University of Michigan Ann Arbor

6 ARTICLES PUBLISHED IN JoVE

Medicine

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Anda-Alexandra Calinescu¹, Felipe Javier Núñez¹, Carl Koschmann², Bradley L. Kolb¹, Pedro R. Lowenstein^1,3, Maria G. Castro^1,3

¹Department of Neurosurgery, University of Michigan School of Medicine, ²Department of Pediatrics, Division of Hematology-Oncology, University of Michigan School of Medicine, ³Department of Cell and Developmental Biology, University of Michigan

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Immunology and Infection

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Gregory J. Baker^1,2, Maria G. Castro^1,2, Pedro R. Lowenstein^1,2

¹Department of Neurosurgery, University of Michigan, ²Department of Cell and Developmental Biology, University of Michigan

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Cancer Research

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Flor M. Mendez¹, Felipe J. Núñez^1,2, Rocío I. Zorrilla-Veloz^3,4, Pedro R. Lowenstein^1,2, Maria G. Castro^1,2

¹Department of Cell and Developmental Biology, University of Michigan Medical School, ²Department of Neurosurgery, University of Michigan Medical School, ³Cancer Research Summer Internship Program (CARSIP), Cancer Biology Program, University of Michigan Medical School, ⁴Department of Biology, University of Puerto Rico-Río Piedras Campus

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Cancer Research

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Felipe J. Núñez^1,2, Flor M. Mendez², Maria B. Garcia-Fabiani^1,2, Joaquín Pardo^1,3, Marta Edwards^1,2, Pedro R. Lowenstein^1,2, Maria G. Castro^1,2

¹Department of Neurosurgery, University of Michigan Medical School, ²Department of Cell & Developmental Biology, University of Michigan, ³INIBIOLP, Histology B-Pathology B, School of Medicine, UNLP

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Neuroscience

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

Andrea Comba^1,2,4, Patrick J. Dunn^1,2,4, Phillip E. Kish^1,3, Padma Kadiyala^1,2,4, Alon Kahana³, Maria G. Castro^1,2,4, Pedro R. Lowenstein^1,2,4

¹Dept. of Neurosurgery, University of Michigan Medical School, ²Dept. of Cell and Developmental Biology, University of Michigan Medical School, ³Dept. of Ophthalmology & Visual Science, University of Michigan Medical School, ⁴Rogel Cancer Center, University of Michigan Medical School

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Cancer Research

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

C. Ryan Oliver^*1,2, Trisha M. Westerhof^*1,2, Maria G. Castro^2,3,4, Sofia D. Merajver^1,2

¹Department of Internal Medicine, University of Michigan Ann Arbor, ²Rogel Cancer Center, University of Michigan Ann Arbor, ³Department of Neurosurgery, University of Michigan Ann Arbor, ⁴Department of Cell and Developmental Biology, University of Michigan Ann Arbor

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).