Platelet-derived Growth Factor Receptor Research Articles

A method to isolate human dermal microvascular pericytes without the use of magnetic beads sorting in vitro

Human dermal microvascular pericytes (HDMPCs) are a critical component of the skin flap microvasculature and play a role in regulating flap blood flow and integrity. Pericytes were isolated mostly via magnetic bead sorting in the published literature. In this study, we discuss in detail how to separate and concentrate pericytes from human facial flaps using enzyme digestion and differential adherence instead of magnetic bead sorting. Cultured HDMPCs were seen to have well-spread irregular edges, with most cells having two longitudinal pericytic processes. The phalloidin staining revealed that HDMPCs had prominent stress fibers, and the nucleus deviated to the side that interacted with the neighboring pericytic processes. Flow cytometry analysis showed that the positive rates of NG2 in the first and second passages were 91.2% ± 0.7% and 98.2% ± 0.1% separately. And the immunofluorescence and western blot results demonstrated a positive expression of α smooth muscle actin (αSMA), platelet-derived growth factor receptor β (PDGFRβ), and NG-2, while the endothelial cell marker CD31 was negatively expressed. In summary, we established a straightforward methodology for selectively isolating and identifying HDMPCs as well as generating high-purity cell cultures in vitro without the use of magnetic bead sorting.

Hypereosinophilic Syndrome with Endomyocarditis: Identification by Next-Generation Sequencing of the JAK2-V617F Mutation.

Hypereosinophilic syndrome requires a peripheral absolute eosinophil count of ≥1.5 × 109 /L with clinical manifestations attributable to peripheral or tissue hypereosinophilia. Clinical manifestations can vary greatly, with the majority of patients relatively asymptomatic and the eosinophilia detected incidentally. However, in a minority of hypereosinophilia cases, they may present with severe lifethreatening organ dysfunction affecting skin, lung, heart, gastrointestinal tract, and nervous system. A case of hypereosinophilia with potentially life-threatening cardiovascular involvement is discussed. Initial laboratory investigations showed an elevated white blood cell count with 60% eosinophils. An endomyocardial biopsy revealed eosinophilic endomyocarditis with granuloma, rare giant cells, and no vasculitis, microorganisms, or malignancy. Her presentation met the criteria for either hypereosinophilic syndrome or eosinophilic granulomatosis with polyangitis. Molecular genetic analysis was negative for myelodysplastic syndrome panel/ Platelet Derived Growth Factor Receptor Beta (PDGFRB) (5q32)/Fibroblast Growth Factor Receptor 1 (FGFR1) Fluorescence In Situ Hybridization (FISH), Feline McDonough Sarcoma-related Tyrosine Kinase 3 (FLT3) Internal Tandem Duplication (ITD) mutation, Calregulin (CALR) exon 9 mutation, and T-cell gene rearrangement/polymerase chain reaction. Bone marrow biopsy revealed a mildly hypocellular marrow with multilineage hematopoiesis,+ megakaryocyte dysplasia, and focal eosinophilia. No excess blasts, no monotypic B-cell population, and no discrete pan T-cell aberrancies were found. Bone marrow cytogenetic studies showed a normal signal pattern for myeloproliferative neoplasms panel/Sec1 Family Domain Containing 2 (SCFD2)-Ligand of Numb Protein-X (LNX)-Platelet-derived Growth Factor Receptor Alpha (PDGFRA) fluorescence in situ hybridization with a normal karyotype of 46 XX. Next-generation sequencing, however, was positive for the JAK2-V617F mutation, a rare molecular abnormality in hypereosinophilic syndrome. The prevalence ranges from approximately 0% to 4%. The JAK2 point mutation leads to aberrant tyrosine phosphorylation and increased cytokine activation. The case demonstrates the complexity and challenging nature of advanced diagnostic opportunities in hypereosinophilia and the potential use, in select subsets, of targeted treatments such as tyrosine kinase inhibitors.

Metanephric stromal tumor in an adult with PDGFRA mutation: a case report

BackgroundMetanephric stromal tumors (MST) are rare benign renal tumors that mainly occur in infants and children. Approximately 72% of MST in children have the B-Raf proto-oncogene serine/threonine kinase (BRAF) V600E mutation. To date, only five cases of adult MSTs have been reported and no clear genetic alterations have been found.Case presentationWe report a case of MST in a 45-year-old woman who complained of left lower back pain for a week, accompanied by hypertension (150/79 mmHg). Magnetic resonance imaging (MRI) showed an abnormally enhanced nodule (1.1 cm in the middle of the left kidney), which was histopathologically consistent with an MST. The BRAF V600E mutation was not detected in tumor cells using PCR and next-generation sequencing (NGS). However, a platelet-derived growth factor receptor alpha (PDGFRA) mutation was detected in this case using NGS. The patient showed no recurrence or metastasis nine months after partial nephrectomy, and her blood pressure was consistently normal.ConclusionThis is the first report of alterations in PDGFRA in MSTs. This result advances our knowledge of genetic variations in adult MSTs, which may have different gene alterations from MSTs in children.

Evaluation of Ki-67 as prognostic factor for pediatric neuroblastoma and the possibility of molecular-targeted drugs with VEGF and PDGFR.

Neuroblastoma (NB) is a pediatric malignant solid tumor characterized as refractory cancer with poor prognosis. The Mitosis-Karyorrhexis Index (MKI) is a prognostic factor but is prone to observer bias. The usefulness of MKI with Ki-67, as a marker of malignancy, was investigated. The efficacy of molecular-targeted therapeutic agents with fewer side effects in tumors has been studied. Molecular-targeted therapy targets include vascular endothelial growth factor (VEGF), involved in tumor angiogenesis; c-Kit, receptor of Kit/stem cells involved in tumor growth, vasculature, and lymphangiogenesis; platelet-derived growth factor receptor (PDGFR); and B-Raf proto-oncogene, serine/threonine kinase (BRAF), involved in the RAS protein-mediated mitogen-activated protein kinase pathway. Therefore, expression profiles of these factors and growth inhibitory effects of molecular-targeted drugs against NB were investigated. Ten frozen NB tissue samples collected from January 1993 to December 2017 were evaluated immunohistochemically for Ki-67 and VEGF. c-Kit, PDGFR, and BRAF expression levels were evaluated using enzyme-linked immunosorbent assays; relationships between these factors and clinicopathological parameters of NB were analyzed. Eight patients with NB showed no amplification of MYCN (MYCN proto-oncogene, bHLH transcription factor). There were two cases of ganglioneuroblastoma (GNB). More NB cells were positive for Ki-67 than for GNB cells. VEGF expression was observed in all NB specimens and was stronger in stage IIB and higher. No BRAF or c-Kit activity was observed; PDGFR activity was greater in NB than in GNB (P=0.02). Thus, Ki-67 may help evaluate NB malignancy. As the first therapy for NB prevents amplification of MYCN, agents targeting PDGFR as well as VGFG can inhibit NB cell proliferation.

Molecular Docking Study of New Sorafenib Analogues as Platelet-Derived Growth Factor Receptor Inhibitors for the Treatment of Cancer.

Cancer is a disease triggered by an uncontrolled growth of a group of cells usually from a single cell. Chemotherapy is a common and systematic therapy that involves the use of anticancer drugs also known as chemotherapeutical agents to treat cancer. Tyrosine kinases are a subset of protein kinases that are a family of over 90 enzymes that selectively phosphorylate tyrosine residues in various substrates. Receptors with internal tyrosine kinase activity mediate the actions of several growth factors, differentiation factors, and hormones, resulting in the reproduction and differentiation of the affected cells. In the fight against cancer, the platelet-derived growth factor receptor has emerged as a novel target via inhibition of this receptor resulting in the inhibition of tyrosine kinase cascade. Docking investigations were conducted using the Genetic Optimization for Ligand Docking (GOLD) Suite (v. 5.7.1) from the Cambridge Crystallographic Data Center. A high-definition X-ray crystallography of the platelet-derived growth factor protein [Protein Data Bank (PDB) ID 6JOL] was downloaded from the website PDB with a resolution of 2 A. Compounds II, III, VII, and VIII have greater binding energies than the GOLD standard medication sorafenib, which gives Piecewise Linear Potential (PLP) fitness value (85.3). Other ligands exhibit good inhibitory action and docking scores comparable to that of the reference ligand sorafenib.

Heterogeneity of the Tumor Microenvironment Across Molecular Subtypes of Breast Cancer.

Breast cancer is a heterogenous disease at the molecular level thus, it can be hypothesized that different molecular subtypes differ in their tumor microenvironment (TME) also. Understanding the TME heterogeneity may provide new prognostic biomarkers and new targets for cancer therapy. For deciphering heterogeneity in the TME, immunohistochemistry for immune markers (CD3, CD4, CD8, CD68, CD163, and programmed death-ligand 1), Cancer-associated fibroblast markers [anti-fibroblast activating protein α (FAP-α), platelet-derived growth factor receptor α (PDGFR-α), S100A4, Neuron-glial antigen 2, and Caveolin-1], and angiogenesis (CD31) was performed on tissue microarrays of different molecular subtypes of breast cancer. High CD3 + T cells were noted in the Luminal B subtype ( P =0.002) of which the majority were CD8 + cytotoxic T cells. Programmed death-ligand 1 expression in immune cells was highest in the human epidermal growth factor receptor 2 (Her-2)-positive and Luminal B subtypes compared with the triple-negative breast cancer (TNBC) subtype ( P =0.003). Her-2 subtype is rich in M2 tumor-associated macrophages ( P =0.000) compared with TNBC and Luminal B subtypes. M2 immune microenvironment correlated with high tumor grade and high Ki-67. Her-2 and TNBC subtypes are rich in extracellular matrix remodeling (FAP-α, P =0.003), angiogenesis-promoting (PDGFR-α; P =0.000) and invasion markers (Neuron-glial antigen 2, P =0.000; S100A4, P =0.07) compared with Luminal subtypes. Mean Microvessel density showed an increasing trend: Luminal A>Luminal B>Her-2 positive>TNBC; however, this difference was not statistically significant. The cancer-associated fibroblasts (FAP-α, PDGFR-α, and Neuron-glial antigen 2) showed a positive correlation with lymph node metastasis in specific subtypes. Immune cells, tumor-associated macrophage, and cancer-associated fibroblast-related s tromal markers showed higher expression in Luminal B, Her-2 positive, and TNBC respectively. This differential expression of different components of TME indicates heterogeneity of the TME across molecular subtypes of breast cancer.

An effective kinase inhibition strategy for metastatic recurrent childhood medulloblastoma.

Medulloblastomas (MBs) constitute the most common malignant brain tumor in children and adolescents. MYC-amplified Group 3 MBs are characterized by disease recurrence, specifically in the leptomeninges, whereby patients with these metastatic tumors have a mortality rate nearing 100%. Despite limited research on such tumors, studies on MB metastases at diagnosis suggest targeting kinases to be beneficial. To identify kinase inhibitors that eradicate cells driving therapy evasion and tumor dissemination, we utilized our established patient-derived xenograft (PDX) mouse-adapted therapy platform that models human MB metastatic recurrences following standard chemoradiotherapy. High-throughput screens of 640 kinase inhibitors were conducted against cells isolated from mouse spines in the PDX model and human fetal neural stem cells to reveal compounds that targeted these treatment-refractory, metastatic cells, whilst sparing healthy cells. Blood-brain barrier permeability assays and additional in vitro experimentation helped select top candidates for in vivo studies. Recurrent Group 3MB PDX spine cells were therapeutically vulnerable to a selective checkpoint kinase 1 (CHK1) inhibitor and small molecular inhibitor of platelet-derived growth factor receptor beta (PDGFRβ). Inhibitor-treated cells showed a significant reduction in MB stem cell properties associated with treatment failure. Mice also demonstrated survival advantage when treated with a CHK1 inhibitor ex vivo. We identified CHK1 and PDGFRβ inhibitors that effectively target MB cells fueling treatment-refractory metastases. With limited research on effective therapies for Group 3MB metastatic recurrences, this work highlights promising therapeutic options to treat these aggressive tumors. Additional studies are warranted to investigate these inhibitors' mechanisms and recommended in vivo administration.

Cardiac Pericytes Acquire a Fibrogenic Phenotype and Contribute to Vascular Maturation After Myocardial Infarction.

Pericytes have been implicated in tissue repair, remodeling, and fibrosis. Although the mammalian heart contains abundant pericytes, their fate and involvement in myocardial disease remains unknown. We used NG2Dsred;PDGFRαEGFP pericyte:fibroblast dual reporter mice and inducible NG2CreER mice to study the fate and phenotypic modulation of pericytes in myocardial infarction. The transcriptomic profile of pericyte-derived cells was studied using polymerase chain reaction arrays and single-cell RNA sequencing. The role of transforming growth factor-β (TGF-β) signaling in regulation of pericyte phenotype was investigated in vivo using pericyte-specific TGF-β receptor 2 knockout mice and in vitro using cultured human placental pericytes. In normal hearts, neuron/glial antigen 2 (NG2) and platelet-derived growth factor receptor α (PDGFRα) identified distinct nonoverlapping populations of pericytes and fibroblasts, respectively. After infarction, a population of cells expressing both pericyte and fibroblast markers emerged. Lineage tracing demonstrated that in the infarcted region, a subpopulation of pericytes exhibited transient expression of fibroblast markers. Pericyte-derived cells accounted for ~4% of PDGFRα+ infarct fibroblasts during the proliferative phase of repair. Pericyte-derived fibroblasts were overactive, expressing higher levels of extracellular matrix genes, integrins, matricellular proteins, and growth factors, when compared with fibroblasts from other cellular sources. Another subset of pericytes contributed to infarct angiogenesis by forming a mural cell coat, stabilizing infarct neovessels. Single-cell RNA sequencing showed that NG2 lineage cells diversify after infarction and exhibit increased expression of matrix genes, and a cluster with high expression of fibroblast identity markers emerges. Trajectory analysis suggested that diversification of infarct pericytes may be driven by proliferating cells. In vitro and in vivo studies identified TGF-β as a potentially causative mediator in fibrogenic activation of infarct pericytes. However, pericyte-specific TGF-β receptor 2 disruption had no significant effects on infarct myofibroblast infiltration and collagen deposition. Pericyte-specific TGF-β signaling was involved in vascular maturation, mediating formation of a mural cell coat investing infarct neovessels and protecting from dilative remodeling. In the healing infarct, cardiac pericytes upregulate expression of fibrosis-associated genes, exhibiting matrix-synthetic and matrix-remodeling profiles. A fraction of infarct pericytes exhibits expression of fibroblast identity markers. Pericyte-specific TGF-β signaling plays a central role in maturation of the infarct vasculature and protects from adverse dilative remodeling, but it does not modulate fibrotic remodeling.

Partial Mural Cell Ablation Disrupts Coronary Vasculature Integrity and Induces Systolic Dysfunction.

Background Although the critical role of pericytes in maintaining vascular integrity has been extensively demonstrated in the brain and the retina, little is known about their role in the heart. We aim to investigate structural and functional consequences of partial pericyte depletion (≈60%) in the heart of adult mice. Methods and Results To deplete pericytes in adult mice, we used platelet-derived growth factor receptor β-Cre/ERT2; RosaDTA mice and compared their phenotype with that of control mice (RosaDTA) chosen among their littermates. Cardiac function was assessed via echocardiography and left ventricular catheterization 1 month after the first tamoxifen injection. We found mice depleted with pericytes had a reduced left ventricular ejection fraction and an increased end-diastolic pressure, demonstrating both systolic and diastolic dysfunction. Consistently, mice depleted with pericytes presented a decreased left ventricular contractility and an increased left ventricular relaxation time (dP/dtmin). At the tissue level, mice depleted of pericytes displayed increased coronary endothelium leakage and activation, which was associated with increased CD45+ cell infiltration. Consistent with systolic dysfunction, pericyte depletion was associated with an increased expression of myosin heavy chain 7 and decreased expression of ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 and connexin 43. More important, coculture assays demonstrated, for the first time, that the decreased expression of connexin 43 is likely attributable to a direct effect of pericytes on cardiomyocytes. Besides, this study reveals that cardiac pericytes may undergo strong remodeling on injury. Conclusions Cardiac pericyte depletion induces both systolic and diastolic dysfunction, suggesting that pericyte dysfunction may contribute to the occurrence of cardiac diseases.

Plasma polymerized nanoparticles are a safe platform for direct delivery of growth factor therapy to the injured heart.

Introduction: Heart failure due to myocardial infarction is a progressive and debilitating condition, affecting millions worldwide. Novel treatment strategies are desperately needed to minimise cardiomyocyte damage after myocardial infarction and to promote repair and regeneration of the injured heart muscle. Plasma polymerized nanoparticles (PPN) are a new class of nanocarriers which allow for a facile, one-step functionalization with molecular cargo. Methods: Here, we conjugated platelet-derived growth factor AB (PDGF-AB) to PPN, engineering a stable nano-formulation, as demonstrated by optimal hydrodynamic parameters, including hydrodynamic size distribution, polydisperse index (PDI) and zeta potential, and further demonstrated safety and bioactivity in vitro and in vivo. We delivered PPN-PDGF-AB to human cardiac cells and directly to the injured rodent heart. Results: We found no evidence of cytotoxicity after delivery of PPN or PPN-PDGFAB to cardiomyocytes in vitro, as determined through viability and mitochondrial membrane potential assays. We then measured contractile amplitude of human stem cell derived cardiomyocytes and found no detrimental effect of PPN on cardiomyocyte contractility. We also confirmed that PDGF-AB remains functional when bound to PPN, with PDGF receptor alpha positive human coronary artery vascular smooth muscle cells and cardiac fibroblasts demonstrating migratory and phenotypic responses to PPN-PDGF-AB in the same manner as to unbound PDGF-AB. In our rodent model of PPN-PDGF-AB treatment after myocardial infarction, we found a modest improvement in cardiac function in PPN-PDGF-AB treated hearts compared to those treated with PPN, although this was not accompanied by changes in infarct scar size, scar composition, or border zone vessel density. Discussion: These results demonstrate safety and feasibility of the PPN platform for delivery of therapeutics directly to the myocardium. Future work will optimize PPN-PDGF-AB formulations for systemic delivery, including effective dosage and timing to enhance efficacy and bioavailability, and ultimately improve the therapeutic benefits of PDGF-AB in the treatment of heart failure cause by myocardial infarction.

18F] AlF-NOTA-FAPI-04 PET/CT can predict treatment response and survival in patients receiving chemotherapy for inoperable pancreatic ductal adenocarcinoma.

We investigated whether uptake of [18F] AlF-NOTA-FAPI-04 on positron emission tomography/computed tomography (PET/CT) could predict treatment response and survival in patients with pancreatic ductal adenocarcinoma (PDAC). We prospectively evaluated 47 patients with histopathologically confirmed primary PDAC who provided pretreatment [18F] AlF-NOTA-FAPI-04 scans to detect fibroblast activation protein (FAP) on the tumor surface by uptake of [18F] AlF-NOTA-FAPI-04. PDAC specimens were immunohistochemically stained with cancer-associated fibroblast (CAF) markers. We obtained a second PET scan after one cycle of chemotherapy to study changes in FAPI uptake variables from before to during treatment. Correlations between baseline PET variables and CAF-related immunohistochemical markers were assessed with Spearman's rank test. Cox regression and Kaplan-Meier methods were used to assess relationships between disease progression and potential predictors. Receiver operating characteristic (ROC) curve analysis was used to define the optimal cut-off points for distinguishing patients according to good response vs. poor response per RECIST v.1.1. The FAPI PET variables maximum and mean standardized uptake values (SUVmax, SUVmean), metabolic tumor volume (MTV), and total lesion FAP expression (TLF) were positively correlated with CAF markers (FAP, α-smooth muscle actin, vimentin, S100A4, and platelet-derived growth factor receptor α/β, all P < 0.05). MTV was associated with survival in patients with inoperable PDAC (all P < 0.05). Cox multivariate regression showed that MTV was associated with overall survival (MTV hazard ratio [HR] = 1.016, P = 0.016). Greater changes from before to during chemotherapy in SUVmax, MTV, and TLF were associated with good treatment response (all P < 0.05). ΔMTV, ΔTLF, and ΔSUVmax had larger areas under the curve than ΔCA19-9 for predicting treatment response. Kaplan-Meier analysis showed that the extent of change in MTV and TLF from before to after treatment predicted progression-free survival, with cut-off values (based on medians) of - 4.95 for ΔMTV (HR = 8.09, P = 0.013) and - 77.83 for ΔTLF (HR = 4.62, P = 0.012). A higher baseline MTV on [18F] AlF-NOTA-FAPI-04 scans was associated with poorer survival in patients with inoperable PDAC. ΔMTV was more sensitive for predicting response than ΔCA19-9. These results are clinically meaningful for identifying patients with PDAC who are at high risk of disease progression.

PDGF-BB-Dependent Neurogenesis Buffers Depressive-Like Behaviors by Inhibition of GABAergic Projection from Medial Septum to Dentate Gyrus.

Hippocampal circuitry stimulation is sufficient to regulate adult hippocampal neurogenesis and ameliorate depressive-like behavior, but its underlying mechanism remains unclear. Here, it is shown that inhibition of medial septum (MS)-dentate gyrus (DG) circuit reverses the chronic social defeat stress (CSDS)-induced depression-like behavior. Further analysis exhibits that inhibition of gamma-aminobutyric acidergic neurons in MS projecting to the DG (MSGABA+ -DG) increases the expression of platelet-derived growth factor-BB (PDGF-BB) in somatostatin (SOM) positive interneurons of DG, which contributes to the antidepressant-like effects. Overexpression of the PDGF-BB or exogenous administration of PDGF-BB in DG rescues the effect of chronic stress on the inhibition of neural stem cells (NSCs) proliferation and dendritic growth of adult-born hippocampal neurons, as well as on depressive-like behaviors. Conversely, knockdown of PDGF-BB facilitates CSDS-induced deficit of hippocampal neurogenesis and promotes the susceptibility to chronic stress in mice. Finally, conditional knockdown platelet-derived growth factor receptor beta (PDGFRβ) in NSCs blocks an increase in NSCs proliferation and the antidepressant effects of PDGF-BB. These results delineate a previously unidentified PDGF-BB/PDGFRβ signaling in regulating depressive-like behaviors and identify a novel mechanism by which the MSGABA+ -DG pathway regulates the expression of PDGF-BB in SOM-positive interneurons.

#3685 PERICYTE AND CXCL12 PLAY CRUCIAL ROLE IN THE RECOVERY OF ACUTE KIDNEY INJURY

Abstract Background and Aims Pericytes are mesenchyme-derived perivascular cells attached to the abluminal surface of capillaries as well as the major origin of myofibroblasts in the animal model of unilateral ureteral obstruction (UUO) and ischemia-reperfusion injury (IRI). Although previous studies reported that myofibroblasts may be beneficial during acute kidney injury (AKI) via growth factor secretion, reconstitution and stabilization of the collagen framework for tubular cell regeneration, lack of solid evidence and definite mechanism make the role of pericyte/myofibroblast in AKI remain controversial. Method To clarify this, we used platelet-derived growth factor receptor β (PDGFRβ)-specific antibody following AKI in C57BL/6 mice to inhibit pericytes. In the meantime, We used Gli1-CreERT2;Cxcl12fl/fl genetically modified mice to conditionally knockout CXCL12 specifically in pericytes before IRI. Results The expression of PDGFRβ on pericytes increased after IRI. In group of administration of PDGFRβ antibody, proliferation and transition to myofibroblasts of pericytes were inhibited. Inflammation became worse and renal recovery was impeded after AKI. The chemokine stromal cell-derived factor-1 (SDF-1) which is also known as the C-X-C-type chemokine CXCL12 decreased as well. Furthermore, we demonstrated that CXCL12 was secreted mainly by pericytes and its receptor CXCR4 was expressed on tubular cell during IRI by in situ hybridization. When knockout of CXCL12 was performed before IRI, the renal microvascular rarefaction was noted after AKI and the recovery of renal function was impaired. Renal tubular cell proliferation decreased and apoptosis increased as well. Conclusion In summary, these findings demonstrated that pericytes contribute to renal repair after IRI through CXCL12 secretion.

#5531 MOLECULAR IMAGING OF PDGFR-β IN KIDNEY FIBROSIS

Abstract Background and Aims Chronic kidney disease affects &amp;gt;10% of the world's population and is associated with high mortality and morbidity. The best predictor of CKD progression is the extent of fibrosis, i.e., pathological deposition of extracellular matrix (ECM) and loss of functional kidney parenchyme. Currently, there are no specific treatment options and invasive biopsies remain the gold standard for diagnosis. Multiple signaling pathways are involved in fibrosis, including platelet-derived growth factor (PDGF) signaling. Mesenchymal stromal cells express both PDGF receptors PDGFR-α and PDGFR-β, whose activation drives proliferation, migration and production of extracellular matrix, i.e., key processes involved in fibrosis initiation and progression. Based on these notions, we set out to study the bicyclic PDGFR-β-binding peptide (BiPPB) for diagnosis, quantitative imaging and treatment monitoring of kidney fibrosis. Method The biodistribution and kidney accumulation of Cy7-labeled BiPPB and a scrambled peptide control were visualized and quantified using in vivo computed tomography - fluorescence molecular tomography (CT-FMT), ex vivo fluorescence reflectance imaging (FRI) and microscopy. This was done in three different mouse models of kidney fibrosis: the unilateral ischemia-reperfusion injury (I/R), the adenine-induced nephropathy (Adenine) and a transgenic model with constitutive PDGFR-β activation specifically in renal mesenchymal cells leading to increased PDGFR-β expression and expansion of PDGFR-β+ cells (Mutant). In the latter model, we also monitored pharmacological PDGFR-β inhibition with imatinib. Kidney fibrosis and therapeutic efficacy findings were verified by immunohistochemistry (IHC). Results In vivo 3D CT-FMT imaging and ex vivo 2D FRI showed strong accumulation in fibrotic kidneys for Cy7-labeled BiPPB, whereas only moderate amounts accumulated in the contralateral (I/R) and healthy (Adenine) kidney over 48 hours after i.v. injection. The accumulation of the scrambled BiPPB was also significantly lower compared to the specific BiPPB. In transgenic Mutant mice with specific activation of mesenchymal cells and primary renal scaring significantly more Cy7-BiPPB accumulated in the kidneys in comparison to healthy wildtype littermates. In combination with imatinib treatment for three weeks (Mutant imatinib) the accumulation of Cy7-BiPPB was decreased (Fig. 1a-c). This was confirmed ex vivo by significantly increased probe accumulation (2D FRI; Fig. 1d) and PDGFR-β expression (immunohistochemistry (IHC) Fig. 1e) in fibrotic kidneys, which was diminished by imatinib treatment. Conclusion Our findings demonstrate the potential of PDGFR-β-based imaging agents for non-invasive and quantitative monitoring of renal fibrosis progression and therapy response.

TRLS-10. ROVER: A PHASE 1/2 STUDY OF AVAPRITINIB IN PEDIATRIC PATIENTS WITH SOLID TUMORS DEPENDENT ON KIT OR PDGFRA SIGNALING

Abstract Pediatric patients with advanced relapsed/refractory (R/R) solid (including central nervous system [CNS]) tumors have poor prognoses. KIT alterations are common in germ cell tumors and high-grade glioma (HGG); platelet-derived growth factor receptor alpha (PDGFRA) alterations are common in sarcoma and HGG. Diffuse midline gliomas with H3K27-altered (DMG-H3K27-altered) depend on PDGFRA signaling for tumor growth. However, no KIT-/PDGFRA-targeted therapies are currently approved for pediatric patients with R/R solid or CNS tumors, including DMG-H3K27-altered. The selective KIT and PDGFRA inhibitor avapritinib has demonstrated potent activity against KIT activation-loop (exon 17) and juxtamembrane (exon 11) mutants (IC50&amp;lt;2 nM), and PDGFRA activation-loop (D842V) mutants (IC50=0.24 nM). Cellular IC50 of wild-type PDGFRA was 95 nM. CNS penetration in preclinical models (steady-state brain-to-plasma ratios from 0.74–1.00) indicates potential for CNS antitumor activity. Avapritinib is approved to treat adults with advanced systemic mastocytosis in the USA, and in Europe after ≥1 prior systemic therapy. Avapritinib is also approved for the treatment of adults with unresectable/metastatic gastrointestinal stromal tumors harboring PDGFRA exon 18 mutations (including D842V) in the USA, and PDGFRA D842V mutations in Europe. ROVER, a 2-part phase 1/2, multicenter, open-label study (NCT04773782), is investigating avapritinib in pediatric patients aged 2 to &amp;lt;18 years with R/R solid tumors dependent on KIT or PDGFRA signaling, including DMG-H3K27-altered. Objectives include safety, efficacy, and pharmacokinetics. Part 1 will enroll ≥12 patients; the primary endpoint is to determine the recommended Part 2 dose (RP2D). Part 2 will enroll ≥25 patients at the RP2D; the primary endpoint is objective response rate per RECIST v1.1 for solid tumors and Response Assessment in Neuro-Oncology for CNS tumors. Avapritinib once daily will be administered in continuous 28-day cycles. Study enrollment is planned at 26 sites in 9 countries, including North America, Europe, and Asia/Pacific.

Mass Spectrometry and Pharmacological Approaches to Measuring Cooption and Reciprocal Activation of Receptor Tyrosine Kinases.

Receptor tyrosine kinases (RTKs) can show extensive crosstalk, directly and indirectly. Elucidating RTK crosstalk remains an important goal in the clinical combination of anti-cancer therapies. Here, we present mass spectrometry and pharmacological approaches showing the hepatocyte growth factor receptor (MET)-promoting tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and other membrane receptors in MET-amplified H1993 NSCLC cells. Conversely, in H292 wt-EGFR NSCLC cells, EGFR promotes the tyrosine phosphorylation of MET. Reciprocal regulation of the EGFR and insulin receptor (IR) was observed in the GEO CRC cells, where inhibition of the EGFR drives tyrosine phosphorylation of the insulin receptor. Similarly, in platelet-derived growth factor receptor (PDGFR)-amplified H1703 NSCLC cells, inhibition of the EGFR promotes the tyrosine phosphorylation of the PDGFR. These RTK interactions are used to illustrate basic principles applicable to other RTK signaling networks. More specifically, we focus on two types of RTK interaction: (1) co-option of one RTK by another and (2) reciprocal activation of one receptor following the inhibition of a distinct receptor.

Mature and Myelinating Oligodendrocytes Are Specifically Vulnerable to Mild Fluid Percussion Injury in Mice.

Myelin loss and oligodendrocyte death are well documented in patients with traumatic brain injury (TBI), as well as in experimental animal models after moderate-to-severe TBI. In comparison, mild TBI (mTBI) does not necessarily result in myelin loss or oligodendrocyte death, but causes structural alterations in the myelin. To gain more insight into the impact of mTBI on oligodendrocyte lineage in the adult brain, we subjected mice to mild lateral fluid percussion injury (mFPI) and characterized the early impact (1 and 3 days post-injury) on oligodendrocytes in the corpus callosum using multiple oligodendrocyte lineage markers (platelet-derived growth factor receptor [PDGFR]-α, glutathione S-transferase [GST]-π, CC1, breast carcinoma-amplified sequence 1 [BCAS1], myelin basic protein [MBP], myelin-associated glycoprotein [MAG], proteolipid protein [PLP], and FluoroMyelin™). Two regions of the corpus callosum in relation to the impact site were analyzed: areas near (focal) and anterior (distal) to the impact site. mFPI did not result in oligodendrocyte death in either the focal or distal corpus callosum, nor impact on oligodendrocyte precursors (PDGFR-α+) and GST-π+ oligodendrocyte numbers. In the focal but not distal corpus callosum, mFPI caused a decrease in CC1+ as well as BCAS1+ actively myelinating oligodendrocytes and reduced FluoroMyelin intensity without altering myelin protein expression (MBP, PLP, and MAG). Disruption in node-paranode organization and loss of Nav1.6+ nodes were observed in both the focal and distal regions, even in areas without obvious axonal damage. Altogether, our study shows regional differences in mature and myelinating oligodendrocyte in response to mFPI. Further, mFPI elicits a widespread impact on node-paranode organization that affects regions both close to and remotely located from the site of injury.

Screening and Analysis of Possible Drugs Binding to PDGFRα: A Molecular Modeling Study.

The platelet-derived growth factor receptor (PDGFR) is a membrane tyrosine kinase receptor involved in several metabolic pathways, not only physiological but also pathological, as in tumor progression, immune-mediated diseases, and viral diseases. Considering this macromolecule as a druggable target for modulation/inhibition of these conditions, the aim of this work was to find new ligands or new information to design novel effective drugs. We performed an initial interaction screening with the human intracellular PDGFRα of about 7200 drugs and natural compounds contained in 5 independent databases/libraries implemented in the MTiOpenScreen web server. After the selection of 27 compounds, a structural analysis of the obtained complexes was performed. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses were also performed to understand the physicochemical properties of identified compounds to increase affinity and selectivity for PDGFRα. Among these 27 compounds, the drugs Bafetinib, Radotinib, Flumatinib, and Imatinib showed higher affinity for this tyrosine kinase receptor, lying in the nanomolar order, while the natural products included in this group, such as curcumin, luteolin, and epigallocatechin gallate (EGCG), showed sub-micromolar affinities. Although experimental studies are mandatory to fully understand the mechanisms behind PDGFRα inhibitors, the structural information obtained through this study could provide useful insight into the future development of more effective and targeted treatments for PDGFRα-related diseases, such as cancer and fibrosis.

The activity and safety of anlotinib for patients with recurrent malignant glioma: A single-center, retrospective study.

e14032 Background: Anlotinib is a novel tyrosine kinase inhibitor that targets vascular endothelial growth factor receptor (VEGFR) 1/2/3, platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptors (FGFR) 1/2/3/4, c-Kit, and Ret. We report results from this retrospective study to determine the efficacy and tolerability of Anlotinib as a treatment for recurrent malignant glioma (rMG). Methods: A total of 26 eligible patients who relapsed from the standard chemoradiotherapy regimen (TMZ and radiotherapy) after surgery because of tumor located in the eloquent brain areas were enrolled in this study between May 2020 and August 2022. Patients were subjected to a concurrent treatment of Anlotinib (12mg qd) until disease progression or intolerable toxicity. Efficacy was evaluated using Response Assessment in Neuro-Oncology criteria for high-grade glioma. Safety was assessed using NCI-CTCAE 5.0. Survival was estimated with the Kaplan-Meier curve and log-rank test. Results: In total, 26 (11 male, 15 female) patients with recurrent malignant glioma were enrolled. The disease control rate was 84.6% (95%CI 36.9%-76.7%) and the objective response rate was 57.7%(95%CI 65.1%-95.6%). The median progress-free survival (PFS) for all patients was 9.0 months (95%CI 2.88-15.19). The most common treatment-related adverse events were hand-foot syndrome (38.5%), Weakness (38.5%), hypertension 30.8%) and there were no Grade ≥3 toxicities. Conclusions: The present study showed that anlotinib had a promising efficacy and favorable tolerability in treat rMG. Further randomized controlled clinical studies are needed to confirm the efficacy of Anlotinib for the treatment of rMG.

A pilot study of lenvatinib plus pembrolizumab in patients with advanced sarcoma.

11517 Background: New treatment options are needed for sarcomas. Lenvatinib (L) is an oral, multi-tyrosine kinase inhibitor with significant inhibitory activity against VEGF receptor (VEFGR) 1-3, FGF receptor (FGFR) 1-3, KIT, platelet-derived growth factor receptor (PDGFR) alpha/beta, and RET. Experimental models suggest that L can favorably alter the tumor immune environment, and the combination of L and pembrolizumab (P) has proven to be synergistic and effective across multiple solid tumor types, providing rationale for this study. Methods: This is a pilot study evaluating the efficacy of L and P in select sarcomas. Patients who had at least 1 prior regimen but ≤ 3 were enrolled into one of five cohorts (n = 10 each) A: leiomyosarcoma (LMS); B: undifferentiated pleomorphic sarcoma (UPS); C: angiosarcoma and epithelioid hemangioendothelioma (EHE); D: synovial sarcoma (SS) and malignant peripheral nerve sheath tumor (MPNST); and E: osteosarcoma (OS) and chondrosarcoma (CS). Patients were treated with an initial 2-week run-in of L 20 mg orally daily. Subsequently, P was administered at 200 mg IV every 21 days. The primary endpoint for each cohort was the best objective response rate (ORR) documented by RECIST v1.1 by 27 weeks. The combination was considered worthy of further study if 2 or more responses were observed in a cohort. Secondary endpoints included progression-free survival (PFS), overall survival, duration of response, and safety of the combination. Archival tissue and on-treatment biopsies were collected. Results: As of January 31st, 2023, cohorts A, D and E have completed accrual. The best response in the LMS cohort was stable disease (SD). In cohort D there were 3 partial responses (PR): 2 SS; 1 radiation associated high grade MPNST. In cohort E one of 6 patients with OS had a PR. To date, of the 6 patients with angiosarcoma, there was 1 PR in a patient with adrenal primary that occurred after the prespecified 27-week timepoint. One of 5 evaluable patients in Cohort B had a PR. Among the 44 patients evaluable for safety, most common AEs were hypertension (56.8%), diarrhea (45.5%), proteinuria (45.5%), fatigue (40.9%), headache (36.4%) and nausea (36.4%). Most common ≥ grade 3 AEs were hypertension (13.6%), dyspnea (6.8%), non-cardiac chest pain (6.8%), syncope (6.8%). Conclusions: A signal of activity was noted in this pilot study for patients with OS, MPNST, angiosarcoma and SS. In the LMS cohort, there were no responses, and PFS was poor. Enrollment to cohorts B and C is ongoing. Clinical trial information: NCT04784247 . [Table: see text]