laser-capture-microdissection-glioma-subregions-for-spatial-molecular

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

University of Michigan Medical School

Purpose: One likely cause of treatment failure in glioblastoma is the persistence of glioma stem-like cells (GSLCs) which are highly resistant to therapies currently employed. We found that CXCL12 has highest expression in glioma cells derived from neural progenitor cells (NPC). The development and molecular signature of NPC-derived glioblastomas were analyzed and the therapeutic effect of blocking CXCL12 was tested.Experimental Design: Tumors were induced by injecting DNA into the lateral ventricle of neonatal mice, using the Sleeping Beauty transposase method. Histology and expression of GSLC markers were analyzed during disease progression. Survival upon treatment with pharmacologic (plerixafor) or genetic inhibition of CXCR4 was analyzed. Primary neurospheres were generated and analyzed for proliferation, apoptosis, and expression of proteins regulating survival and cell-cycle progression.Results: Tumors induced from NPCs display histologic features of human glioblastoma and express markers of GSLC. In vivo, inhibiting the CXCL12/CXCR4 signaling axis results in increased survival of tumor-bearing animals. In vitro, CXCR4 blockade induces apoptosis and inhibits cell-cycle progression, downregulates molecules regulating survival and proliferation, and also blocks the hypoxic induction of HIF-1α and CXCL12. Exogenous administration of CXCL12 rescues the drug-induced decrease in proliferation.Conclusions: This study demonstrates that the CXCL12/CXCR4 axis operates in glioblastoma cells under hypoxic stress via an autocrine-positive feedback mechanism, which promotes survival and cell-cycle progression. Our study brings new mechanistic insight and encourages further exploration of the use of drugs blocking CXCL12 as adjuvant agents to target hypoxia-induced glioblastoma progression, prevent resistance to treatment, and recurrence of the disease. Clin Cancer Res; 23(5); 1250-62. ©2016 AACR.

Survival and Proliferation of Neural Progenitor-Derived Glioblastomas Under Hypoxic Stress is Controlled by a CXCL12/CXCR4 Autocrine-Positive Feedback Mechanism.

Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro, inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy.

CXCR4 increases in-vivo glioma perivascular invasion, and reduces radiation induced apoptosis: A genetic knockdown study.

Tumors are associated with expansion of immunosuppressive cells such as tumor associated macrophages (TAMs), regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs). These cells promote tumor growth, angiogenesis, metastasis and immune escape. Cancer patients frequently present symptoms such as anemia, leukocytosis and/or cytopenia; associated with poor prognosis. To uncover tumor-mediated hematopoietic abnormalities and identify novel targets that can be harnessed to improve tumor-specific immune responses, we investigated the hematopoietic stem and progenitor cell compartment in melanoma bearing mice. We show that melanoma growth results in expansion of myeloid lineages such as MDSCs, macrophages and DCs along with a reduction in mature RBCs and platelets. Mature B lymphocytes in the blood and BM of melanoma mice were also reduced. Mice bearing melanoma showed extramedullary hematopoiesis in the spleen. Increased expansion of myeloid lineages occurred directly at the level of stem and progenitor cells. The reduction in mature B lymphocytes resulted from a block at the Pro-B cell stage in the bone marrow. Addition of recombinant IL-3 to bone marrow cells resulted in the expansion of committed myeloid progenitors including common myeloid precursors, granulocyte-monocyte precursors and megakaryocyte-erythrocyte precursors. , IL-3 receptor stimulation in melanoma bearing mice using an IL-3 antibody also resulted in a robust expansion of committed myeloid progenitors and hematopoietic stem cells. Collectively our findings demonstrate that tumor growth plays a pivotal role in reprogramming the host immune system by impacting hematopoiesis directly at the level of stem cell compartment.

Melanoma induced immunosuppression is mediated by hematopoietic dysregulation.

Glioblastoma (GBM) is an aggressive and highly vascular tumor with median survival below 2 years. Despite advances in surgery, radiotherapy, and chemotherapy, survival has improved modestly. To combat glioma vascular proliferation, anti-angiogenic agents targeting vascular endothelial growth factor (VEGF) were introduced. Preclinically these agents were effective, yet they did not improve overall survival in phase III trials. We tested the hypothesis that ganciclovir (GCV)-mediated killing of proliferating endothelial cells expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) would have direct antitumor effects, and whether vessel ablation would affect the antitumor activity of anti-VEGF antibodies and radiotherapy.

Molecular ablation of tumor blood vessels inhibits therapeutic effects of radiation and bevacizumab.

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor, for which there is no cure. Treatment effectiveness for GBM has been limited due to tumor heterogeneity, an immunosuppressive tumor microenvironment (TME), and the presence of the blood-brain barrier, which hampers the transport of chemotherapeutic compounds to the central nervous system (CNS). High-density lipoprotein (HDL)-mimicking nanodiscs hold considerable promise to achieve delivery of bioactive compounds into tumors. Herein, we tested the ability of synthetic HDL nanodiscs to deliver chemotherapeutic agents to the GBM microenvironment and elicit tumor regression. To this end, we developed chemo-immunotherapy delivery vehicles based on sHDL nanodiscs loaded with CpG, a Toll-like receptor 9 (TLR9) agonist, together with docetaxel (DTX), a chemotherapeutic agent, for targeting GBM. Our data show that delivery of DTX-sHDL-CpG nanodiscs into the tumor mass elicited tumor regression and antitumor CD8 T cell responses in the brain TME. We did not observe any overt off-target side effects. Furthermore, the combination of DTX-sHDL-CpG treatment with radiation (IR), which is the standard of care for GBM, resulted in tumor regression and long-term survival in 80% of GBM-bearing animals. Mice remained tumor-free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of anti-GBM immunological memory. Collectively, these data indicate that sHDL nanodiscs constitute an effective drug delivery platform for the treatment of GBM, resulting in tumor regression, long-term survival, and immunological memory when used in combination with IR. The proposed delivery platform has significant potential for clinical translation.

High-Density Lipoprotein-Mimicking Nanodiscs for Chemo-immunotherapy against Glioblastoma Multiforme.

Patients with glioma whose tumors carry a mutation in isocitrate dehydrogenase 1 (IDH1) are younger at diagnosis and live longer.  mutations co-occur with other molecular lesions, such as 1p/19q codeletion, inactivating mutations in the tumor suppressor protein 53 ) gene, and loss-of-function mutations in alpha thalassemia/mental retardation syndrome X-linked gene (). All adult low-grade gliomas (LGGs) harboring ATRX loss also express the IDH1 mutation. The current molecular classification of LGGs is based, partly, on the distribution of these mutations. We developed a genetically engineered mouse model harboring IDH1,  and  inactivating mutations, and activated NRAS G12V. Previously, we established that ATRX deficiency, in the context of wild-type IDH1, induces genomic instability, impairs nonhomologous end-joining DNA repair, and increases sensitivity to DNA-damaging therapies. In this study, using our mouse model and primary patient-derived glioma cultures with IDH1 mutations, we investigated the function of IDH1 in the context of TP53 and ATRX loss. We discovered that IDH1 expression in the genetic context of  and  gene inactivation (i) increases median survival in the absence of treatment, (ii) enhances DNA damage response (DDR) via epigenetic up-regulation of the ataxia-telangiectasia-mutated (ATM) signaling pathway, and (iii) elicits tumor radioresistance. Accordingly, pharmacological inhibition of ATM or checkpoint kinases 1 and 2, essential kinases in the DDR, restored the tumors' radiosensitivity. Translation of these findings to patients with IDH1 glioma harboring TP53 and ATRX loss could improve the therapeutic efficacy of radiotherapy and, consequently, patient survival.

IDH1-R132H acts as a tumor suppressor in glioma via epigenetic up-regulation of the DNA damage response.

High grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. FYN tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinases pathway and is overexpressed in human gliomas. FYN's role in vivo in glioma growth remains unknown. We investigated whether FYN regulates glioma initiation, growth and invasion.

FYN tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates anti-glioma immune responses.

: The field of neuro-oncology has experienced significant advances in recent years. More is known now about the molecular and genetic characteristics of glioma than ever before. This knowledge leads to the understanding of glioma biology and pathogenesis, guiding the development of targeted therapeutics and clinical trials. The goal of this review is to describe the state of basic, translational, and clinical research as it pertains to biological and synthetic pharmacotherapy for gliomas.: Challenges remain in designing accurate preclinical models and identifying patients that are likely to respond to a particular targeted therapy. Preclinical models for therapeutic assessment are critical to identify the most promising treatment approaches.: Despite promising new therapeutics, there have been no significant breakthroughs in glioma treatment and patient outcomes. Thus, there is an urgent need to better understand the mechanisms of treatment resistance and to design effective clinical trials.

Prospects of biological and synthetic pharmacotherapies for glioblastoma.

Despite advances in uncovering the molecular mechanisms that mediate glioma progression and the implementation of novel therapeutic modalities, patients' prognosis remains dismal. This is due to both systemic and local tumor induced immune suppression. We are particularly interested in the role played by infiltrating immunosuppressive myeloid derived suppressor cells (MDSCs) in the glioma tumor microenvironment (TME). This immunosuppressive TME also interferes with the effectiveness of immunotherapies against glioma. Development of multipronged treatment approaches is imperative when aiming to generate a robust anti-glioma immune response. Evaluating the inhibitory potential of MDSCs within the TME is an important aspect for developing effective treatments for glioma. Herein, we discuss methodology to assess the inhibitory effects of MDSCs isolated from the TME using a mouse glioma model.

Functional assay to assess T-cell inhibitory properties of myeloid derived suppressor cells (MDSCs) isolated from the tumor microenvironment of murine glioma models.

Gliomas are the most common malignant brain tumors in the pediatric population. Even though great efforts have been made to understand their distinctive molecular characteristics, there has not been any improvements in the median survival in decades. In children, high-grade glial tumors have a median survival of 9-15 months. It has recently been demonstrated that pediatric high-grade gliomas (pHGG) are biologically and molecularly different from the adult counterparts, which could explain why conventional treatments universally fail. The development of an in vivo pHGG model harboring the specific genetic alterations encountered in pediatric gliomas is imperative in order to study the molecular basis that drives the progression and aggressiveness of these tumors. It would also enable harnessing these results for the development of novel therapeutic approaches. Our lab has implemented a method to induce brain tumors using transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mouse brain. One of the main advantages of this method is that tumors are induced by altering the genome of the host cells, allowing us to recapitulate the salient features of the human disease. In this chapter we describe a method to isolate two cell populations from tumors generated in situ in mice, i.e., one population enriched in tumor cells and another population enriched in CD45+ cells. We also present methodologies as to how tumor infiltrating immune cells can be phenotypically characterized using flow cytometry.

Isolation and characterization of immune cells from the tumor microenvironment of genetically engineered pediatric high-grade glioma models using the sleeping beauty transposon system.

Article	Year
Survival and Proliferation of Neural Progenitor-Derived Glioblastomas Under Hypoxic Stress is Controlled by a CXCL12/CXCR4 Autocrine-Positive Feedback Mechanism. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 27542769	2017
CXCR4 increases in-vivo glioma perivascular invasion, and reduces radiation induced apoptosis: A genetic knockdown study. Oncotarget\| PubMed ID: 27863376	2016
Melanoma induced immunosuppression is mediated by hematopoietic dysregulation. Oncoimmunology\| PubMed ID: 29399415	2018
Molecular ablation of tumor blood vessels inhibits therapeutic effects of radiation and bevacizumab. Neuro-oncology\| PubMed ID: 29660022	2018
High-Density Lipoprotein-Mimicking Nanodiscs for Chemo-immunotherapy against Glioblastoma Multiforme. ACS nano\| PubMed ID: 30721028	2019
IDH1-R132H acts as a tumor suppressor in glioma via epigenetic up-regulation of the DNA damage response. Science translational medicine\| PubMed ID: 30760578	2019
FYN tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates anti-glioma immune responses. Neuro-oncology\| PubMed ID: 31950181	2020
Prospects of biological and synthetic pharmacotherapies for glioblastoma. Expert opinion on biological therapy\| PubMed ID: 31959027	2020
Functional assay to assess T-cell inhibitory properties of myeloid derived suppressor cells (MDSCs) isolated from the tumor microenvironment of murine glioma models. Methods in enzymology\| PubMed ID: 32000897	2020
Isolation and characterization of immune cells from the tumor microenvironment of genetically engineered pediatric high-grade glioma models using the sleeping beauty transposon system. Methods in enzymology\| PubMed ID: 32000905	2020

Padma Kadiyala

Biography

JoVE Journal Publications

Other Publications