Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Summary

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

Introduction

Cancer is a heterogeneous disease; furthermore, the same type of tumor can present different genetic mutations in different patients, and within a patient a tumor is composed by multiple populations of cells¹. Heterogeneity presents a challenge in the identification of cells responsible for initiating and propagating cancer, but their characterization is essential for the development of efficient treatments. The notion of tumor propagating cells (TPC), a rare population of cells that contribute to tumor development, has been previously extensively reviewed². Despite the fact that TPCs have been characterized in multiple ....

Protocol

The housing, treatment, and sacrifice of mice were performed following the approved IACUC protocol of the Sanford Burnham Prebys Medical Discovery Institute.

1. Reagent preparation

1. Prepare 100 mL of cell isolation media: F10 medium supplemented with 10% horse serum (HS).
2. Prepare 50 mL of collagenase type II solution: dissolve 1 g of collagenase type II powder in 50 mL of cell isolation media (note the units of the enzyme per 1 mL of media, since the number of units change.......

Discussion

Multiple methods have been employed for isolation and characterization of TPCs from tumor heterogeneous cell populations: tumor clonogenic assays, FACS isolation, and tumorsphere formation assay. The tumor clonogenic assay was first described in 1971, used for stem cell studies, and only subsequently applied to cancer biology^29,30. This method is based on the cancer stem cells intrinsic property to expand without constraints in soft gels cultures

Materials

Name	Company	Catalog Number	Comments
Accutase cell dissociation reagent	Gibco	A1110501	Detach adherent cells and dissociate tumorspheres
Celigo	Nexcelom	Celigo	Microwell plate based image cytometer for adherent and suspension cells
Collagenase, Type II	Life Technologies	17101015	Tissue digestion enzyme
Dispase II, protease	Life Technologies	17105041	Tissue digestion enzyme
DMEM high glucose media	Gibco	11965092	Component of tumor cells media
DMEM/F12 Media	Gibco	11320033	Component of tumosphere media
EDTA	ThermoFisher	S312500	Component of FACS buffer
EGF recombinant mouse protein	Gibco	PMG8041	Component of tumosphere media
FACSAria II Flow Cytometry	BD Biosciences	650033	Fluorescent activated cell sorter
Fetal Bovine Serum	Omega Scientific	FB-11	Component of tumor cells media
Fluriso (Isofluornae) anesthetic agent	MWI Vet Supply	502017	Anesthetic reagent for animals
FxCycle Violet Stain	Life Technologies	F10347	Discriminate live and dead cells
Goat Serum	Life Technologies	16210072	Component of FACS buffer
Ham's F10 Media	Life Technologies	11550043	Component of FACS buffer
Horse Serum	Life Technologies	16050114	Component of cell isolation media
Lipofectamine 3000 transfection reagent	ThermoFisher	L3000015	Transfection Reagent
Matrigel membrane matrix	Corning	CB40234	Provides support to trasplanted cells
N-2 Supplemtns (100X)	Gibco	17502048	Component of tumosphere media
Neomycin-Polymyxin B Sulfates-Bacitracin Zinc Ophthalmic Ointment	MWI Vet Supply	701008	Eyes ointment
PBS	Gibco	10010023	Component of FACS buffer and used for washing cells
pEGFP-C1	Addgene	6084-1	GFP plasmid
Penicillin - Streptomyocin	Life Technologies	15140163	Component of tumosphere and tumor cells media
Recombinant Human βFGF-basic	Peprotech	10018B	Component of tumosphere media
Recombinant mouse Flt-3 Ligand Protein	R&D Systems	427-FL-005	Recombinant protein
Trypan blue	ThermoFisher	15250061	Discriminate live and dead cells