scholarly journals Endosialin defines human stem Leydig cells with regenerative potential

2020 ◽  
Vol 35 (10) ◽  
pp. 2197-2212
Author(s):  
Kai Xia ◽  
Yuanchen Ma ◽  
Xin Feng ◽  
Rongda Deng ◽  
Qiong Ke ◽  
...  
Keyword(s):  
Single Cell ◽  
Leydig Cells ◽  
Growth Factor Receptor ◽  
Human Testis ◽  
Specific Marker ◽  
Self Renewal ◽  
Immunodeficient Mice ◽  
Single Cell Sequencing

Abstract STUDY QUESTION Is endosialin a specific marker of human stem Leydig cells (SLCs) with the ability to differentiate into testosterone-producing Leydig cells (LCs) in vitro and in vivo? SUMMARY ANSWER Endosialin is a specific marker of human SLCs which differentiate into testosterone-producing LCs in vitro and in vivo. WHAT IS KNOWN ALREADY Human SLCs have been identified and isolated using the marker platelet-derived growth factor receptor α (PDGFRα) or nerve growth factor receptor (NGFR). However, the specificity was not high; thus, LCs and germ cells could be mistakenly sorted as SLCs if PDGFRα or NGFR was used as a marker for human SLCs isolation. STUDY DESIGN, SIZE, DURATION Firstly, we re-evaluated the specificity of PDGFRα and NGFR for SLCs in adult human testes. Then we analysed the previously published single-cell sequencing data and found that endosialin may identify human SLCs. Subsequently, we sorted endosialin+ cells from four human donors and characterized their self-renewal and multipotent properties. To assess whether endosialin+ cells have the potential to differentiate into functional LCs in vitro, these cells were stimulated by differentiation-inducing medium. We next assessed the in vivo regenerative potential of human endosialin+ cells after xenotransplantation into the testes of immunodeficient mice. PARTICIPANTS/MATERIALS, SETTING, METHODS Single-cell sequencing analysis, immunofluorescence and flow cytometry were used to characterize human testis tissues. In vitro colony formation, multipotent differentiation (adipogenic, osteogenic and chondrogenic) and Leydig cell-lineage induction were used to assess stem cell activity. Xenotransplantation into 3-week-old immunodeficient mice was used to determine in vivo regenerative potential. Endpoint measures included testosterone measurements, cell proliferation, immunofluorescence, flow cytometry and quantitative RT–PCR. MAIN RESULTS AND THE ROLE OF CHANCE The results indicate that endosialin is a specific marker of SLCs compared with PDGFRα and NGFR. Additionally, endosialin+ cells isolated from human testes show extensive proliferation and differentiation potential in vitro: their self-renewal ability was inferred by the formation of spherical clones derived from a single cell. Moreover, these cells could differentiate into functional LCs that secreted testosterone in response to LH in a concentration-dependent manner in vitro. These self-renewal and differentiation properties reinforce the proposal that human testicular endosialin+ cells are SLCs. Furthermore, transplanted human endosialin+ cells appear to colonize the murine host testes, localize to peritubular and perivascular regions, proliferate measurably and differentiate partially into testosterone-producing LCs in vivo. LARGE SCALE DATA NA. LIMITATIONS, REASONS FOR CAUTION Owing to the difficulty in collecting human testis tissue, the sample size was limited. The functions of endosialin on SLCs need to be elucidated in future studies. WIDER IMPLICATIONS OF THE FINDINGS A discriminatory marker, endosialin, for human SLCs purification is a prerequisite to advance research in SLCs and logically promote further clinical translation of SLCs-based therapies for male hypogonadism. STUDY FUNDING/COMPETING INTEREST(S) A.P.X. was supported by the National Key Research and Development Program of China (2017YFA0103802 and 2018YFA0107200). C.D. was supported by the National Natural Science Foundation of China (81971314) and the Natural Science Foundation of Guangdong Province, China (2018B030311039). The authors declare no conflict of interest.

scholarly journals Single-cell RNA-seq revealed the role of Alkbh5 in reproduction

2021 ◽  
Author(s):  
Xiaozhong Shen ◽  
Gangcai Xie
Keyword(s):  
Single Cell ◽  
Messenger Rna ◽  
Single Cells ◽  
Rna Seq ◽  
Single Cell Sequencing ◽  
Differential Gene ◽  
Higher Eukaryotes

AbstractN(6)-methyladenosine (m(6)a) is the most common internal modification of messenger RNA (mRNA) in higher eukaryotes. According to previous literature reports, alkbh5, as another demethylase in mammals, can reverse the expression of m(6)a gene in vivo and in vitro. In order to reveal the effect of Alkbh5 deletion on the level of single cells in the testis during spermatogenesis in mice, the data were compared using single-cell sequencing. In this article, we discussed the transcription profile and cell type identification of mouse testis, the expression of mitochondrial and ribosomal genes in mice, the analysis of differential gene expression, and the effects of Alkbh5 deletion, and try to explain the role and influence of Alkbh5 on reproduction at the level of single-cell sequencing.

Ex-Vivo Expansion of Marrow Repopulating Cells by Chromatin Modifying Agents Is Associated with Modulation of Expression of Genes Implicated in HSC Self-Renewal.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1670-1670
Author(s):  
Hiroto Araki ◽  
Kazumi Yoshinaga ◽  
Ronald Hoffman ◽  
Piernicola Boccuni ◽  
Nadim Mahmud
Keyword(s):  
Ex Vivo ◽  
Trichostatin A ◽  
Donor Cell ◽  
Characteristic Functional ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Cell Divisions

Abstract Human hematopoietic stem cells (HSCs) exposed to cytokine combinations in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program which determines the properties of HSC. In order to expand the number of HSC present in a single unit of cord blood (CB) ex vivo, self-renewal type of HSC division must occur. We hypothesize that in vitro culture conditions result in the silencing of genes crucial for HSC maintenance and that silencing of these genes can be circumvented by addition of chromatin modifying agents. We have attempted to reverse the silencing of the genes crucial for HSC self-renewal which apparently occurs during the ex vivo culture by treatment of CD34+ cells with the chromatin modifying agents, 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA). In our current studies, we have investigated the mechanism of expansion of SRC following treatment with chromatin modifying agents in the culture. We demonstrate that all CD34+CD90+ cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to cytokines alone. CD34+CD90+ cells exposed to the chromatin modifying agents are capable of producing greater numbers of primitive multipotential progenitors and also form cobblestone areas. When CD34+CD90+ cells that had undergone extensive number of cell divisions (5–10) in vitro in the presence of cytokines alone were re-isolated by FACS and transplanted into immunodeficient mice, donor cell chimerism was not detectable (0 of 5 mice). By contrast, 5azaD/TSA treated cells that had undergone similar numbers of cell divisions retain their marrow repopulating potential (3 of 6 mice). To test whether chromatin modifying agents treated cells following culture possess long-term in vivo repopulation potential, we have performed secondary NOD/SCID assay. Five of six secondary NOD/SCID mice receiving bone marrow from primary mice engrafted with cells treated with 5azaD/TSA resulted in human cell engraftment, indicating that these cells are capable of secondary reconstitution. To understand the molecular mechanism responsible for the expansion of HSC observed following 5azaD/TSA treatment, we examined transcription levels of several genes and their products (i.e., HOXB4, Bmi-1 and P21) implicated in self-renewal of HSC using real-time quantitative PCR and Western blot. The expression of these genes and their products were up-regulated in CB cells treated with 5azaD/TSA. We have also compared the efficacy of an additional HDAC inhibitor valproic acid (VPA) in order to determine its ability to expand HSC ex vivo. VPA was capable of dramatic expansion of CD34+CD90+ cells as well as progenitor cells but was unable to expand SRC. However, unlike the culture exposed to cytokines alone VPA treatment resulted in maintenance of SRC numbers. Currently, we are investigating key candidate genes accountable for the expansion of SRC using a global microarray approach analyzing cells exposed to various chromatin modifying agents in conjunction with their in vivo functional potential. In summary, our data suggest that the loss of SRC can be circumvented by the use of chromatin modifying agents in the culture which results in a slower rate of cell division and is associated with higher expression of a group of HSC regulatory genes.

Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 248-248
Author(s):  
Xiao Yan ◽  
Heather A. Himburg ◽  
Phuong L. Doan ◽  
Mamle Quarmyne ◽  
Nelson J. Chao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Growth Factor Receptor ◽  
Self Renewal ◽  
Donor Cells ◽  
Growth Factor Receptor Bound

Abstract The mechanisms which regulate HSC regeneration following stress or injury remain poorly understood. Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. Following 550cGy TBI, BM KSL cells were depleted by 7 days post-TBI, whereas KSL cell recovery was evident at day+14. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells. We identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed in regenerating BM KSL cells compared to non-irradiated KSL cells (3.3 fold, p<0.0001). Grb10 is a member of the family of imprinted genes which are predominately expressed in numerous stem cell populations, including embryonic stem cells, skin and muscle stem cells. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) in detected in 7 day culture (p=0.03 and p=0.002, respectively). Furthermore, mice which were competitively transplanted with Grb10-deficient KSL cells had 10-fold decreased donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed > 10-fold deficit in long-term repopulating capacity in Grb10 deficient KSL cells compared to Grb10 expressing KSL cells (p=0.006 for %CD45.1+ donor cells in secondary mice). In order to examine the effect of Grb10-deficiency on HSC fate and hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8-week old Grb10m/+ (1 mutant allele, 1 wild type allele) were found to have 10-fold decreased Grb10 expression in BM lin- cells and had normal range complete blood counts. However, BM CFCs were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006) and competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (1/14, 7% vs. 5/14, 38% of mice with > 0.1% donor CD45.2+ cells). These results suggest that Grb10 regulates HSC self-renewal in vitro and in vivo. Mechanistically, Grb10m/+ mice displayed no alterations in the cell cycle status or frequency of apoptotic cells within BM HSCs compared to Grb10+/+ mice. However, single cytokine functional screening suggested that Grb10 regulates SCF-mediated proliferation of HSCs. Grb10m/+ BM KSL cells generated significantly less CFCs in culture in response to SCF treatment compared to Grb10+/+ KSL cells (p=0.008). Commensurate with this, SCF-mediated activation of mTOR was significantly increased in Grb10m/+ KSL cells compared to that observed in Grb10+/+ KSL cells (p=0.006). These data suggest that cytokine-mediated induction of mTOR signaling, which has been shown to deplete functional HSCs, is antagonized by Grb10, and that Grb10 is necessary to block cytokine-mediated HSC differentiation in vitro and in vivo. Grb10 represents a novel regulator of HSC fate determination and a new mechanistic target to facilitate HSC self-renewal. Studies are underway to determine whether Grb10 is also necessary for HSC regeneration after TBI. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mechanisms of Leukemia Stem Cell Plasticity Revealed By Single Cell Analysis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
R. Grant Rowe ◽  
George Q. Daley ◽  
Trista E. North ◽  
William Marion ◽  
Yana Pikman ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Childhood Leukemia ◽  
Lymphoblastic Leukemia ◽  
Chemotherapy Resistance ◽  
Leukemic Cells ◽  
Publicly Traded ◽  
Immunodeficient Mice

Leukemia stem cells (LSCs) are the subset of leukemic cells that drive leukemia progression, resist therapy, and remain latent to spark disease relapse. LSCs are quantified by efficiency of xenografting immunodeficient mice, a measurement that is predictive of leukemia outcome. Although LSCs have been previously thought to be rare and phenotypically primitive, recent data indicate that LSCs may actually be heterogeneous. Here, we use single-cell transcriptomics combined with limiting dilution xenotransplantation to dissect the ontogeny of MLL-rearranged B-lymphoblastic leukemia (MLL-r B-ALL), an aggressive form of childhood leukemia. Compared to acute myeloid leukemia (AML), LSCs are abundant in MLL-r B-ALL. Recapitulating the unique clinical behavior of this form of leukemia, MLL-r B-ALL cells undergo a B-lymphoid to myeloid lineage switch under chemotherapy pressure consistent with primitive, multipotent transcriptional programs present in LSCs. Although we identify rare, chemotherapy-resistant, primitive LSCs, we also observe LSCs emerging from more differentiated populations. These facultative LSCs self-renew and possess the capability to replenish the full cellular diversity of MLL-r B-ALL. Using CITE-seq, we find that stem cell programs can be fully uncoupled from immunophenotype in MLL-r B-ALL. In mechanistic studies, we find that the phenotypically differentiated LSCs that drive bottom-up reconstitution of the leukemic cellular ontogeny bear signatures of MYC activation and oxidative phosphorylation. We confirm recruitment of these pathways in actively reconstituting, phenotypically differentiated LSCs, and define a pathway by which MYC rewires metabolism in MLL-r B-ALL LSCs. We find that MYC is required for LSC plasticity in vitro and in vivo. Targeting oxidative metabolism impairs LSC engraftment, identifying a potential therapeutic intervention. We conclude that the high LSC content and dual lineage and LSC plasticities of MLL-r B-ALL contribute to its chemotherapy resistance and persistently poor outcomes. Disclosures Shalek: Honeycomb Biotechnologies: Consultancy, Current equity holder in publicly-traded company; Cellarity: Consultancy, Current equity holder in publicly-traded company; Repertoire Immune Medicines: Consultancy, Current equity holder in publicly-traded company; Merck: Consultancy; Orche Bio: Consultancy, Current equity holder in publicly-traded company; Dahlia Biosciences: Consultancy, Current equity holder in publicly-traded company.

scholarly journals The Evidence of Glioblastoma Heterogeneity

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Akio Soeda ◽  
Akira Hara ◽  
Takahiro Kunisada ◽  
Shin-ichi Yoshimura ◽  
Toru Iwama ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Convincing Evidence ◽  
Proliferative Potential ◽  
Phenotypic Characteristics ◽  
Self Renewal ◽  
Clonal Origin ◽  
Epidermal Growth ◽  
Microarray Analyses

Abstract Cancers are composed of heterogeneous combinations of cells that exhibit distinct phenotypic characteristics and proliferative potentials. Because most cancers have a clonal origin, cancer stem cells (CSCs) must generate phenotypically diverse progenies including mature CSCs that can self-renew indefinitely and differentiated cancer cells that possess limited proliferative potential. However, no convincing evidence exists to suggest that only single CSCs are representative of patients' tumors. To investigate the CSCs' diversity, we established 4 subclones from a glioblastoma patient. These subclones were subsequently propagated and analyzed. The morphology, the self-renewal and proliferative capacities of the subclones differed. Fluorescence-activated cell sorting and cDNA-microarray analyses revealed that each subclone was composed of distinct populations of cells. Moreover, the sensitivities of the subclones to an inhibitor of epidermal growth factor receptor were dissimilar. In a mouse model featuring xenografts of the subclones, the progression and invasion of tumors and animal survival were also different. Here, we present clear evidence that a brain tumor contains heterogeneous subclones that exhibit dissimilar morphologies and self-renewal and proliferative capacities. Our results suggest that single cell-derived subclones from a patient can produce phenotypically heterogeneous self-renewing progenies in both in vitro and in vivo settings.

Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Triple Negative ◽  
Growth Factor Receptor ◽  
Engineered Nanoparticles ◽  
Efficacy Evaluation ◽  
Dual Action ◽  
Epidermal Growth ◽  
Laminin 411 ◽  
Immunologic Analysis

Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safetyfor cancer patient's treatment. All-in-one covalent nanodrugs of the third generation have beensynthesized based on a poly(β-L-malic acid) (PMLA) platform, targeting human triple-negativebreast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blockingsynthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumorvascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicityat low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and singleactionprecursor nanoconjugates were assessed under in vitro conditions and in vivo with multipletreatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo withdifferent drugs included blood hematologic and immunologic analysis after multiple intravenousadministrations. The present study demonstrates that the dual-action nanoconju-gate is highlyeffective in preclinical TNBC treatment without side effects, supported by hematologic andimmunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multipletoxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimizedand efficacious for the treatment of cancer patients in the future.

scholarly journals The roles and prognostic significance of ABI1-TSV-11 expression in patients with left-sided colorectal cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yu Zhang ◽  
Zhaohui Zhong ◽  
Mei Li ◽  
Jingyi Chen ◽  
Tingru Lin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Prognostic Significance ◽  
Growth Factor Receptor ◽  
The Cancer Genome Atlas ◽  
Actin Dynamics ◽  
Elevated Expression ◽  
Sw480 Cells ◽  
And Migration

AbstractAbnormally expressed and/or phosphorylated Abelson interactor 1 (ABI1) participates in the metastasis and progression of colorectal cancer (CRC). ABI1 presents as at least 12 transcript variants (TSVs) by mRNA alternative splicing, but it is unknown which of them is involved in CRC metastasis and prognosis. Here, we firstly identified ABI1-TSV-11 as a key TSV affecting the metastasis and prognosis of left-sided colorectal cancer (LsCC) and its elevated expression is related to lymph node metastasis and shorter overall survival (OS) in LsCC by analyzing data from The Cancer Genome Atlas and TSVdb. Secondly, ABI1-TSV-11 overexpression promoted LoVo and SW480 cells adhesion and migration in vitro, and accelerated LoVo and SW480 cells lung metastasis in vivo. Finally, mechanism investigations revealed that ABI1-isoform-11 interacted with epidermal growth factor receptor pathway substrate 8 (ESP8) and regulated actin dynamics to affect LoVo and SW480 cells biological behaviors. Taken together, our data demonstrated that ABI1-TSV-11 plays an oncogenic role in LsCC, it is an independent risk factor of prognosis and may be a potential molecular marker and therapeutic target in LsCC.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy

2021 ◽  
Vol 22 (9) ◽  
pp. 4390
Author(s):  
Jana Horváthová ◽  
Roman Moravčík ◽  
Miroslava Matúšková ◽  
Vladimír Šišovský ◽  
Andrej Boháč ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Progression ◽  
Umbilical Vein ◽  
Cell Metabolism ◽  
High Rate ◽  
Ki 67 ◽  
Immunodeficient Mice ◽  
Inhibition Of Glycolysis

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

scholarly journals MODL-12. DEVELOPMENT OF A NOVEL IMMUNOCOMPETENT MOUSE MODEL FOR DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii413-iii413
Author(s):  
Maggie Seblani ◽  
Markella Zannikou ◽  
Katarzyna Pituch ◽  
Liliana Ilut ◽  
Oren Becher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Growth Factor Receptor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Time Of Application ◽  
Tumor Associated Antigen ◽  
Sarcoma Virus

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor affecting young children. Immunotherapies hold promise however the lack of immunocompetent models recreating a faithful tumor microenvironment (TME) remains a challenge for development of targeted immunotherapeutics. We propose to generate an immunocompetent DIPG mouse model through induced overexpression of interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen overexpressed by glioma cells. A model with an intact TME permits comprehensive preclinical assessment of IL13Rα2-targeted immunotherapeutics. Our novel model uses the retroviral avian leucosis and sarcoma virus (RCAS) for in vivo gene delivery leading to IL13Rα2 expression in proliferating progenitor cells. Transfected cells expressing IL13Rα2 and PDGFB, a ligand for platelet derived growth factor receptor, alongside induced p53 loss via the Cre-Lox system are injected in the fourth ventricle in postnatal pups. We validated the expression of PDGFB and IL13Rα2 transgenes in vitro and in vivo and will characterize the TME through evaluation of the peripheral and tumor immunologic compartments using immunohistochemistry and flow cytometry. We confirmed expression of transgenes via flow cytometry and western blotting. Comparison of survival dynamics in mice inoculated with PDGFB alone with PDGFB+IL13Rα2 demonstrated that co-expression of IL13Rα2 did not significantly affect mice survival compared to the PDGFB model. At time of application, we initiated experiments to characterize the TME. Preliminary data demonstrate establishment of tumors within and adjacent to the brainstem and expression of target transgenes. Preclinical findings in a model recapitulating the TME may provide better insight into outcomes upon translation to clinical application.

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