1Interdepartmental Program in Translational Biology & Molecular Medicine, Baylor College of Medicine, 2Texas Children's Cancer Center, Baylor College of Medicine, 3Department of Pediatrics, Baylor College of Medicine, 4Department of Pathology, The Methodist Hospital Research Institute, 5Department of Ophthalmology, Retinoblastoma Center of Houston, 6Baylor College of Medicine, Center for Cell and Gene Therapy, 7Center for Cell and Gene Therapy, Baylor College of Medicine
Bond, W. S., Wadhwa, L., Perlaky, L., Penland, R. L., Hurwitz, M. Y., Hurwitz, R. L., et al. Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice. J. Vis. Exp. (54), e2644, doi:10.3791/2644 (2011).
Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor.
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation.
Wesley S. Bond and Lalita Wadhwa are co-first authors.
1. Preparation of retinoblastoma tumorspheres
2. Preparation of tumor cells for injection
3. Preparation of animals
4. Injection of tumor cells
5. Monitoring of injected mice and harvesting of tumors
6. Representative Results:
Retinoblastoma tumorspheres will begin to appear from the disaggregated tissue almost immediately as they are liberated from the tumor mass. Within 2-4 days, more tumorspheres will begin to form and will increase in size. The spheres tend to be regular and exhibit a well-defined, secondary membrane surrounding the aggregate (Figure 2).
The animal typically presents with leukocoria within 4 weeks post-injection (Figure 3b), followed by enlargement of the globe and distention of the surrounding tissues 5-8 weeks after injection as the tumor grows (Figure 3c).
Figure 1. Cross-sectional diagram of the mouse eye highlighting features referenced in the protocol.
Figure 2. Culture of human retinoblastoma cells in vitro imaged at a) 4x, and b) 10x objective magnification. Retinoblastoma primary tumor cells produce tumorspheres with a regular spheroid shape and a crisp outer membrane. Scale bars represent a) 500 μm, and b) 200 μm.
Figure 3. Eye of Rag2-/- mouse showing a) normal features, b) leukocoria indicative of a tumor mass in vitreous cavity, and c) a large tumor mass filling the globe with associated periocular distension, intraocular hemorrhage.
The technique described herein facilitates the propagation retinoblastoma tumors in their intraocular, intravitreal milieu. The intraocular injection technique has been used in the past to create tumors from retinoblastoma-derived cell lines1 as well as to deliver viral vectors for intraocular gene therapy2,3. This technique has now been successfully utilized to produce human retinoblastoma tumors by direct injection of cells from the primary tumor and injection of tumorspheres as well as serial propagation of xenograft tumors. Visible evidence of tumor formation (usually leukocoria) is usually first noted within 4 weeks, after which intraocular hemorrhage and distention of the globe and/or tissues surrounding the orbit develop within 5-8 weeks. A minority of injected mice injure the injected eye, leading to permanent closure of the eyelids. In these cases, distention is the only sign of tumor formation.
Establishment of murine xenograft tumors has not been successful with all human retinoblastoma tumors, though propagation of established xenograft tumors is highly successful. This observation suggests that certain characteristics of the primary tumor, such as invasiveness and level of differentiation or some other unknown factor, may be influencing the ability of these tumors to persist in the murine ocular environment.
The amount of tissue that can be acquired from a human retinoblastoma tumor is quite small, and there are significant limitations on the ability to culture human retinoblastoma cells in vitro such as limited life span, loss of solid tumor histology and changes in cellular phenotype. This protocol provides a relatively simple way to propagate the human tumor and establish a murine model of the human disease. This allows further in vitro and in vivo experimentation of the biology of retinoblastoma and longer maintenance of the tumor outside of the patient.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Baylor College of Medicine IACUC Committee.
Funding for this project is provided by the Clayton Foundation for Research and the Retina Research Foundation.
|Phenylephrine HCl 2.5%||Bausch and Lomb||053-11||Ophthalmic solution|
|30-ga Needle||BD Biosciences||305128||Regular bevel|
|10-mL Luer-Lock Syringe||BD Biosciences||309604|
|3/10-cc Insulin Syringe||BD Biosciences||328431|
|Alcohol swabs||BD Biosciences||326895|
|6-Well Plate, Tissue Culture-Treated||BD Biosciences||353046|
|Proparacaine HCl 0.5%||Butler Animal Health Supply||017239||Ophthalmic solution|
|Ketamine HCl 100mg/mL||Fort Dodge Animal Health||4402A|
|10-ÁL Hamilton Syringe||Hamilton Co||7648-01|
|32-ga Hamilton Needle||Hamilton Co||7803-04||Custom length - 0.5"|
|B-27 Supplement Minus Vitamin A, 50X||Invitrogen||12587-010|
|RPMI-1640 Media||Mediatech, Inc.||10-040-CV|
|Non-essential Amino Acid Solution, 100X||Mediatech, Inc.||25-025-CI|
|L-Glutamine, 100X||Mediatech, Inc.||25-005-CI|
|Disposable #11 Scalpel||Miltex Inc.||4-411|
|Rodent Anesthesia Combo||N/A||n/a||In-house pharmacy formulation (ketamine 37.6 mg/mL, xylazine 1.92 mg/mL, acepromazine 0.38 mg/mL)|
|Recombinant Human Epidermal Growth Factor (EGF)||Stem Cell Technologies||02633||Reconstitute at 10 μg/mL stock solution|
|Recombinant Human Basic Fibroblast Growth Factor (bFGF)||Stem Cell Technologies||02634||Reconstitute at 10 μg/mL stock solution|
|OMS-75 Operation Microscope||Topcon Medical Systems||OMS-75||This model has been discontinued|
|10% Formalin||VWR international||95042-908|