreover, APCPs enhanced the phosphorylation of Akt(Ser473), GSK-3β(Ser9) and β-catenin(Ser675), indicating the activation of the GSK-3β/β-catenin signalling pathway. Exvivo culture of human hair follicles (hHFs) tissue and invivo patch assay revealed that APCPs promoted the elongation of hHFs and the induction of new hair shafts. In a mouse model, APCPs significantly promoted the transition from telogen to anagen phase and prolonged anagen phase, resulting in increased hair growth. APCPs also improved the thickness, amino acid content (cystine and methionine) and roughness of mouse hair. Taken together, these findings demonstrate that APCPs accelerate hair growth and contribute to overall hair health. Therefore, APCPs have the potential to be utilized as a food supplement and ingredient for preventing hair loss and maintaining hair health.

Abstract A008: Pre-diabetic D-glucose exposure promotes EOC progression and cisplatin resistance: Role of BAD associated pathway and potential therapeutic strategy

Abstract Prediabetes denotes a condition when blood sugar levels exceed normal thresholds (&amp;gt;7mmol) but have not reached the diagnostic criteria for type 2 diabetes (&amp;lt;11mmol). It positively correlates with diminished progression-free survival and overall survival among women with epithelial ovarian cancer (EOC). This suggests a potential metabolic state associated with prediabetes that may facilitate tumor survival and progression. Moreover, prediabetes is also associated with an increased risk of recurrence and poorer survival outcomes of EOC patients, possibly promoting tumor aggressiveness and resistance to treatment, although the precise mechanisms remain unclear and warrant additional investigation. In this study, we aimed to explore whether prediabetic D-glucose levels accelerate EOC progression and elucidate underlying mechanisms. We also aimed to propose a viable and effective therapy strategy for EOC progression. Various in vitro and ex vivo oncogenic assays were used to assess the effects of prediabetic levels of D-glucose in EOC cells. It was found to stimulate oncogenic phenotypes in EOC cells in a dose-dependent manner. EOC cells exposed to prediabetic levels of D-glucose (8mM) exhibited increased cell survival, enhanced foci formation on monolayer, growth in soft agar, spheroid formation capacity in 3D Matrigel (ex-vivo culture), increased migration and invasion, and resistance to cisplatin compared to those exposed to lower D-glucose doses (4mM). Exposure to 8mM D-glucose led to metabolic alterations associated with cisplatin resistance, including increased D-glucose consumption, elevated ATP production, increased thermogenesis, enhanced glycolytic capacity, and augmented mitochondrial activity. RNA sequencing analysis showed the BCL-2-associated death promoter (BAD) pathway positively correlates with metabolic alterations of EOC cells in 8mM. Prediabetic levels of D-glucose upregulated phosphorylation of BAD at serine (S) 99 residue along with key metabolic enzymes such as ALDH1A1, HK2, PFKP, G6PD, and LDHA. Inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) attenuated 8mM D-glucose effects. Furthermore, forced expression of phosphorylated BADS99 increased oncogenic phenotypes of EOC. The elevated activities of metabolic enzymes associated with BADS99 phosphorylation were observed in EOCs, while this effect was diminished with dephosphorylated BADS99. High-throughput screening identified a combination therapy involving a BADS99 phosphorylation inhibitor and HDAC inhibitors demonstrated higher synergy to stimulate apoptosis in EOC cells. The combined BADS99-HDAC inhibition synergistically enhanced the efficacy of HDAC inhibitors, significantly reducing their IC50 values. It impaired cell survival, viability, anchorage-independent growth, mitochondrial activity, energy production, growth in 3D Matrigel and successfully overcome cisplatin resistance of EOC cell lines and a patient-derived cell line (AFC). This synergistic therapeutic approach offers a promising strategy for exploiting synthetic lethality in treating EOC patients. Citation Format: Jing Huang, Xi Zhang, Peng Huang, Basappa Basappa, Tao Zhu, Peter E. Lobie, Vijay Pandey. Pre-diabetic D-glucose exposure promotes EOC progression and cisplatin resistance: Role of BAD associated pathway and potential therapeutic strategy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A008.