Manoj Kumar

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. While in vitro patient-derived xenografts (PDX) lines are useful for studying GBM, they often exclude astrocytes and macrophages, which contribute significantly to tumor growth, invasion, and chemoradioresistance. Integrating these cells into tumor models is difficult due to their need for serum, which triggers GBM-PDX lines to lose their stem-like properties. The aim of this study was to develop a serum-free triculture model of GBM-PDX lines, normal human astrocytes (NHAs), and macrophages. Serum-free media alternatives were formulated for NHAs and identified for THP-1 macrophages, then combined with GBM PDX media to establish “PSX,” an experimental maintenance media. Cells were transitioned to serum-free media alternatives and functionally assessed through several parameters unique to each cell type. In addition to assessing GBM “stemness,” a custom 350-gene NanoString chip was used to assess differential gene expression in monocultured PDX cells versus PDX cells exposed to NHAs and macrophages. PSX maintained canonical function in astrocytes and macrophages while preserving the stem-like properties of GBM-PDX cells. Tri-culturing all three cells increased the expression of stemness-associated transcription factors and increased the expression of genes related to stemness and hypoxia in GBM cells. GBM PDX cells exposed to NHAs and macrophages in direct triculture exhibit increases in markers of stemness and hypoxia. These findings suggest that the serum-free triculture model presented herein may better recapitulate the tumoral heterogeneity of GBM in vitro, providing a novel model to utilize in current research.

The gold standard assay for radiation response is the clonogenic assay, a normalized colony formation assay (CFA) that can capture a broad range of radiation-induced cell death mechanisms. Traditionally, this assay relies on two-dimensional (2D) cell culture conditions with colonies counted by fixing and staining protocols. While some groups have converted these to three-dimensional (3D) conditions, these models still utilize 2D-like media compositions containing serum that are incompatible with stem-like cell models such as brain tumor initiating cells (BTICs) that form self-aggregating spheroids in neural stem cell media. BTICs are the preferred patient-derived model system for studying glioblastoma (GBM) as they tend to better retain molecular and phenotypic characteristics of the original tumor tissue. As such, it is important that preclinical radiation studies should be adapted to BTIC conditions. In this study, we describe a series of experimental approaches for performing CFA experiments with BTIC cultures. Our results indicate that serum-free clonogenic assays are feasible for combination drug and radiation testing and may better facilitate translatability of preclinical findings.

Poly (ADP‐ribose) polymerase (PARP) is a superfamily of 18 proteins characterized by the PARP homology domain, the catalytic domain. This catalytic domain helps in the ADP‐ribosylation of various acceptor proteins using nicotinamide adenine dinucleotide (NAD+) as a donor for ADP‐ribose. PARP‐1 and PARP‐2 carry out 80% of poly‐ADP‐ribosylation of cellular protein. Hence, their combined knockout results in embryonic lethality of mice. PARP‐1 consists of three major domains, namely, DNA binding domain, automodification domain, and a catalytic domain. These domains further consist of subdomains and motifs, which helps PARP‐1 in a diverse function. PARP‐1 is mainly involved in DNA damage detection and repair, but emerging evidence suggests its role in many other functions such as DNA synthesis, replication, apoptosis, necrosis, and cancer progression. Herein, we review the current state of the PARP‐1 role in DNA damage repair and other biological processes including epithelial to mesenchymal transition (EMT). We have also observed the role of PARP‐1 in modulating EMT regulators like E‐cadherin, Vimentin, Claudin‐1, Snail, Smad‐4, Twist‐1, and β‐catenin. Here, we have also attempted to relate the role of PARP‐1 in EMT of cancer cells.