Name: Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas (Video) | JoVE Journal
Uploaded: 2019-09-13T13:30:00.000+00:00
Duration: 9 min 21 s
Description: Learn tumorsphere derivation and treatment from mouse rhabdomyosarcoma primary cells for oncology research. This method enables drug screening and identification of tumor-originating cells.

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5 minutes

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.

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The authors have nothing to disclose.

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<sup class='xref'...

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 types of cancer, the identification of markers for their reliable isolation remains a challenge for several tumor types3,4,5,6,7,8,9. Thus, a method that does not rely on molecular markers but rather on TPC functional properties (high self-renewal and the ability to grow in low-attachment conditions), known as the tumorsphere formation assay, can be widely applied for the identification of TPCs from most tumors. Importantly, this assay can also be employed for expansion of TPCs and thus directly applied to cancer drug screening and studies on cancer resistance1,10.
Rhabdomyosarcoma (RMS) is a rare form of soft tissue sarcoma most common in young children11. Althoug RMS can be histologically identified through assessment of expression of myogenic markers, the RMS cell of origin has not been univocally characterized due to the multiple tumor subtypes and high heterogeneity of the tumor developmental stimuli. Indeed, recent studies have generated significant scientific discussion about whether RMS is of myogenic or non-myogenic origins, suggesting that RMS may derive from different cells types depending on the context12,13,14,15,16,17. Numerous studies on RMS cell lines have been performed employing the tumorsphere formation assay for the identification of pathways involved in tumor development and characterization of markers associated with highly self-renewal populations18,19,20,21.
However, despite the tumorsphere formation assay&#39;s potential for identifying RMS cells of origin, a reliable method that can be employed on primary RMS cells has not yet been described. In this context, a recent study from our group employed an optimized tumorsphere formation assay for the identification of the RMS cells of origin in a Duchenne muscular dystrophy (DMD) mouse model22. Multiple pre-tumorigenic cell types, isolated from muscle tissues, are tested for their ability to grow in low-attachment conditions, allowing the identification of muscle stem cells as cells of origin for RMS in dystrophic contexts. Described here is a reproducible and reliable protocol for the tumorsphere formation assay (Figure 1), which has been successfully employed for the identification of extremely rare cell populations that are responsible for mouse RMS development.

<table><tbody><tr><td>Accutase cell dissociation reagent</td><td>Gibco</td><td>A1110501</td><td>Detach adherent cells and dissociate tumorspheres</td></tr><tr><td>Celigo</td><td>Nexcelom</td><td>Celigo</td><td>Microwell plate based image cytometer for adherent and suspension cells</td></tr><tr><td>Collagenase, Type II</td><td>Life Technologies</td><td>17101015</td><td>Tissue digestion enzyme</td></tr><tr><td>Dispase II, protease</td><td>Life Technologies</td><td>17105041</td><td>Tissue digestion enzyme</td></tr><tr><td>DMEM high glucose media</td><td>Gibco</td><td>11965092</td><td>Component of tumor cells media</td></tr><tr><td>DMEM/F12 Media</td><td>Gibco</td><td>11320033</td><td>Component of tumosphere media</td></tr><tr><td>EDTA</td><td>ThermoFisher</td><td>S312500</td><td>Component of FACS buffer</td></tr><tr><td>EGF recombinant mouse protein</td><td>Gibco</td><td>PMG8041</td><td>Component of tumosphere media</td></tr><tr><td>FACSAria II Flow Cytometry</td><td>BD Biosciences</td><td>650033</td><td>Fluorescent activated cell sorter</td></tr><tr><td>Fetal Bovine Serum</td><td>Omega Scientific</td><td>FB-11</td><td>Component of tumor cells media</td></tr><tr><td>Fluriso (Isofluornae) anesthetic agent</td><td>MWI Vet Supply</td><td>502017</td><td>Anesthetic reagent for animals</td></tr><tr><td>FxCycle Violet Stain</td><td>Life Technologies</td><td>F10347</td><td>Discriminate live and dead cells</td></tr><tr><td>Goat Serum</td><td>Life Technologies</td><td>16210072</td><td>Component of FACS buffer</td></tr><tr><td>Ham&#039;s F10 Media</td><td>Life Technologies</td><td>11550043</td><td>Component of FACS buffer</td></tr><tr><td>Horse Serum</td><td>Life Technologies</td><td>16050114</td><td>Component of cell isolation media</td></tr><tr><td>Lipofectamine 3000 transfection reagent</td><td>ThermoFisher</td><td>L3000015</td><td>Transfection Reagent</td></tr><tr><td>Matrigel membrane matrix</td><td>Corning</td><td>CB40234</td><td>Provides support to trasplanted cells</td></tr><tr><td>N-2 Supplemtns (100X)</td><td>Gibco</td><td>17502048</td><td>Component of tumosphere media</td></tr><tr><td>Neomycin-Polymyxin B Sulfates-Bacitracin Zinc Ophthalmic Ointment</td><td>MWI Vet Supply</td><td>701008</td><td>Eyes ointment</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010023</td><td>Component of FACS buffer and used for washing cells</td></tr><tr><td>pEGFP-C1</td><td>Addgene</td><td>6084-1</td><td>GFP plasmid</td></tr><tr><td>Penicillin - Streptomyocin</td><td>Life Technologies</td><td>15140163</td><td>Component of tumosphere and tumor cells media</td></tr><tr><td>Recombinant Human &amp;beta;FGF-basic</td><td>Peprotech</td><td>10018B</td><td>Component of tumosphere media</td></tr><tr><td>Recombinant mouse Flt-3 Ligand Protein</td><td>R&amp;amp;D Systems</td><td>427-FL-005</td><td>Recombinant protein</td></tr><tr><td>Trypan blue</td><td>ThermoFisher</td><td>15250061</td><td>Discriminate live and dead cells</td></tr></tbody></table>

tumorsphere derivation and treatment from primary tumor cells isolated from mouse rhabdomyosarcomas

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

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.

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 performed...

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 ...

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In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Learn tumorsphere derivation and treatment from mouse rhabdomyosarcoma primary cells for oncology research. This method enables drug screening and identification of tumor-originating cells.

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

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