PDGFRα Expression Research Articles

CTNI-45. PHASE 2 STUDY OF SORAFENIB, VALPROIC ACID, AND SILDENAFIL IN THE TREATMENT OF RECURRENT HIGH-GRADE GLIOMA

Abstract PURPOSE Here we report the results of a single-center phase 2 clinical trial combining sorafenib tosylate, valproic acid, and sildenafil for the treatment of patients with recurrent high-grade glioma. PATIENTS AND METHODS Eligible patients were 18 years of age and older with an Eastern Cooperative Oncology Group performance status of 0-2 and pathologically confirmed high-grade glioma (WHO grade 3 or 4), with documented CT or MRI progression or recurrence and measurable or evaluable disease. The treatment schedule was a combination of sorafenib, valproic acid, and sildenafil, each agent administered orally, twice daily continuously. A cycle consisted of 4 weeks. RESULTS Clinical toxicities were grade 1 and grade 2, with one grade 3 toxicity for maculopapular rash (6.4%). For all evaluable patients, the median progression-free survival was 3.65 months and overall survival (OS) 10.0 months. There was promising evidence showing clinical activity and benefit. In the 33 evaluable patients, low protein levels of the chaperone GRP78 (HSPA5) was significantly associated with a better OS (p < 0.0026). A correlation between the expression of PDGFRa and OS approached significance (p < 0.0728). Five patients presently have a mean OS of 73.6 months and remain alive. CONCLUSIONS This is the first therapeutic intervention glioblastoma trial to significantly associate GRP78 expression to OS. Our data suggest that the combination of sorafenib tosylate, valproic acid, and sildenafil requires additional clinical development in the recurrent glioma population.

Patterns of expression of VEGFR2, PDGFRs and c-Kit in pediatric patients with high grade non-rhabdomyosarcoma soft tissue sarcoma.

Activated vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs) and c-Kit have been shown to be involved in the growth, invasion and metastasis of non-rhabdomyosarcoma soft tissue sarcoma tumor (NRSTS) with promising results for targeted therapy. Our aim was to assess the expression of these markers among different histological types and correlate with outcomes. This retrospective study included pediatric patients aged ≤ 18 years diagnosed with high-grade NRSTS who were treated at Children Cancer Hospital Egypt 57357 as per the COG NRSTS protocol (ARST0332). Expression of VEGFR2, PDGFRs (α and β) and c-Kit in tumor tissue was assessed by immunohistochemistry and correlated with clinical outcome. Of 113 patients, 96 were eligible for the analysis with a median age of 11 years. Overall, 32.3% demonstrated high expression of PDGFRα, 17.7% for PDGFRβ, 19.8% for VEGFR2 and 8.3% exhibited positive expression for c-kit on the tumor cells. Most cases of synovial sarcoma (45.8%) and 43.7% of patients with undifferentiated sarcoma exhibited high expression of PDGFRα while 41.6% of MPNST showed high expression to PDGFRβ. The 5-year overall survival (OS), event free survival and relapse free survival (RFS) for the whole cohort were 59%, 54% and 60% respectively. In univariate analyses, only PDGFRα had a negative prognostic impact on relapse free survival (RFS) (p=0.03). In multivariate analyses, VEGFR2 was found to have a negative prognostic impact for OS (p = 0.02). Our findings indicated that tyrosine kinase receptors are upregulated in NRSTS and exhibited a distinct expression pattern within various subgroups. High expression of VEGFR2 and PDGFRα significantly correlated with reduced survival and may guide targeted therapy approaches for this poor prognosis group of patients.

Colon Cancer-Derived Exosomal LncRNA-XIST Promotes M2-like Macrophage Polarization by Regulating PDGFRA.

Colon cancer ranks second in overall cancer-related deaths and poses a serious risk to human life and health. In recent years, exosomes are believed to play an important and significant role in cancer, especially tumor-derived exosomes (TDEs). Previous studies have highlighted the pivotal role of exosomes in tumor development, owing to their ability to mediate communication between tumor cells and macrophages, induce macrophage M2 polarization, and facilitate the progression of tumorigenesis. In this study, we revealed that colon cancer-derived exosomes promoted M2-like macrophage polarization. Moreover, exosome-induced M2-like macrophages, in turn, promoted the proliferation, migration, and invasion abilities of colon cancer cells. Specifically, CT26- and HCT116-derived exosomes led to the activation of AKT, ERK, and STAT3/6 signaling pathways in THP-1(Mφ) cells. Furthermore, our findings showed that colon cancer-derived exosomes secreted lncXIST to sponge miR-17-5p, which, in turn, promoted the expression of PDGFRA, a common gene found in all three signaling pathways, to facilitate M2-like macrophage polarization. Dual-luciferase reporter assays confirmed the binding relationship between lncXIST and miR-17-5p, as well as miR-17-5p and PDGFRA. Collectively, our results highlight the novel role of lncXIST in facilitating macrophage polarization by sponging miR-17-5p and regulating PDGFRA expression.

Protective effects of PDGF-AB/BB against cellular senescence in human intervertebral disc.

Cellular senescence, characterized by a permanent state of cell cycle arrest and a secretory phenotype contributing to inflammation and tissue deterioration, has emerged as a target for age-related interventions. Accumulation of senescent cells is closely linked with intervertebral disc (IVD) degeneration, a prevalent age-dependent chronic disorder causing low back pain. Previous studies have highlighted that platelet-derived growth factor (PDGF) mitigated IVD degeneration through anti-apoptosis, anti-inflammation, and pro-anabolism. However, its impact on IVD cell senescence remains elusive. In this study, human NP and AF cells derived from aged, degenerated IVDs were treated with recombinant human (rh) PDGF-AB/BB for 5 days and changes of transcriptome profiling were examined through mRNA sequencing. NP and AF cells demonstrated similar but distinct responses to the treatment. However, the effects of PDGF-AB and BB on human IVD cells were comparable. Specifically, PDGF-AB/BB treatment resulted in downregulation of gene clusters related to neurogenesis and response to mechanical stimulus in AF cells while the downregulated genes in NP cells were mainly associated with metabolic pathways. In both NP and AF cells, PDGF-AB and BB treatment upregulated the expression of genes involved in cell cycle regulation, mesenchymal cell differentiation, and response to reduced oxygen levels, while downregulating the expression of genes related to senescence associated phenotype, including oxidative stress, reactive oxygen species (ROS), and mitochondria dysfunction. Network analysis revealed that PDGFRA and IL6 were the top hub genes in treated NP cells. Furthermore, in irradiation-induced senescent NP cells, PDGFRA gene expression was significantly reduced compared to non-irradiated cells. However, rhPDGF-AB/BB treatment increased PDGFRA expression and mitigated the senescence progression through increased cell population in the S phase, reduced SA-β-Gal activity, and decreased expression of senescence related regulators including P21, P16, IL6, and NF-κB. Our findings reveal a novel anti-senescence role of PDGF in the IVD, demonstrating its ability to alleviate the senescent phenotype and protect against the progression of senescence. This makes it a promising candidate for preventing or treating IVD degeneration by targeting cellular senescence.

The role of long non‐coding RNA Maternally Expressed Gene 3 in cancer‐associated fibroblasts at single cell pan‐cancer level

AbstractLong non‐coding RNAs (lncRNAs) can crucially regulate activation and transformation of cancer‐associated fibroblasts (CAFs) but have not been systematically investigated at single cell resolution. Here, by utilizing integrated single‐cell sequencing datasets, we screened the aberrantly expressed lncRNAs in CAFs, which are the major component of tumor microenvironment. Our findings revealed a consistent CAF‐specific downregulation of Maternally Expressed Gene 3 (MEG3) expression and increased MEG3+ proportion at the pan‐cancer level, which may be attributed to m6A‐related post‐transcriptional modifications. Through activation trajectory analysis of the major CAF subtypes, it was determined that elevated MEG3 expression in CAFs leads to an increase in PDGFRA expression. This, in turn, promotes CAF activation and transformation into an MEG3+ adipogenic CAF (MACAF) subtype, which is more sensitive to Dasatinib. MACAF‐related cell–cell interactions highlighted that MACAF could enhance the epithelial‐mesenchymal transition process in tumor cells via the TGF‐β pathway, promoting tumor cell migration and possibly contributing to tumor progression and invasiveness. Notably, patients with higher MACAF scores experience unfavorable prognoses and poor response rates to checkpoint inhibitor‐based immunotherapy, suggesting a correlation between MACAF and immunosuppressive microenvironment shaping. Our findings provide novel insights of the MEG3 in CAF activation and highlight the potential value of the MACAF score for therapeutic strategies design involving Dasatinib and immunotherapy.

Biophysically stressed vascular smooth muscle cells express MCP-1 via a PDGFR-β-HMGB1 signaling pathway.

Vascular smooth muscle cells (VSMCs) under biophysical stress play an active role in the progression of vascular inflammation, but the precise mechanisms are unclear. This study examined the cellular expression of monocyte chemoattractant protein 1 (MCP-1) and its related mechanisms using cultured rat aortic VSMCs stimulated with mechanical stretch (MS, equibiaxial cyclic stretch, 60 cycles/ min). When the cells were stimulated with 10% MS, MCP-1 expression was markedly increased compared to those in the cells stimulated with low MS intensity (3% or 5%). An enzyme-linked immunosorbent assay revealed an increase in HMGB1 released into culture media from the cells stimulated with 10% MS compared to those stimulated with 3% MS. A pretreatment with glycyrrhizin, a HMGB1 inhibitor, resulted in the marked attenuation of MCP-1 expression in the cells stimulated with 10% MS, suggesting a key role of HMGB1 on MCP-1 expression. Western blot analysis revealed higher PDGFR-α and PDGFR-β expression in the cells stimulated with 10% MS than 3% MS-stimulated cells. In the cells deficient of PDGFR-β using siRNA, but not PDGFR-α, HMGB1 released into culture media was significantly attenuated in the 10% MS-stimulated cells. Similarly, MCP-1 expression induced in 10% MS-stimulated cells was also attenuated in cells deficient of PDGFR-β. Overall, the PDGFR-β signaling plays a pivotal role in the increased expression of MCP-1 in VSMCs stressed with 10% MS. Therefore, targeting PDGFR-β signaling in VSMCs might be a promising therapeutic strategy for vascular complications in the vasculatures under excessive biophysical stress.

Directing Rat Hair Follicle Stem Cells Toward Neuronal Lineage With Enhanced Trophic Factor Expression.

Hair follicle stem cells (HFSCs) are promising candidates for cell-based therapies in neurodegenerative diseases because of their ability to differentiate into neural lineages and exert paracrine effects in damaged tissues. However, their clinical application faces challenges, particularly in efficiently guiding them toward neural lineages. This study explores using chick embryo extract (CEE) to enhance HFSCs' secretory capacity and neuronal differentiation. HFSCs from rat whisker pads were cultured in growth medium supplemented with either 20% FBS or a combination of 10% FBS and 10% CEE, transitioning to 20% FBS after the first subculture. We conducted gene expression profiling of lineage commitment markers and neurotrophic factors in both experimental groups, alongside morphological assessments and protein expression analyses. CEE supplementation during migration increased neuronal differentiation, evidenced by more cells with neurites and higher MAP2 expression at both the gene and protein levels. CEE also inhibited the expression of PDGFR-α, indicating a suppression of differentiation toward Schwann cells. Furthermore, we observed increased levels of trophic factors such as BDNF and VEGF at passage 3 induced by CEE supplementation. Enhancing the neuronal lineage commitment of hair follicle stem cells (HFSCs) and boosting the expression of trophic and angiogenic factors through short-term CEE preconditioning during their migratory stage presents a compelling approach. This strategy holds great promise in enhancing the effectiveness of stem cell-based therapies for neurological disorders.

Pleiotropy in FOXC1-attributable phenotypes involves altered ciliation and cilia-dependent signaling

Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld–Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes.

Avapritinib efficacy in primary hepatic neuroendocrine carcinoma with elevated PDGFRA expression: Insights from a PDX model study

Background & aimsPrimary Hepatic Neuroendocrine Carcinoma (PHNEC) is a rare and aggressive tumor with high recurrence rates. Surgical resection remains the only therapeutic strategy. The effectiveness of tyrosine kinase inhibitors (TKIs) for PHNEC remains unclear due to limited research. MethodsWe employed immunohistochemical staining to diagnose PHNEC and assess the expression of eight tyrosine kinase receptors in tumor tissues, including VEGFRs, PDGFRA, EGFR, FGFRs et al. A patient-derived xenograft (PDX) model was established using PHNEC tumor tissues to test the efficacy of TKIs. PDX mice bearing tumors were treated with Avapritinib, an FDA-approved PDGFRA-targeting drug, at a daily oral dose of 10 mg/kg for 2 weeks. ResultsPathological analysis confirmed the diagnosis of PHNEC with positive expression of Neural cell adhesion molecule (NCAM/CD56), Synaptophysin (Syn), and Somatostatin receptor 2 (SSTR-2), and negative expression of Hep (Hepatocyte Paraffin 1), a biomarker for Hepatocellular carcinoma. Notably, PDGFRA was significantly overexpressed in PHNEC tumor tissues compared to other tyrosine kinases. Avapritinib treatment significantly reduced tumor growth in PDX mice by 73.9 % (p = 0.008). Additionally, Avapritinib treatment led to a marked decrease in PDGFRA and Ki-67 expression, suggesting that it inhibits tumor cell proliferation by suppressing PDGFRA. ConclusionOur findings suggest that PDGFRA is a potential therapeutic target for PHNEC, and its inhibition with Avapritinib may offer clinical benefits to patients with this rare malignancy.

CD34+stromal cells/telocytes and their role in mouse lung development: Light microscopy, immunofluorescence, ultrastructural and scanning electron microscopy evidence.

Telocytes (TCs), a novel type of mesenchymal or interstitial cell with specific, very long and thin cellular prolongations, have been found in various mammalian organs and have potential biological functions. However, their existence during lung development is poorly understood. This study aimed to investigate the existence, morphological features, and role of CD34+ SCs/TCs in mouse lungs from foetal to postnatal life using primary cell culture, double immunofluorescence, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The immunofluorescence double staining profiles revealed positive expression of CD34 and PDGFR-α, Sca-1 or VEGFR-3, and the expression of these markers differed among the age groups during lung development. Intriguingly, in the E18.5 stage of development, along with the CD34+ SCs/TCs, haematopoietic stem cells and angiogenic factors were also significantly increased in number compared with those in the E14.5, E16.5, P0 and P7. Subsequently, TEM confirmed that CD34+ SCs/TCs consisted of a small cell body with long telopodes (Tps) that projected from the cytoplasm. Tps consisted of alternating thin and thick segments known as podomers and podoms. TCs contain abundant endoplasmic reticulum, mitochondria and secretory vesicles and establish close connections with neighbouring cells. Furthermore, SEM revealed characteristic features, including triangular, oval, spherical, or fusiform cell bodies with extensive cellular prolongations, depending on the number of Tps. Our findings provide evidence for the existence of CD34+ SCs/TCs, which contribute to vasculogenesis, the formation of the air‒blood barrier, tissue organization during lung development and homoeostasis.

IL-10 Enhances the Inhibitory Effect of Adipose-Derived Stromal Cells on Insulin Resistance/Liver Gluconeogenesis by Treg Cell Induction.

The modulation of cellular phenotypes within adipose tissue provides a potential means for therapeutic intervention for diabetes. Endogenous interleukin-10 (IL-10) protects against diet-induced insulin resistance. We examined the effects and mechanisms of action of IL-10-treated adipose-derived stromal cells on diabetes-induced insulin resistance and liver gluconeogenesis. We harvested stromal vascular fractions (SVFs) from the adipose tissue of diabetic (Leprdb/db) mice and treated them with IL-10 in vitro. SVFs treated with 10 or 100 ng of IL-10 were injected into the inguinal adipose tissue of Leprdb/db mice. IL-10 treatment suppressed the mRNA expression of IL-6, IL-33, CCL2, TNF-α, and IL-1β. Additionally, it suppressed the protein expression of IL-6, pmTOR, pJNK, and pNF-κB but enhanced Foxp3 mRNA expression in SVFs from diabetic mice. Meanwhile, IL-10 treatment repressed CCL2 and PDGFRα expression in adipose tissue macrophages (ATMs) and IL-6 expression in non-ATMs but increased the Foxp3 and IL-10 mRNA expression of ATMs from diabetic mice. Injection of IL-10-treated SVFs decreased the IL-6, IL-33, CCL2, IL-1β, and CCL2 but enhanced the Foxp3 and IL-10 mRNA expression of adipose tissue from Leprdb/db mice. Furthermore, injection of IL-10-treated SVFs increased CD4+ regulatory T cells (Tregs) in SVFs and adipose IL-10 levels and suppressed plasma adiponectin levels and DPP4 activity in diabetic mice. Injection of IL-10-treated SVFs decreased hepatic G6PC and PCK1 mRNA expression and increased Akt activation, STAT3 phosphorylation in the liver, and glucose tolerance in diabetic mice. Our data suggest that IL-10 treatment decreases inflammation in adipose SVFs of diabetic mice. Injection of IL-10-treated SVFs into the adipose tissue decreased diabetes-induced gluconeogenesis gene expression, DPP4 activity, and insulin resistance by enhancing Treg cells in diabetic mice. These data suggest that IL-10-treated adipose stromal vascular cells could be a promising therapeutic strategy for diabetes mellitus.

Unveiling the Differentiation Potential of Ovarian Theca Interna Cells from Multipotent Stem Cell-like Cells.

Theca interna cells (TICs) are an indispensable cell source for ovarian follicle development and steroidogenesis. Recent studies have identified theca stem cells (TSCs) in both humans and animals. Interestingly, TSCs express mesenchymal stem cell (MSC)-related markers and can differentiate into mesenchymal lineages. MSCs are promising for tissue engineering and regenerative medicine due to their self-renewal and differentiation abilities. Therefore, this study investigated the potential origin of TICs from MSCs. Whole ovaries from postmenopausal organ donors were obtained, and their cortex was cryopreserved prior to the isolation of stromal cells. These isolated cells were differentiated in vitro to TICs using cell media enriched with various growth factors and hormones. Immunocytochemistry, an enzyme-linked immunosorbent assay, flow cytometry, and reverse transcription-quantitative polymerase chain were employed at different timepoints. Data were analyzed using one-way ANOVA. Immunocytochemistry showed an increase in TIC markers from day 0 to day 8 and a significant rise in MSC-like markers on day 2. This corresponds with rising androstenedione levels from day 2 to day 13. Flow cytometry identified a decreasing MSC-like cell population from day 2 onwards. The CD13+ cell population and its gene expression increased significantly over time. NGFR and PDGFRA expression was induced on days 0 and 2, respectively, compared to day 13. This study offers insights into MSC-like cells as the potential origin of TICs. Differentiating TICs from these widely accessible MSCs holds potential significance for toxicity studies and investigating TIC-related disorders like polycystic ovary syndrome (PCOS).

Identifying and validating angiogenesis-related genes remodeling tumor microenvironment and suppressing immunotherapy response in gastric cancer

Angiogenesis significantly correlates with tumor microenvironment remodeling and immunotherapy response. Our study aimed to construct a prognostic angiogenesis-related model for gastric cancer. Using public database, a angiogenetic related five-gene (FGF1, GRB14, PAK3, PDGFRA, and PRKD1) model was identified. The top 25 % of patients were defined as high-risk, and the remaining as low-risk. The area under the curve for 1-, 3-, and 5-year overall survival (OS) were 0.646, 0.711, and 0.793, respectively. Survival analysis showed a better 10-year OS in low-risk patients in the construction (HR = 0.57, p = 0.002) and validation cohorts. GO and GSEA revealed that DEGs were enriched in extracellular matrix receptor interactions, dendritic cell antigen processing/presentation regulation, and angiogenesis pathways. CIBERSORT analysis revealed abundant naïve B cells, resting mast cells, resting CD4+ memory T cells, M2 macrophages, and monocytes in high-risk subgroups. The TIMER database showed strong positive correlations between PAK3, FGF1, PRKD1, and PDGFRA expression levels and the infiltration of CD4+ T cells and macrophages. The IOBR analysis revealed an immunosuppressive environment in the high-risk subgroup. Low-risk patients show a higher response rate to anti-PD1 treatment. TMA showed that FGF1 overexpression was associated with poor prognosis and CD4+ T cells and macrophage infiltration. In vivo study based on the 615 mice indicated that inhibiting FGF1 function could suppress tumor growth and enhance anti-PD1 therapeutic efficacy. In summary, we established a five-angiogenesis-related gene model to predict survival outcomes and immunotherapy responses in patients with gastric cancer and identified FGF1 as a prognostic gene and potential target for improving immune treatment.

High-intensity interval training stimulates remyelination via the Wnt/β-catenin pathway in cuprizone-induced demyelination mouse model

ABSTRACT Objectives This study aims to investigate the role of high-intensity interval training (HIIT) in promoting myelin sheath recovery during the remyelination phase in cuprizone (CPZ)-induced demyelination mice and elucidate the mechanisms involving the Wnt/β-catenin pathway. Methods After 5 weeks of a 0.2% CPZ diet to induce demyelination, a 4-week recovery phase with a normal diet was followed by HIIT intervention. Mice body weight was monitored. Morris water maze (MWM) gauged spatial cognition and memory, while the open field test (OFT) assessed anxiety levels. Luxol fast blue (LFB) staining measured demyelination, and immunofluorescence examined myelin basic protein (MBP) and platelet-derived growth factor receptor-alpha (PDGFR-α). Western blotting analyzed protein expression, including MBP, PDGFR-α, glycogen synthase kinase-3β (GSK3β), β-catenin, and p-β-catenin. Real-time PCR detected mRNA expression levels of CGT and CST. Results HIIT promoted remyelination in demyelinating mice, enhancing spatial cognition, memory, and reducing anxiety. LFB staining indicated decreased demyelination in HIIT-treated mice. Immunofluorescence demonstrated increased MBP fluorescence intensity and PDGFR-α+ cell numbers with HIIT. Western blotting revealed HIIT reduced β-catenin levels while increasing p-β-catenin and GSK3β levels. Real-time PCR demonstrated that HIIT promoted the generation of new myelin sheaths. Additionally, the Wnt/β-catenin pathway agonist, SKL2001, decreased MBP expression but increased PDGFR-α expression. Discussion HIIT promotes remyelination by inhibiting the Wnt/β-catenin pathway and is a promising rehabilitation training for demyelinating diseases. It provides a new theoretical basis for clinical rehabilitation and care programs.

P-786 Mesenchymal stem cells as a potential source of ovarian theca interna cell differentiation

Abstract Study question Are mesenchymal stem cells (MSCs) the cell source of origin during the differentiation of ovarian stromal cells into theca interna cells (TICs)? Summary answer MSC-like cells were observed during in vitro TIC differentiation, particularly on day 2, suggesting their potential role in the origin of TICs. What is known already Folliculogenesis is the process during which a follicle develops from its primordial towards its antral stage. Follicles in the antral stage consist of an oocyte, granulosa and theca cells. While the oocyte is surrounded by granulosa cells in the primordial stage, theca cells differentiate from their precursor cell from the secondary stage onwards. Recent studies identified the existence of theca stem cells (TSCs) in both humans and animals. These TSCs express both platelet-derived growth factor receptor (PDGFR) and nerve growth factor receptor (NGFR), and demonstrate the ability to differentiate into mesenchymal cell lineages, resembling characteristics of MSCs. Study design, size, duration Ovarian stromal cells were isolated from cryopreserved cortex fragments, and cultured in vitro for 13 days, using cell media enriched with various growth factors and hormones, as previously established by our group. Flow cytometry, immunocytochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were employed to analyze the cells and their media at days 0, 2, 8 and 13. Participants/materials, setting, methods Whole ovaries from three postmenopausal organ donors were obtained (50, 54, and 64 years) and the cortex was cryopreserved in small 5x5 fragments prior to the isolation and differentiation into TICs. Main results and the role of chance Immunocytochemistry results showed an increase in the proportion of cells positive for TIC markers from day 0 to day 8 (12.13 ± 2.11% vs. 57.73 ± 49.99%), while the proportion of MSC-like cell markers was increased significantly from day 0 to day 2 (4.54 ± 1.71% vs. 57.34 ± 18.05%). This TIC proportion aligns with the rising androstenedione levels from day 2 to day 13 (81.31 ± 25.09 pg/ml vs. 384.29 ± 245.82 pg/ml). Flow cytometry analysis identified a CD73+ CD90+ CD105+ CD34- CD45- HLA-DR- cell population, expanding from less than 1% on day 0, to 15.30 ± 7.9% on day 2. A CD13+ cell population and its corresponding gene expression increased significantly from the beginning towards the end of the culture (13.52 ± 7.9% vs. 84.13 ± 11.08, and 1.38 ± 0.97 vs. 285.80 ± 27.43, flow cytometry and gene expression analysis respectively). Moreover, NGFR and PDGFRA expression was induced on day 0 and day 2, respectively, compared to day 13 (1.12 ± 0.60 vs. 0.08 ± 0.08, and 2.03 ± 1.11 vs. 0.24 ± 0.14). In conclusion, our study provides valuable insights into the differentiation potential of TICs, suggesting a possible origin from MSCs. Limitations, reasons for caution Due to the limited accessibility of a large quantity of human ovarian tissue from postmenopausal women, this study is restricted to three biological replicates. When examining statistical differences, it is crucial to bear in mind the considerable biological variabilities that exist among humans. Wider implications of the findings Characterizing the origin of TICs will allow for a comprehensive study of TICs, leading to a broader knowledge of these cells. The in vitro differentiation to TICs from easily accessible cells, like MSCs, holds potential significance in toxicity studies and investigating TIC-related disorders, such as polycystic ovary syndrome (PCOS). Trial registration number not applicable

Mesenchymal-specific Alms1 knockout in mice recapitulates metabolic features of Alström syndrome

ObjectiveAlström Syndrome (AS), caused by biallelic ALMS1 mutations, includes obesity with disproportionately severe insulin resistant diabetes, dyslipidemia, and fatty liver. Prior studies suggest that hyperphagia is accounted for by loss of ALMS1 function in hypothalamic neurones, whereas disproportionate metabolic complications may be due to impaired adipose tissue expandability. We tested this by comparing the metabolic effects of global and mesenchymal stem cell (MSC)-specific Alms1 knockout. MethodsGlobal Alms1 knockout (KO) mice were generated by crossing floxed Alms1 and CAG-Cre mice. A Pdgfrα-Cre driver was used to abrogate Alms1 function selectively in MSCs and their descendants, including preadipocytes. We combined metabolic phenotyping of global and Pdgfrα+ Alms1-KO mice on a 45% fat diet with measurements of body composition and food intake, and histological analysis of metabolic tissues. ResultsAssessed on 45% fat diet to promote adipose expansion, global Alms1 KO caused hyperphagia, obesity, insulin resistance, dyslipidaemia, and fatty liver. Pdgfrα-cre driven KO of Alms1 (MSC KO) recapitulated insulin resistance, fatty liver, and dyslipidaemia in both sexes. Other phenotypes were sexually dimorphic: increased fat mass was only present in female Alms1 MSC KO mice. Hyperphagia was not evident in male Alms1 MSC KO mice, but was found in MSC KO females, despite no neuronal Pdgfrα expression. ConclusionsMesenchymal deletion of Alms1 recapitulates metabolic features of AS, including fatty liver. This confirms a key role for Alms1 in the adipose lineage, where its loss is sufficient to cause systemic metabolic effects and damage to remote organs. Hyperphagia in females may depend on Alms1 deficiency in oligodendrocyte precursor cells rather than neurones. AS should be regarded as a forme fruste of lipodystrophy.

The relationships of platelet-derived growth factor, microvascular proliferation, and tumor cell proliferation in canine high-grade oligodendrogliomas: Immunohistochemistry of 45 tumors and an AFOB-01 xenograft mouse model.

High-grade oligodendroglioma (HGOG) is the most common type of glioma in dogs and expresses platelet-derived growth factor receptor-α (PDGFR-α). Microvascular proliferation is often observed in HGOG. Therefore, the present study investigated the functional relationships between PDGFR-α, microvascular proliferation, and tumor cell proliferation in canine HGOG. The expression of PDGFR-α and PDGF-subunit A (PDGF-A) in tumor cells, as well as endothelial cells and pericytes of tumor-associated microvascular proliferations, in 45 canine HGOGs were examined immunohistochemically. Microvascular proliferation was observed in 24/45 cases (53%). PDGFR-α expression in tumor cells and microvascular proliferations was observed in 45/45 (100%) and 2/24 cases (8%), respectively. Furthermore, PDGF-A expression in tumor cells and microvascular proliferations was detected in 13/45 (29%) and 24/24 cases (100%), respectively. In vitro, stimulation of the canine HGOG cell line AOFB-01 with PDGF-A showed that the doubling time of AOFB-01 cells was significantly shorter with PDGF-A than without PDGF-A. Crenolanib (a PDGFR inhibitor) inhibited AOFB-01 cell proliferation. In vivo, the AOFB-01 xenograft mouse model was treated with crenolanib. Tumor xenografts were smaller in crenolanib-treated mice than in untreated control mice. PDGFR-α expression in tumor cells and PDGF-A expression in microvascular proliferations and tumor cells suggest autocrine and paracrine effects of PDGF-A in canine HGOG. The results of in vitro assays indicate that canine HGOG expresses functional PDGFR-α, which responds to PDGF-A. Therefore, PDGF-A produced by microvascular proliferations and tumor cells may promote the proliferation of PDGFR-α-expressing tumor cells in canine HGOG. PDGFR-α signaling has potential as a therapeutic target.

Abstract 6945: Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma

Abstract The five-year survival rate of glioblastoma multiforme (GBM) is 5.8%, and its standard of care has not changed since 2005. This stagnation in therapeutic development is due, in part, to its characteristic heterogeneity–multiforme–highlighting a critical need to understand the biological processes underlying this GBM hallmark. Despite the well-documented individual relationships between extrachromosomal DNA (ecDNA), GBM, and heterogeneity, the interplay between the three is not well understood. The Furnari lab has generated a library of cancer avatars, or human induced pluripotent stem cells (hiPSCs) engineered to harbor cancer driving mutations. Single cell RNA sequencing (scRNA-seq) was used to characterize the longitudinal evolution of the first avatars engineered to have TP53−/−;PDGFRAΔ8-9 and PTEN−/−;NF1−/− genotypes. The TP53−/−;PDGFRAΔ8-9 avatar acquired ecDNA, while the PTEN−/−; NF1−/− avatar did not. Re-analysis of the scRNA-seq data identified a subpopulation of cells in the TP53−/−;PDGFRAΔ8-9 avatar that had high expression of CDK4 and PDGFRA, two genes that are commonly co-amplified, found on ecDNA, and drive cellular states. This subpopulation of cells also had highly expressed genes near the CDK4 locus indicating a structural variant and possibly ecDNA. This population diverged transcriptionally from other cells and was associated with the OPC-like cellular state, which has been shown to be associated with high levels of PDGFRA and, at times, co-expression with CDK4. While TP53−/−;PDGFRAΔ8-9 tumors contained cells across all four GBM cellular states, PTEN−/−;NF1−/− tumors had a significantly less diverse composition of cellular states suggesting that ecDNA may facilitate cellular state diversity. With the eventual goal to directly test whether ecDNA enables transcriptional state heterogeneity compared to chromosomally inherited amplifications, CRISPR-Cas9 and CRISPR-C were implemented to engineer EGFRvIII ecDNA in U87. While inverse PCR confirmed the successful generation of ecDNA in U87, ecDNA was lost following long term in vitro cell passaging. Current follow up work aims to identify different intrinsic (oncogene amplified, genetic background, cell type, cell line) and extrinsic (in vitro conditions, orthotopic engraftment) contexts that promote the selection and amplification of engineered ecDNA, as it would be would be valuable to have genotypically identical cancer models with different classes of amplifications (i.e., engineered ecDNA vs. lentiviral overexpression, which is chromosomally inherited). Together, these results highlight the potential to use hiPSC-derived models and single cell methods to investigate gliomagenesis and cell identity. Citation Format: Brett Taylor, Brandon Jones, Daisuke Kawauchi, Yohei Miyake, Raghav Vadla, Nathan Jameson, Shunichiro Miki, Tomoyuki Koga, Bing Ren, Frank Furnari. Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma [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 6945.

Morphological and Molecular Biological Characteristics of Experimental Rat Glioblastoma Tissue Strains Induced by Different Carcinogenic Chemicals.

We estimated different gene expressions by qPCR-RT and conducted Western blotting and histological and morphometric analysis of three different tissue strains of rat GBM. GBM 101.8 was characterized by the shortest period of tumor growth and the greatest number of necroses and mitoses; overexpression of Abcb1, Sox2, Cdkn2a, Cyclin D, and Trp53; and downregulated expression of Vegfa, Pdgfra, and Pten; as well as a high level of HIF-1α protein content. GBM 11-9-2 and GBM 14-4-5 were relevant to low-grade gliomas and characterized by downregulated Mgmt expression; furthermore, a low content of CD133 protein was found in GBM 11-9-2. GBM 101.8 is a reliable model for further investigation due to its similarity to high-grade human GBMs, while GBM 11-9-2 and GBM 14-4-5 correspond to Grade 2-3 gliomas.

Abstract PR-008: Modeling epigenetic lesions that cause gliomas

Abstract PR-008: Modeling epigenetic lesions that cause gliomas