Stem Cell Assay Research Articles

Abstract 3682: Therapeutic targeting of stem cell self-renewal in childhood medulloblastoma: Strategies for blocking recurrence

Abstract Introduction: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Group 3 MB patients face the highest incidence of metastasis and poor overall patient survival. The early onset and highly aggressive nature of MB suggest a stem cell origin, where a highly self-renewing transformed cell of the postnatal cerebellum drives MB tumorigenesis. In this work, we explore how WNT signaling and other essential drivers of self-renewal, BMI1 and MSI1, promote MB progression. We subsequently generate new strategies to therapeutically target mechanisms of MB stem cell self-renewal that drive treatment resistance and relapse in Group 3 MB. Experimental procedures: We apply stem cell assays, patient-derived human-mouse xenograft (PDX) models, and genomic and bioinformatic profiling of recurrent patient-derived MB. Our established brain tumor initiating cell (BTIC) model provides an excellent tool for the examination of developmental pathways implicated in MB. New Unpublished Data: A small molecule Bmi1 inhibitor, PTC-028, induced a remarkable decrease in self-renewal as well as reduction of local and spinal metastatic disease in recurrent MB, which is striking as no prior drug has shown efficacy against recurrent Group 3 MB. Although mouse and human neural stem cells (NSCs) express Bmi1 and are mildly sensitive to Bmi1 inhibitors, no significant toxicity was observed in either mouse or human NSCs upon PTC-028 treatment, at doses that induced efficacious killing of MB cells. Another novel therapeutic paradigm includes activating Wnt signaling in otherwise non-Wnt MB, which abrogates self-renewal and tumorigenicity of these highly aggressive tumors. For safe and non-toxic activation of Wnt in preclinical models, we identified L807mts, a novel inhibitor that functions through a substrate-to-inhibitor conversion mechanism within the catalytic site of GSK. A final therapeutic strategy to target self-renewal lies in the discovery of the targetable MB-specific interactome of the RNA binding protein (RBP) Musashi1, another key regulator of stem cell self-renewal. Msi1 is overexpressed in Group 3 MB compared to normal cerebellum, and is associated with poor patient prognosis. shRNA knockdown of Msi1 decreased the self-renewal capacity of MB stem cells and significantly decreased tumor burden and increased survival in our PDX model. Finally, comparative eCLIP (enhanced cross-linking and immunoprecipitation) of MB stem cells and normal NSCs, combined with mass spectrometry and RNA-sequencing of shMsi1 MB cells, has elucidated novel therapeutic targets in the RBP interactome of Msi1 Conclusion: Characterization and therapeutic targeting of self-renewal mechanisms unique to MB BTICs may provide an opportunity to limit treatment-resistant stem cell populations from driving patient relapse in recurrent Group 3 MB, a disease currently lacking any targeted therapies. Note: This abstract was not presented at the meeting. Citation Format: David Bakhshinyan, Michelle Kameda-Smith, Branavan Manoranjan, Ashley Adile, Chitra Venugopal, Sheila Kumari Singh. Therapeutic targeting of stem cell self-renewal in childhood medulloblastoma: Strategies for blocking recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3682.

Cancer Stem Cell Assay Directed Chemotherapy in Recurrent Platinum Resistant Ovarian Cancer

Cancer Stem Cell Assay Directed Chemotherapy in Recurrent Platinum Resistant Ovarian Cancer

Photosensitive Hydrogel Creates Favorable Biologic Niches to Promote Spinal Cord Injury Repair.

Photochemistry is considered to be a promising strategy for hydrogels to mimic the complex and dynamic properties of natural extracellular matrix. However, it is seldom applied in 3D tissue engineering and regenerative medicine due to the attenuation of light. In this study, phenyl azide photchemistry and optical fiber technology are first used to localize adhesive protein on the inner surface of the nerve guidance conduit in a 3D hydrogel scaffold. In vitro coculture assay of neural stem cells (NSCs) shows that photoimmobilization of collagen significantly improves the adhesion and survival of NSCs in the conduit, and exhibits synergistic effect with the sustainable release of growth factor. After implantation in transected spinal cord, the optimized hydrogel scaffold is found to improve the locomotion recovery of rats 12 weeks after spinal cord injury (SCI). Histological analysis suggests that the designed hydrogel scaffold provides a favorable biological niche for neuronal regeneration, thus producing directional neuron tissue and promoting the repair of SCI. This study demonstrates a promising hydrogel scaffold for SCI repair and provides the first understanding of the photoimmobilization of adhesive protein in a 3D hydrogel conduit concerning its functions on spinal cord tissue restoration.

Long-Term Combinatorial Exposure to Trichloroethylene and Inorganic Arsenic in Genetically Heterogeneous Mice Results in Renal Tubular Damage and Cancer-Associated Molecular Changes.

Trichloroethylene (TCE) and inorganic arsenic (iAs) are environmental contaminants that can target the kidney. Chronic exposure to TCE is associated with increased incidence of renal cell carcinoma, while co-exposure to TCE and iAs likely occurs in exposed human populations, such as those near Superfund sites. In order to better understand the kidney health consequences of TCE and/or iAs exposure, a genetically heterogeneous mouse population derived from FVB/NJ and CAST/EiJ mouse strains and deficient for multidrug resistance genes (Abcb1atm1Bor, Abcb1btm1Bor) was chronically exposed for 52-weeks to varying concentrations of TCE and iAs. Although no exposure group resulted in primary renal cell tumors, kidneys from exposed mice did have significant increases in histologic and biochemical evidence of renal tubular disease with each toxicant alone and with combined exposure, with males having significantly higher levels of damage. Although no added increase in tubular disease was observed with combination exposure compared to single toxicants, molecular changes in kidneys from mice that had the combined exposure were similar to those previous observed in an embryonic stem cell assay for the P81S TCE-induced renal cell carcinoma mutation in the Von Hippel-Lindau syndrome (VHL) gene. While this model more accurately reflects human exposure conditions, development of primary renal tumors observed in humans following chronic TCE exposure was not reproduced even after inclusion of genetic heterogeneity and co-carcinogenic iAs.

Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

The role of stem cell metabolites derived from placenta for skin regeneration: An in vitro study

Background: The role of stem cells in skin aging is to repair injured tissue or replace other cells in programmed cell death. Stem cell metabolites are rich in growth factors including IL-10, IL-4, EGF, GM-CSF, and TGF-β that can induce the skin production of protein and elastic fbers, leading to the improvement of skin appearance. Aim: This study aimed to assess the characteristics of stem cell metabolites in vitro. Methods: Cytotoxicity assay was performed using MTT reagents and optical cell densities were determined using ELISA reader to fnd the percentage of living cells. Cytokine detection assay was performed by analyzing the cytokine levels in the peripheral blood mononuclear cell (PBMC) and mesenchymal stem cell (MSC) using ELISA. Apoptosis assay was performed using the double staining method with the markers identifed were Hsp70, p53, and caspase-3. Results: All samples showed the percentage of living cells that exceed 70%. Cytokine detection assay showed a decrease of IL- 12 and IFN-γ in both PBMC and MSC groups. The apoptosis assay of human adipose mesenchymal stem cells using a fluorescence microscope showed most of the green light was lost in control cells without metabolites. We found that the expressions of Hsp70 were increased while the expression of p53 and caspase-3 were decreased in the stem cell metabolites samples. Conclusion: These results showed that stem cell metabolites are non-toxic, do not cause a systemic immune response to surrounding tissue, and able to inhibit the occurrence of apoptosis.

Impact of the Trivedi Effect ® - Energy of Consciousness Healing on the Proliferation of Plant, Mouse and Human Stem Cells

The objective of the present study was to evaluate the effect of Biofield Energy Healing on the plant callus, mouse and human derived Mesenchymal Stem Cells (MSCs) for their cell count and cellular proliferation. All the three type stem cells and DMEM media for the stem cell culture were divided into two parts, one received Biofield Energy Treatment (known as The Trivedi Effect®-Energy of Consciousness Healing Treatment) by a renowned Biofield Energy Healer, Mahendra Kumar Trivedi through the Healer’s unique Energy Transmission process remotely and defined as Biofield Energy Treated (BT) group. On day 9 after treatment, the plant callus flasks of mandukparni, amla, and Centella were observed and visualized which showed an increased percentage in weight of the callus by 11.4%, 24.9%, and 51.7%, respectively as compared with the untreated groups. In addition, stem cell assay in murine bone marrow derived Mesenchymal Stem Cells (MSCs) showed an increased cellular proliferation by 115.2%, 127.6%, 161.9%, and 123.6%, in Biofield Energy Treated DMEM in the passage 1, 2, 3, and 4, respectively. Similarly, human derived Mesenchymal Stem Cells, Adipose-Derived (AD-MSC) cells showed a significant increased cell count by 139%, 119%, and 182% in the Biofield Energy Treated DMEM group with the passage 1, 2, and 3, respectively. Similarly, bone marrow (BM-MSC) cells showed a significant increased cell count by 102%, 170%, and 141% in the Biofield Energy Treated DMEM group with the passage 1, 2, and 3, respectively. However, BrdU assay for % cellular proliferation in the AD-MSC cells showed a significant increased cellular proliferation by 122%, 120%, and 184% in the passage 1, 2, and 3, respectively. On the other hand, BM-MSC cells showed a significant increased cellular proliferation by 125%, 146%, and 157% in the passage 1, 2, and 3, respectively. Hence, the results suggest that there was a significant growth in plant callus weight, mouse and human stem cell growth and proliferation after The Trivedi Effect®-Energy of Consciousness Treatment. It can be concluded that The Trivedi Effect® can be used as a complementary and alternate therapy for the stem cells regeneration, improvement of tissue injury that might be every effective against various autoimmune human diseases such as arthritis, osteoarthritis, and different neurodegenerative disorders.

Combining stem cell-derived hepatocytes with impedance sensing to better predict human drug toxicity

ABSTRACTBackground: The liver plays a central role in human drug metabolism. To model drug metabolism, the major cell type of the liver, the hepatocyte, is commonly used. Hepatocytes can be derived from human and animal sources, including pluripotent stem cells. Cell-based models have shown promise in modeling human drug exposure. The assays used in those studies are normally ‘snap-shot’ in nature, and do not provide the complete picture of human drug exposure.Research design and methods: In this study, we employ stem cell-derived hepatocytes and impedance sensing to model human drug toxicity. This impedance-based stem cell assay reports hepatotoxicity in real time after treatment with compounds provided by industry.Results: Using electric cell-substrate impedance Sensing (ECIS), we were able to accurately measure drug toxicity post-drug exposure in real time and more quickly than gold standard biochemical assays.Conclusions: ECIS is robust and non-destructive methodology capable of monitoring human drug exposure with superior performance to current gold standard ‘snapshot’ assays. We believe that the methodology presented within this article could prove valuable in the quest to better predict off-target effects of drugs in humans.

Sirolimus Augments Hematopoietic Stem and Progenitor Cell Recovery from Hematopoietic Stress

Sirolimus is an approved drug to prevent solid organ transplant rejection and to treat lymphangioleiomyomatosis. Sirolimus also has been employed in a broad range of clinical circumstances, including induction of tolerance in nonmyeloablative HLA-mismatched bone marrow (BM) transplantation and treatment of graft-versus-host disease following allogeneic stem cell transplantation. Sirolimus suppresses mammalian target of rapamycin (mTOR), a serine-threonine protein kinase that responds to multiple signals to regulate cellular metabolism, survival, and growth. We recently reported the application of sirolimus on murine models of immune-mediated BM failure (Feng X, Haematologica, 2017;102:1691-1703) in which sirolimus alleviated pancytopenia and attenuated BM destruction by depleting active cytotoxic T cells and expanding regulatory T cells. Controls for these experiments suggested that sirolimus might also act directly to preserve hematopoietic stem and progenitor cells (HSPCs) in non-immune BM damage and hematopoietic cell injury. In the current study, we systematically tested the effects of sirolimus on HSPC regeneration following irradiation and exposure to cytotoxic drugs. B6 mice subjected to 5 Gys total body irradiation (TBI) showed large declines in HSPC numbers, which were abrogated by sirolimus at 2 mg/Kg/day by intraperitoneal (i.p.) injection for 13 days: we observed significant increases (2-4-fold) in the proportions and absolute numbers of HSPCs relative to TBI controls when animals were evaluated at 14 days (Fig. 1A). Sirolimus did not alter peripheral blood cell counts or BM cellularity. Functionally, BM cells from sirolimus-treated TBI mice contained 2-3-fold more colonies than did marrow of TBI controls. In a competitive repopulation stem cell assay, recipients of sirolimus-treated TBI donor BM cells had enhanced donor engraftment, as measured in recipient animals' peripheral blood and BM. There was also an increased recovery of donor HSPCs in recipient BM, relative to recipients of TBI-only donor BM cells, evaluated at 7 months after donor cell infusion. In other experiments, sirolimus at the same dose following busulfan (10 mg/kg i.p. three times weekly for 10 injections; Fig. 1B) or 5-fluorouracil (150 mg/Kg on days 0, 15, and 29 respectively; Fig. 1C) induced similar improved HSPC regeneration. Sirolimus-mediated HSPC regeneration was associated with c-Kitup-regulation in BM cells. In a PCR-based transcriptome assay of hematopoiesis genes performed on sorted Lin-CD150+ BM cells, sirolimus-treated TBI mice showed a 90% increase in c-Kit mRNA relative to TBI controls. Expression of other stem cell and transcription factor genes, including Cd34, Gata2,Angpt1, and Etv6, was also increased, while expression of T cell differentiation and Notch signaling genes, such as Cd2, Cd4, Cd8a, Tnfsf11, Jag1a, and Il-1a, was decreased in sirolimus-treated TBI mice relative to TBI controls. In parallel experiments, sirolimus had no effect on HSPCs in steady-state B6 mice that were not subjected to hematopoietic injury. Sirolimus stimulated HSPC expansion in mice carrying the Wv mutation in the c-Kit locus (Fig. 1D). To test the potential utility of sirolimus in humans, we pre-irradiated human BM CD34+ cells with 5 Gys to induce injury, then transplanted the radiated cells into immunodeficient NSG mice, followed by sirolimus treatment (2 mg/Kg/day i.p. for 4 injections). Sirolimus significantly increased engraftment of human CD45+ cells in recipient blood and BM at 5 weeks relative to the mice without sirolimus treatment (Fig. 1E). In summary, sirolimus augments c-Kit expression and HSPC recovery in response to hematopoietic stress. This novel functional property detected in murine models and xenotransplantation of human cells suggests potential clinical applicability of sirolimus to ameliorate hematopoietic cell injury and accelerate HSPC regeneration after BM hematopoietic injury. [Display omitted] DisclosuresYoung:CRADA with Novartis: Research Funding; GlaxoSmithKline: Research Funding; National Institute of Health: Research Funding.

Neural Differentiation of Mouse Neural Stem Cells as a Tool to Assess Developmental Neurotoxicity of Drinking Water in Taihu Lake

In this study, we used neural stem cells (NSCs) as a toxicology tool to assess the potential developmental neurotoxicity of drinking water from Taihu Lake. We found that the condensed drinking water could inhibit the proliferation and differentiation of NSCs, especially the tap water. Inductively coupled plasma mass spectrometry and high-performance liquid chromatography analysis showed that nickel was detected in the tap water with a high concentration. Our study revealed that nickel could inhibit NSCs proliferation and differentiation, which is induced not only by the intracellular reactive oxygen species generation, but also by the protein levels upregulation of p-c-Raf, p-MEK1/2 and p-Erk1/2 through the axon guidance signal pathways. These findings will provide a new way of research insight for investigation of nickel-induced neurotoxicity. Meanwhile, our test method confirmed the feasibility and reliability of stem cell assays for developmental neurotoxicity testing.

C-Terminal Residue of Ultrashort Peptides Impacts on Molecular Self-Assembly, Hydrogelation, and Interaction with Small-Molecule Drugs

Single molecular changes on a tripeptide can have dramatic effects on their self-assembly and hydrogelation. Herein, we explore C-terminal residue variation on two consistent ultrashort peptide backbones, i.e. acetylated-Leu-Ile-Val-Ala-Gly-Xaa and acetylated-Ile-Val-Xaa (Xaa = His, Arg, Asn). The objective of this study is to identify candidates that can form hydrogels for small-molecule drug (SMD) delivery. Haemolysis and cytotoxicity (with human adipose-derived mesenchymal stem cells) assays showed that the new soluble peptides (Xaa = His, Arg) are cytocompatible. Gelation studies showed that all but acetylated-Ile-Val-Arg could gel under physiological conditions. Longer peptidic backbones drive self-assembly more effectively as reflected in field emission scanning electron microscopy (FESEM) and circular dichroism spectroscopy studies. Rheological studies revealed that the resultant hydrogels have varying stiffness and yield stress, depending on the backbone and C-terminal residue. Visible spectroscopy-based elution studies with SMDs (naltrexone, methotrexate, doxorubicin) showed that besides the C-terminal residue, the shape of the SMD also determines the rate and extent of SMD elution. Based on the elution assays, infrared spectroscopy, and FESEM, we propose models for the peptide fibril-SMD interaction. Our findings highlight the importance of matching the molecular properties of the self-assembling peptide and SMD in order to achieve the desired SMD release profile.

Integrin-Rac signalling for mammary epithelial stem cell self-renewal

BackgroundStem cells are precursors for all mammary epithelia, including ductal and alveolar epithelia, and myoepithelial cells. In vivo mammary epithelia reside in a tissue context and interact with their milieu via receptors such as integrins. Extracellular matrix receptors coordinate important cellular signalling platforms, of which integrins are the central architects. We have previously shown that integrins are required for mammary epithelial development and function, including survival, cell cycle, and polarity, as well as for the expression of mammary-specific genes. In the present study we looked at the role of integrins in mammary epithelial stem cell self-renewal.MethodsWe used an in vitro stem cell assay with primary mouse mammary epithelial cells isolated from genetically altered mice. This involved a 3D organoid assay, providing an opportunity to distinguish the stem cell- or luminal progenitor-driven organoids as structures with solid or hollow appearances, respectively.ResultsWe demonstrate that integrins are essential for the maintenance and self-renewal of mammary epithelial stem cells. Moreover integrins activate the Rac1 signalling pathway in stem cells, which leads to the stimulation of a Wnt pathway, resulting in expression of β-catenin target genes such as Axin2 and Lef1.ConclusionsIntegrin/Rac signalling has a role in specifying the activation of a canonical Wnt pathway that is required for mammary epithelial stem cell self-renewal.

Injectable negatively charged gelatin microsphere-based gels as hemostatic agents for intracavitary and deep wound bleeding in surgery

Gelatin, as natural macromolecular material, has been used in biomedical fields widely. In this study, various injectable gelatins A, B, and their compound AB microsphere-based gels (A-GMGs, B-GMGs and AB-GMGs) were prepared through water-in-oil emulsion method for hemostasis, and the effects of blood coagulation in vitro and surgical hemostasis (a deep liver wound model) in vivo were evaluated. Furthermore, the influences of gelatin sorts, the size of microsphere, zeta potential (ZP) and viscoelastic properties on hemostasis were also assessed. Results showed that the gelatin microspheres (GMs) exhibited smooth surface, good sphericity and the particle size of a rough normal distribution. GMs carried negative charges and their electronegativity was stronger than that of gelatin A (GA) and gelatin B (GB) raw materials. Rheological analysis showed that a decreasing particle size of the microspheres led to stronger gel strength, and solid-like gels were exhibited under low stress conditions and liquid-like gels were exhibited under high stress conditions. The blood clotting time of B-GMGs was within 60 s, which exhibited a significantly higher blood clotting effect compared with control groups. The hemostasis assay in vivo showed that the gels had better hemostatic effect on a deep liver wound bleeding model compared with control groups, especially B-GMGs. However, in vivo and vitro hemostatic experiments, particle size of GMs had no obvious influence on the hemostatic effect of the gels. In addition, the CCK-8 assay of bone marrow mesenchymal stem cells of murine (mMSCs) indicated non-cytotoxicity of GMs for cells. These results demonstrated that the gelatin microsphere-based gels (GMGs) had potential to be an effective hemostatic material for intracavitary and deep wound bleeding in surgery.

In Vitro Self-Renewal Assays for Brain Tumor Stem Cells.

Early development of human organisms relies on stem cells, a population of non-specialized cells that can divide symmetrically to give rise to two identical daughter cells, or divide asymmetrically to produce one identical daughter cell and another more specialized cell. The capacity to undergo cellular divisions while maintaining an undifferentiated state is termed self-renewal and is responsible for the maintenance of stem cell populations during development. In addition, self-renewal plays a crucial role in the homeostasis of developed organism through replacement of defective cells.Similar to their non-malignant counterparts, it has been postulated that tumor cells follow a differentiation hierarchy, with the least differentiated cells termed cancer stem cells (CSCs) at the apex. These tumor stem cells possess the ability to self-renew, have a higher capacity to initiate tumor growth when xenografted into an animal model, and can recapitulate the cell heterogeneity of the tumor they originate from. Hence, further investigation of mechanisms governing the self-renewal in cancer can lead to development of novel therapies targeting CSCs.In this chapter, we described the soft agar assay and the limiting dilution assay (LDA) as two easy-to-implement and inexpensive assays to measure the stemness properties of brain tumor stem cells (BTSCs). These techniques constitute useful tools for the preclinical evaluation of therapeutic strategies targeting BTSCs clonogenicity.

Imetelstat, a telomerase inhibitor, is capable of depleting myelofibrosis stem and progenitor cells

AbstractClinical trials of imetelstat therapy have indicated that this telomerase inhibitor might have disease-modifying effects in a subset of patients with myelofibrosis (MF). The mechanism by which imetelstat induces such clinical responses has not been clearly elucidated. Using in vitro hematopoietic progenitor cell (HPC) assays and in vivo hematopoietic stem cell (HSC) assays, we examined the effects of imetelstat on primary normal and MF HSCs/HPCs. Treatment of CD34+ cells with imetelstat reduced the numbers of MF but not cord blood HPCs (colony-forming unit–granulocyte/macrophage, burst-forming unit–erythroid, and colony-forming unit–granulocyte/erythroid/macrophage/megakaryocyte) as well as MF but not normal CD34+ALDH+ cells irrespective of the patient's mutational status. Moreover, imetelstat treatment resulted in depletion of mutated HPCs from JAK2V617F+ MF patients. Furthermore, treatment of immunodeficient mice that had been previously transplanted with MF splenic CD34+ cells with imetelstat at a dose of 15 mg/kg, 3 times per week for 4 weeks had a limited effect on the degree of chimerism achieved by normal severe combined immunodeficiency repopulating cells but resulted in a significant reduction in the degree of human MF cell chimerism as well as the proportion of mutated donor cells. These effects were sustained for at least 3 months after drug treatment was discontinued. These actions of imetelstat on MF HSCs/HPCs were associated with inhibition of telomerase activity and the induction of apoptosis. Our findings indicate that the effects of imetelstat therapy observed in MF patients are likely attributable to the greater sensitivity of imetelstat against MF as compared with normal HSCs/HPCs as well as the intensity of the imetelstat dose schedule.

Abstract 1911: Uncovering novel targets of recurrent glioblastoma using transcriptomic profiling in a patient-derived xenograft model

Abstract Glioblastoma (GBM) is the most common and aggressive adult primary brain tumor feared for its near uniformly fatal prognosis despite advances in multimodal therapy including surgical resection, chemotherapy and radiation. Poor patient survival due to tumor relapse is thought to be linked to intratumoral heterogeneity (ITH), driven by various environmental cues including chemotherapy and radiation treatment. ITH can be explained at the cellular level by the existence of multiple populations of cancer cells, including cancer stem cells (CSCs), which have acquired stemness properties like self-renewal, proliferation, and multilineage differentiation. In brain tumors, CSCs or brain tumor initiating cells (BTICs), have been shown to be resistant to both chemotherapy and radiation treatment, allowing them to escape therapy and allowing for tumor recurrence. To profile ITH as it evolves through therapy delivery, we have developed a novel and dynamic BTIC patient-derived xenograft (PDX) model of treatment-refractory human GBM, allowing for multimodal profiling of GBM BTICs through tumor engraftment, remission, and recurrence. In this study, we present the transcriptomic profiling at each stage, and novel target selection and validation through CRISPR/Cas9 knockouts, well-established in vitro stem cell assays, and in vivo characterization of their tumor initiation, development, and maintenance properties. Despite the fact that the BTIC population is responsible for GBM recurrence and thus patient demise, it remains a largely unknown landscape. Consequently, therapies that focus on targeting the BTIC compartment within the bulk tumor would provide better treatment and prognosis for patients with brain tumors. The study we present provides a unique therapeutic window into the recurrence of GBM, which drives patient mortality, yet is profiled far less than primary treatment-naïve GBM. Citation Format: Nicolas Yelle, Chirayu Chokshi, Parvez Vora, Kevin R. Brown, Maleeha A. Qazi, Mohini Singh, Jarrett J. Adams, Chitra Venugopal, Sachdev Sidhu, Jason Moffat, Sheila K. Singh. Uncovering novel targets of recurrent glioblastoma using transcriptomic profiling in a patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1911.

An in vitro approach for prioritization and evaluation of chemical effects on glucocorticoid receptor mediated adipogenesis

Rising obesity rates worldwide have socio-economic ramifications. While genetics, diet, and lack of exercise are major contributors to obesity, environmental factors may enhance susceptibility through disruption of hormone homeostasis and metabolic processes. The obesogen hypothesis contends that chemical exposure early in development may enhance adipocyte differentiation, thereby increasing the number of adipocytes and predisposing for obesity and metabolic disease. We previously developed a primary human adipose stem cell (hASC) assay to evaluate the effect of environmental chemicals on PPARG-dependent adipogenesis. Here, the assay was modified to determine the effects of chemicals on the glucocorticoid receptor (GR) pathway. In differentiation cocktail lacking the glucocorticoid agonist dexamethasone (DEX), hASCs do not differentiate into adipocytes. In the presence of GR agonists, adipocyte maturation was observed using phenotypic makers for lipid accumulation, adipokine secretion, and expression of key genes. To evaluate the role of environmental compounds on adipocyte differentiation, progenitor cells were treated with 19 prioritized compounds previously identified by ToxPi as having GR-dependent bioactivity, and multiplexed assays were used to confirm a GR-dependent mode of action. Five chemicals were found to be strong agonists. The assay was also modified to evaluate GR-antagonists, and 8/10 of the hypothesized antagonists inhibited adipogenesis. The in vitro bioactivity data was put into context with extrapolated human steady state concentrations (Css) and clinical exposure data (Cmax). These data support using a human adipose-derived stem cell differentiation assay to test the potential of chemicals to alter human GR-dependent adipogenesis.

Epimorphin regulates the intestinal stem cell niche via effects on the stromal microenvironment.

Stem cell therapy is a potential therapeutic approach for disorders characterized by intestinal injury or loss of functional surface area. Stem cell function and proliferation are mediated by the stem cell niche. Stromal cells such as intestinal subepithelial myofibroblasts (ISEMFs) are important but poorly studied components of the stem cell niche. To examine the role of ISEMFs, we have previously generated mice with deletion of epimorphin ( Epim), an ISEMF protein and member of the syntaxin family of intracellular vesicle docking proteins that regulate cell secretion. Herein we explore the mechanisms for previous observations that Epim deletion increases gut crypt cell proliferation, crypt fission, and small bowel length in vivo. Stem cell-derived crypt culture techniques were used to explore the interaction between enteroids and myofibroblasts from Epim-/- and WT mice. Enteroids cocultured with ISEMFS had increased growth and crypt-like budding compared with enteroids cultured without stromal support. Epim deletion in ISEMFs resulted in increased enteroid budding and surface area compared with cocultures with wild-type (WT) ISEMFs. In primary crypt cultures, Epim-/- enteroids had significantly increased surface area and budding compared with WTs. However, stem cell assays comparing the number of Epim-/- vs. WT colony-forming units after first passage showed no differences in the absence of ISEMF support. Epim-/- vs. WT ISEMFs had increased Wnt4 expression, and addition of Wnt4 to WT cocultures enhanced budding. We conclude that ISEMFs play an important role in the stem cell niche. Epim regulates stem cell proliferation and differentiation via stromal contributions to the niche microenvironment. NEW & NOTEWORTHY The role of subepithelial intestinal myofibroblasts (ISEMFs) in the gut stem cell niche is controversial. We provide novel evidence supporting ISEMFs as important niche contributors. We show that the in vivo intestinal effects of deletion of myofibroblast Epim can be recapitulated in crypt stem cell cultures in vitro. ISEMFs support cocultured stem cell proliferation and enteroid growth, and these effects are augmented by deletion of Epim, a syntaxin that regulates myofibroblast cell secretion.

Abstract P6-07-04: Rapamycin inhibits the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

Abstract Rapamycin, a well-studied mTOR inhibitor, has been demonstrated to inhibit mammary carcinogenesis at multiple stages, including initiation, invasion, and metastasis, in preclinical animal models. Nevertheless, the cancer preventative potential and underlying mechanisms remain unclear, especially in individual breast cancer subtypes like ErbB2/Her2-positive breast cancers. ErbB2 amplification/overexpression is a particular clinical concern because it occurs in approximately one-third of human breast cancers and is associated with poor prognosis. Therefore, we used MMTV-ErbB2 transgenic mice as our model system to test the efficacy of rapamycin in the prevention of ErbB2-mediated mammary tumor development. Our initial data provided proof of concept regarding the anti-cancer effects of rapamycin in vivo. Indeed, rapamycin (1.5 mg/kg/day for 12 days) significantly reduced the volume and weight of syngeneic 78617 cell-derived mammary tumors in MMTV-ErbB2 mice, despite observed decreases in CD4+ and CD8+ immune cells. Since advanced mammary gland development can serve as an indicator of breast cancer risk, we investigated the effects of rapamycin on mammary gland development in MMTV-ErbB2 mice that were treated with low-dose rapamycin (1 mg/kg/day) between weeks 10 and 20 of age. As such, rapamycin significantly attenuated mammary morphogenesis at 20 weeks of age, as indicated by decreased branching density, ductal elongation, and proliferative index of the premalignant mammary glands. Flow cytometric analysis of isolated primary mammary epithelial cells (MECs) was performed using CD24 and CD49f markers to identify MEC populations. We found that rapamycin has a significant impact on MEC stemness based on changes in luminal (CD24highCD49flow), mammary stem cell (MaSC)-enriched (CD24highCD49fhigh), and myoepithelial/basal (CD24low/highCD49fhigh) MEC populations. We also used CD61 and CD49f markers to identify a population enriched with luminal progenitor cells (CD61highCD49fhigh) that was selectively inhibited by rapamycin. Consistent with our flow cytometric analyses, rapamycin inhibited the luminal progenitor cell-enriched population, self-renewal, and anchorage-independent cell growth of primary MECs, as demonstrated by colony-forming cell, mammosphere, and 3D culture assays, respectively. These functional stem cell assays further corroborate that rapamycin suppresses the stemness of primary MECs. Molecular analysis of MECs demonstrated that rapamycin inhibited mTOR signaling, as expected. Importantly, rapamycin also significantly suppressed the receptor tyrosine kinase/ErbB2, estrogen receptor, Wnt/β-catenin, and TGFβ/Smad3 signaling pathways prior to malignant transformation. Collectively, our study provides evidence that rapamycin has potential cancer preventative effects in the mammary glands of MMTV-ErbB2 mice during the premalignant risk window. These rapamycin-induced anti-cancer effects ultimately highlight the promising clinical significance of rapamycin for the prevention and treatment of human ErbB2-overexpressing breast cancers. Citation Format: Yang X, Zhao Q, Parris AB, Howard EW, Zhao M, Guo Z, Xing Y, Ma Z. Rapamycin inhibits the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-04.

Severe hypoxia selects hematopoietic progenitors with stem cell potential from primary Myelodysplastic syndrome bone marrow cell cultures.

Myelodysplastic Syndromes (MDS) are clonal neoplasms where stem/progenitor cells endowed with self-renewal and capable of perpetuating the disease have been demonstrated. It is known that oxygen tension plays a key role in driving normal hematopoiesis and that hematopoietic stem cells are maintained in hypoxic areas of the bone marrow (BM). Hypoxia could also regulate leukemic/dysplastic hematopoiesis. We evaluated the stem cell potential of MDS cells derived from the BM of 39 MDS patients and selected under severe hypoxia. MDS cells rescued from hypoxia-incubated cultures were subjected to stem and progenitor cell assays in vitro, as well as to hematopoietic reconstitution assay in NOD-SCID mice. Incubation in severe hypoxia of cells explanted from MDS patients selected a cell subset endowed with stem cell potential, as determined in vitro. This occurred only from the BM of patients classified as IPSS low/INT-1 risk. Transplantation into NOD-SCID mice confirmed using an in vivo model that severe hypoxia selects a cell subset endowed with stem cell potential from bone marrow mononuclear cells (BMMC). derived from patients belonging to the IPSS low/int-1 risk group. Data here reported show that cells endowed with stem cell potential and capable of adapting to hypoxia and escaping hypoxia-induced apoptosis exist within MDS cell populations.