Delyan P. Ivanov

Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

In vitro models of medulloblastoma: choosing the right tool for the job

The recently-defined four molecular subgroups of medulloblastoma have required updating of our understanding of in vitro models to include molecular classification and risk stratification features from clinical practice. This review seeks to build a more comprehensive picture of the in vitro systems available for modelling medulloblastoma. The subtype classification and molecular characterisation for over 40 medulloblastoma cell-lines has been compiled, making it possible to identify the strengths and weaknesses in current model systems. Less than half (18/44) of established medulloblastoma cell-lines have been subgrouped. The majority of the subgrouped cell-lines (11/18) are Group 3 with MYC-amplification. SHH cell-lines are the next most common (4/18), half of which exhibit TP53 mutation. WNT and Group 4 subgroups, accounting for 50% of patients, remain underrepresented with 1 and 2 cell-lines respectively. In vitro modelling relies not only on incorporating appropriate tumour cells, but also on using systems with the relevant tissue architecture and phenotype as well as normal tissues. Novel ways of improving the clinical relevance of in vitro models are reviewed, focusing on 3D cell culture, extracellular matrix, co-cultures with normal cells and organotypic slices. This paper champions the establishment of a collaborative online-database and linked cell-bank to catalyse preclinical medulloblastoma research.

Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D

We describe and share a device, methodology and image analysis algorithms, which allow up to 66 spheroids to be arranged into a gel-based array directly from a culture plate for downstream processing and analysis. Compared to processing individual samples, the technique uses 11-fold less reagents, saves time and enables automated imaging. To illustrate the power of the technology, we showcase applications of the methodology for investigating 3D spheroid morphology and marker expression and for in vitro safety and efficacy screens. First, spheroid arrays of 11 cell-lines were rapidly assessed for differences in spheroid morphology. Second, highly-positive (SOX-2), moderately-positive (Ki-67) and weakly-positive (βIII-tubulin) protein targets were detected and quantified. Third, the arrays enabled screening of ten media compositions for inducing differentiation in human neurospheres. Last, the application of spheroid microarrays for spheroid-based drug screens was demonstrated by quantifying the dose-dependent drop in proliferation and increase in differentiation in etoposide-treated neurospheres.

Separating chemotherapy-related developmental neurotoxicity from cytotoxicity in monolayer and neurosphere cultures of human fetal brain cells.

Chemotherapy-induced neurotoxicity can reduce the quality of life of patients by affecting their intelligence, senses and mobility. Ten percent of safety-related late-stage clinical failures are due to neurological side effects. Animal models are poor in predicting human neurotoxicity due to interspecies differences and most in vitro assays cannot distinguish neurotoxicity from general cytotoxicity for chemotherapeutics. We developed in vitro assays capable of quantifying the paediatric neurotoxic potential for cytotoxic drugs. Mixed cultures of human fetal brain cells were differentiated in monolayers and as 3D-neurospheres in the presence of non-neurotoxic chemotherapeutics (etoposide, teniposide) or neurotoxicants (methylmercury). The cytotoxic potency towards dividing progenitors versus differentiated neurons and astrocytes was compared using: (1) immunohistochemistry staining and cell counts in monolayers; (2) through quantitative Western blots in neurospheres; and (3) neurosphere migration assays. Etoposide and teniposide, were 5-10 times less toxic to differentiated neurons compared to the mix of all cells in monolayer cultures. In contrast, the neurotoxicant methylmercury did not exhibit selectivity and killed all cells with the same potency. In 3D neurospheres, etoposide and teniposide were 24 to 10 times less active against neurons compared to all cells. These assays can be used prioritise drugs for local drug delivery to brain tumours.

Data on the number and frequency of scientific literature citations for established medulloblastoma cell lines.

This article collates information about the number of scientific articles mentioning each of the established medulloblastoma cell lines, derived through a systematic search of Web of Science, Scopus and Google Scholar in 2016. The data for each cell line have been presented as raw number of citations, percentage share of the total citations for each search engine and as an average percentage between the three search engines. In order to correct for the time since each cell line has been in use, the raw citation data have also been divided by the number of years since the derivation of each cell line. This is a supporting article for a review of in vitro models of medulloblastoma published in “in vitro models of medulloblastoma: choosing the right tool for the job” (D.P. Ivanov, D.A. Walker, B. Coyle, A.M. Grabowska, 2016) [1].

High-throughput spheroid screens using volume, resazurin reduction and acid phosphatase activity

Mainstream adoption of physiologically relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price, and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily available reagents and open-source software to analyze spheroid volume, metabolism, and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination-for both single images and images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyze plate uniformity (such as edge effects and systematic seeding errors), detect outliers, and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time.

Synthesis and properties of a biodegradable polymer-drug conjugate: Methotrexate-poly(glycerol adipate)

Polymer-drug conjugates have been actively developed as potential anticancer drug delivery systems. In this study, we report the first polymer-anticancer drug conjugate with poly(glycerol adipate) (PGA) through the successful conjugation of methotrexate (MTX). MTX-PGA conjugates were controllably and simply fabricated by carbodiimide-mediated coupling reaction with various high molar ratios of MTX. The MTX-PGA conjugate self-assembled into nanoparticles with size dependent on the amount of conjugated MTX and the pH of medium. Change in particle size was attributed to steric hindrance and bulkiness inside the nanoparticle core and dissociation of free functional groups of the drug. The MTX-PGA nanoparticles were physically stable in media with pH range of 5-9 and ionic strength of up to 0.15 M NaCl and further chemically stable against hydrolysis in pH 7.4 medium over 30 days but enzymatically degradable to release unchanged free drug. Although 30%MTX-PGA nanoparticles exhibited only slightly less potency than free MTX in 791T cells in contrast to previously reported human serum albumin-MTX conjugates which had >300 times lower potency than free MTX. However, the MTX nanoparticles showed 7 times higher toxicity to Saos-2 cells than MTX. Together with the enzymic degradation experiments, these results suggest that with a suitable biodegradable polymer a linker moiety is not a necessary component. These easily synthesised PGA drug conjugates lacking a linker moiety could therefore be an effective new pathway for development of polymer drug conjugates.

Highlights of Children with Cancer UK's Workshop on Drug Delivery in Paediatric Brain Tumours

The first Workshop on Drug Delivery in Paediatric Brain Tumours was hosted in London by the charity Children with Cancer UK. The goals of the workshop were to break down the barriers to treating central nervous system (CNS) tumours in children, leading to new collaborations and further innovations in this under-represented and emotive field. These barriers include the physical delivery challenges presented by the blood–brain barrier, the underpinning reasons for the intractability of CNS cancers, and the practical difficulties of delivering cancer treatment to the brains of children. Novel techniques for overcoming these problems were discussed, new models brought forth, and experiences compared.