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This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.
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.
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 ....
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
Tumorspheres detection
Cell isolation was optimized to obtain the maximum heterogeneity of cell populations present in the tumor tissue. First, since isolated tissues presented morphologically dissimilar areas, to enhance the chances of isolating uniform rare cell populations, sampling was performed from multiple areas of the tumor (Figure 1A, first panel on the left). Second, mechanical dissociation of the harvested samples was perform.......
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 biology29,30. This method is based on the cancer stem cells intrinsic property to expand without constraints in soft gels cultures
This work was supported by the Ellison Medical Foundation grant AG-NS-0843-11, and the NIH Pilot Grant within the NCI Cancer Center Support Grant P30CA030199 to A.S.
....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 |
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