Cell Culture Models

A lack of cellular model systems was identified as a major gap in cutaneous neurofibroma (cNF) research, slowing investigators’ progress due to reliance on costly, time-consuming animal systems to identify agents that might be effective against these tumors.

The NTAP Cell Culture Models initiative addressed a basic need in cNF research: a rapid, cost effective way to predict the efficacy of potential drug candidates alone and in combination. The range of cell-lines being generated support multiple applications from quantifiable high throughput screening to secondary assay systems that model the importance of the micro-environment in cNF and the heterogeneity of these tumors.

Projects were focused on building cell culture models that can be used for the following applications:
• Preclinical drug screening
• Exploration of the effects of the mutation type within the NF1 gene on cNF disease pathophysiology
• Elucidation of the critical steps and cell types involved in cNF tumor formation

Historical Cell-Culture Initiative NTAP-funded projects:

Development of a Novel Plexiform Neurofibroma Farming System for 96-well Plate Drug Screening

Karlyne M. Reilly, Ph.D., National Cancer Institute

Dr. Reilly and her team explored the development of culture systems composed of various extracellular matrix (ECM) components using MAPTRix in a 96-well format to identify the preferred substrate for NF1 null Schwann cells. Simultaneously, they proposed to design a rig of microcylinders for Schwann cell attachment to mimic radial growth of cNFs around axons. They then compared the growth characteristics of the MAPTrix ECM in 2D, 3D and the guitar rig. The goals of this project were to: [1] establish the optimal conditions to encourage maintenance in culture over long passages; [2] investigate the factors in the extracellular matrix that NF1 null Schwann cells are most dependent on; and [3] investigate the physical structures that Schwann cells prefer for growth.

Narrative: Read about the project’s background, goals, specific aims, and preliminary results (auspices of Dr. Karlyne Reilly, reillyk@mail.nih.gov)

Development of Plexiform Neurofibroma Cellular Assay for High-Throughput Screening

Margaret (Peggy) Wallace, Ph.D., University of Florida
Dr. Wallace and her team generated three (semi)-immortalized NF1 null Schwann cell culture models derived from human tissue. They fully characterized these cell lines and generated additional lines to represent the diversity of cNF and to include control Schwann cell cultures. The primary goals of her proposal were to [1] complete the creation and characterization of at least 5 independent cNF (semi)immortalized Schwann cell lines from human cNF; [2] fully characterize the cell lines and identify phenotypes that recapitulate the original tumor tissue; and [3] scale selected cell culture models for assays for high-throughput screening.

Narrative: Read this exciting summary of the first report of immortalization and detailed characterization of multiple human NF1 normal nerve and neurofibroma-derived Schwann cell lines (auspices of Dr. Wallace, UFL, peggyw@mgm.ufl.edu)

Publication: Li H, Chang LJ, Neubauer D, Muir D, Wallace MR, Immortalization of human normal and NF1 neurofibroma schwann cells. Laboratory Investigation 2016 Oct; 96:1105–1115. PMID: 27617404

Perpetuating NF1+/- and NF1-/- plexiform neurofibroma-derived tumor cells through the generation of induced pluripotent stem (iPS) cells

Eduard Serra, Ph.D., Institute of Predictive and Personalized Medicine of Cancer (Barcelona, Spain)
Using patient-derived cells from cutaneous neurofibromas (cNFs), Dr. Serra explored the degree of genomic, molecular and cellular variation within regions taken from individual cNFs in order to better understand the variability within these tumors. He also interrogated the expression profile of cell cultures in isolation and in co-culture conditions in order to authenticate and characterize NF1+/- and NF1-/- induced pluripotent stem cells (iPSCs) to allow exploration of the functional status of the NF1 gene on biologic properties of the cells. The primary goals of their proposal were to: [1] explore genomic, molecular and cellular variation within a single cNF; [2] investigate the effects of co-culture on cell behavior in NF1-/- Schwann cells and NF1+/- fibroblast cells; and [3] create and characterize iPSCs from human tumors to investigate the influence of the NF1 gene status on cell behavior.

Narrative: Read about the project’s background, goals, specific aims, methods, and results (auspices of Dr. Eduard Serra, eserra@igtp.cat)

A primary plexiform neurofibroma cell culture model for use in cell-based high-throughput screens.

Luis Parada, Ph.D., Memorial Sloan Kettering Cancer Center
Dr. Parada and his team applied the techniques optimized for cell-based high-throughput screens (HTS) from pooled tumor-derived cells for GBM to the development of primary cell culture models of cNF from the mouse models of mCNF generated in their lab. They attempted to optimize the growth conditions for these cells and expand the cells to allow for HTS. The goals of this project were to: [1] generate and validate a physiologically-relevant cell culture model of cNF, derived from genetically engineered mouse tissue; [2] optimize growth conditions for the culture model; and [3] expand the cells to allow high throughput chemical and siRNA screens, with the long term goal of conducting these screens.

Narrative: Read about the project’s background, goals, specific aims, methods, and results (auspices of Dr. Luis Parada, Paradal@mskcc.org)

Plexiform Neurofibroma Model Systems for Preclinical Drug Screening

Ray Mattingly, Ph.D., Wayne State University
Dr. Mattingly’s laboratory applied their expertise in co-culture of breast ductal carcinoma to develop co-culture conditions for patient-derived tumor-associated cells. They executed this in collaboration with Dr. Wallace who has several NF1 null and heterozygous cells including Schwann cells, fibroblasts endothelial cells and macrophages. He also collaborated with Dr. Reilly for the development of optimal conditions for co-culture. The goals of this project were to: [1] optimize 3D co-culture conditions for cNF inclusive of the cell type found in cNF for use as confirmatory/secondary drug screens.

Narrative: Read about the project’s background, rationale, goals, specific aims, methods, results, and impact! (auspices of Dr. Ray Mattingly, rmatting@med.wayne.edu)

Publication: Brock EJ, Ji K, Reiners JJ Jr. & Mattingly RR. How to Target Activated Ras Proteins: Direct Inhibition vs. Induced Mislocalization. Mini Rev Med Chem. 2016 ; 16(5): 358–369. PMID: 26423696.

Transition to confirmatory and secondary screening of plexiform neurofibroma models in 96-well format

Ray Mattingly, Ph.D., Wayne State University
Dr. Mattingly’s laboratory applied their expertise in co-culture to prove that cells grown in 3D matrices, as compared to those growing in 2D (on plastic) exhibit drug sensitivities that serve as better clinical predictors. They will performed confirmatory and secondary assays in collaboration with NCATS.

Narrative: Read about the project’s background, goals, methods, results, and impact (auspices of Dr. Ray Mattingly, rmatting@med.wayne.edu)

Cell Culture Activities:

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CIDr/NTAP Collaboration: (1) Genetic characterization studies (Cell Line Authentication, SNP Array, Exome Sequencing, and RNA Sequencing) of NF1-/-, NF1-/+, and NF1+/+ cell-lines to be available as tool-set for community, and (2) Analyses conducted by Sage Bionetworks.

Sage Analysis: Bioinformatics analyses of cNF cell lines’ high throughput screening data.

Screening (Single Agent MIPEs, combo): As a result of a collaboration between UFL, NTAP, and NCATS, a panel of cell culture systems that represented cNF complexity was used to screen new compounds (as single and combination agents) and identify novel therapeutic targets.

Validation Studies: The goal of these experiments (conducted at IU) was to validate the results of preliminary HTS at NIH/NCATS by Dr. Ferrer.