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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative Results

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.......

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 biology29,30. This method is based on the cancer stem cells intrinsic property to expand without constraints in soft gels cultures

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Accutase cell dissociation reagentGibcoA1110501Detach adherent cells and dissociate tumorspheres
CeligoNexcelomCeligoMicrowell plate based image cytometer for adherent and suspension cells
Collagenase, Type IILife Technologies17101015Tissue digestion enzyme
Dispase II, proteaseLife Technologies17105041Tissue digestion enzyme
DMEM high glucose mediaGibco11965092Component of tumor cells media
DMEM/F12 MediaGibco11320033Component of tumosphere media
EDTAThermoFisherS312500Component of FACS buffer
EGF recombinant mouse proteinGibcoPMG8041Component of tumosphere media
FACSAria II Flow CytometryBD Biosciences650033Fluorescent activated cell sorter
Fetal Bovine SerumOmega ScientificFB-11Component of tumor cells media
Fluriso (Isofluornae) anesthetic agentMWI Vet Supply502017Anesthetic reagent for animals
FxCycle Violet StainLife TechnologiesF10347Discriminate live and dead cells
Goat SerumLife Technologies16210072Component of FACS buffer
Ham's F10 MediaLife Technologies11550043Component of FACS buffer
Horse SerumLife Technologies16050114Component of cell isolation media
Lipofectamine 3000 transfection reagentThermoFisherL3000015Transfection Reagent
Matrigel membrane matrixCorningCB40234Provides support to trasplanted cells
N-2 Supplemtns (100X)Gibco17502048Component of tumosphere media
Neomycin-Polymyxin B Sulfates-Bacitracin Zinc Ophthalmic OintmentMWI Vet Supply701008Eyes ointment
PBSGibco10010023Component of FACS buffer and used for washing cells
pEGFP-C1Addgene6084-1GFP plasmid
Penicillin - StreptomyocinLife Technologies15140163Component of tumosphere and tumor cells media
Recombinant Human βFGF-basicPeprotech10018BComponent of tumosphere media
Recombinant mouse Flt-3 Ligand ProteinR&D Systems427-FL-005Recombinant protein
Trypan blueThermoFisher15250061Discriminate live and dead cells

References

  1. Dagogo-Jack, I., Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. Nature Reviews Clinical Oncology. 15 (2), 81-94 (2018).
  2. Wicha, M. S., Liu, S., Dontu, G. Cancer stem cells: an old idea--a paradigm shift.

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