Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models

Summary

We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P₀-GGFβ3 mouse model.

Abstract

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that subsequently transform into highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). Understanding the process by which a PN transforms into an MPNST would be facilitated by the availability of genetically engineered mouse (GEM) models that accurately replicate the PN-MPNST progression seen in humans with NF1. Unfortunately, GEM models with Nf1 ablation do not fully recapitulate this process. This led us to develop P₀-GGFβ3 mice, a GEM model in which overexpression of the Schwann cell mitogen neuregulin-1 (NRG1) in Schwann cells results in the development of PNs that progress to become MPNSTs with high frequency. However, to determine whether tumorigenesis and neoplastic progression in P₀-GGFβ3 mice accurately model the processes seen in NF1 patients, we had to first prove that the pathology of P₀-GGFβ3 peripheral nerve sheath tumors recapitulates the pathology of their human counterparts.

Here, we describe the specialized methodologies used to accurately diagnose and grade peripheral nervous system neoplasms in GEM models, using P₀-GGFβ3 and P₀-GGFβ3;Trp53^+/- mice as an example. We describe the histologic, immunohistochemical, and histochemical methods used to diagnose PNs and MPNSTs, how to distinguish these neoplasms from other tumor types that mimic their pathology, and how to grade these neoplasms. We discuss the establishment of early-passage cultures from GEM MPNSTs, how to characterize these cultures using immunocytochemistry, and how to verify their tumorigenicity by establishing allografts. Collectively, these techniques characterize the pathology of PNs and MPNSTs that arise in GEM models and critically compare the pathology of these murine tumors to their human counterparts.

Introduction

Over the last three decades, numerous laboratories have attempted to create mouse models of human cancers by introducing human cancer-associated mutations into the mouse genome or by overexpressing a gene product that is overexpressed in human cancers. The resulting genetically engineered mouse (GEM) models can be used for a variety of purposes such as establishing that the newly introduced genomic modification initiates tumorigenesis, identifying other subsequently occurring genetic or epigenetic changes that contribute to tumor progression, and defining the key signaling pathways that drive tumor initiation and progression. Unlike orthotopic xenograft models, which ....

Protocol

The procedures described here were approved by the Medical University of South Carolina's IACUC and were performed by properly trained personnel in accordance with the NIH Guide for the Care and Use of Laboratory Animals and MUSC's institutional animal care guidelines.

1. Determining tumor penetrance and survival in P₀-GGFβ3 mice and identifying tumors in these animals for further characterization

1. Generate the cohort of mice that will be ass.......

Discussion

The histological and biochemical methods presented here provide a framework for diagnosing and characterizing GEM models of neurofibroma and MPNST pathogenesis. Over the years, we have found these methodologies to be quite useful for assessing the pathology of peripheral nerve sheath tumors arising in GEM models^21,25,26. However, while the protocols outlined here are useful for determining how accurately tumors in the GEM models.......

Materials

Name	Company	Catalog Number	Comments
100 mm Tissue Culture Plates	Corning Falcon	353003
3, 3'- Diaminobensidine (DAB)	Vector Laboratories	SK-400
6- well plates	Corning Costar	3516
Acetic Acid	Fisher Scientific	A38-212
Alexa Fluor 488 Secondary (Goat Anti-Mouse)	Invitrogen	A11029
Alexa Fluor 568 Secondary (Goat Anti-Mouse)	Invitrogen	A21043 or A11004
Alexa Fluor 568 Secondary (Goat Anti-Rabbit)	Invitrogen	A11036
Ammonium Chloride (NH4Cl)	Fisher Scientific	A661-500
BCA Protein Assay Kit	Thermo Scientific	23225
Bovine Serum Albumin	Fisher Scientific	BP1600-100
Caldesmon	ABCAM	E89, ab32330
CD117	Cell Marque	117R-18-ASR
CD163	Leica	NCL-L-CD163
CD31	ABCAM	ab29364
CD34	ABCAM	ab81289
CD86	ABCAM	ab53004
Cell Scraper	Sarstedt	83.183
Cell Stripper	Corning	25-056-CI
Circle Coverslip	Fisher Scientific	12-545-100
Citrisolve Hybrid (d-limonene-based solvent)	Decon Laboratories	5989-27-5
Critic Acid	Fisher Scientific	A104-500
Cytokeratin	ABCAM	C-11, ab7753
Desmin	Agilent Dako	clone D33 (M0760)
Diaminobensizdine (DAB) Solution	Vector Laboratories	SK-4100
DMEM	Corning	15-013-CV
Eosin Y	Thermo Scientific	7111
Ethanol (200 Proof)	Decon Laboratories	2716
Fetal Calf Serum	Omega Scientific	FB-01
Forksolin	Sigma-Aldrich	F6886
Glycerol	Sigma-Aldrich	G6279
Hank's Balanced Salt Solution (HBSS)	Corning	21-022-CV
Harris Hematoxylin	Fisherbrand	245-677
Hemacytometer	Brightline-Hauser Scientific	1490
Hydrochloric Acid	Fisher Scientific	A144-212
Hydrogen Peroxide	Fisher Scientific	327-500
Iba1	Wako Chemicals	019-19741
ImmPRESS HRP (Peroxidase) Polymer Kit ,Mouse on Mouse	Vector Laboratories	MP-2400
ImmPRESS HRP (Peroxidase) Polymer Kit, Horse Anti-Rabbit	Vector Laboratories	MP-7401
Incubator	Thermo Scientific	Heracell 240i CO2 incubator
Isoflurane	Piramal	NDC 66794-017-25
Isopropanol	Fisher Scientific	A415
Ki-67	Cell Signaling	12202
Laminin	Thermo Fisher Scientific	23017015
Liquid Nitrogen
MART1	ABCAM	M2-9E3, ab187369
Microtome
Nestin	Millipore	Human: MAD5236 (10C2), Human:MAB353 (Rat-401)
Neuregulin 1 beta	In house	Made by S.L.C. (also available as 396-HB-050/CF from R&D Systems)
Neurofibromin	Santa Cruz Biotechnology	sc-67
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice	Jackson Laboratory	5557
Nonfat Dry Milk	Walmart	Great Value Brand
P0-GGFβ3 mice	In house
Paraffin Wax	Leica	Paraplast 39601006
Parafilm M	Sigma-Aldrich	PM-999
Paraformaldehyde (4%)	Thermo Scientific	J19943-K2
Permount (Xylene Mounting Medium)	Fisher Scientific	SP15-100
pH Meter	Mettler Toldedo	Seven Excellence, 8603
Phosphate Buffered Saline (Dulbecco's)	Corning	20-031-CV
PMEL	ABCAM	EP4863(2), ab137078
Poly-L-Lysine Hydrobromide	Sigma-Aldrich	P5899-5MG
Portable Isoflurance Machine	VetEquip Inhalation Anesthesia Systems
PVA-DABCO (Aqueous Mounting Medium)	Millipore Sigma	10981100ML
Rice Cooker	Beech Hamilton
S100B	Agilent Dako	Z0311  (now GA504)
SMA	Ventana Medical Systems	clone 1A4
Sodium Chloride	Fisher Scientific	S640
Sodium Citrate (Dihydrate)	Fisher Scientific	BP327-1
Sox10	ABCAM	ab212843
Steel histology mold
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
TCF4/TCFL2	Cell Signaling	(CH48H11) #2569
Tissue Cassette
Toluidine Blue	ACROS Organics	348600050
Triton X-100	Fisher Scientific	BP151-500
TRIzol	Invitrogen	15596026
Trypsin	Corning	25-051-31