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Animal Model |
,
**¶**§¶**
From the Departments of Pathology,*
Ophthalmology,
Pediatrics,
and Molecular and Cellular
Biology,§
the Center for Cell and Gene
Therapy,¶
The Texas Children’s Cancer
Center,**
and the Retinoblastoma
Center,
Baylor College of
Medicine, Houston, Texas
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human retinoblastomas exhibit four patterns of invasion and metastasis.1 The first, direct invasive spread along the optic nerve to the brain, can also seed the orbital tissue and adjacent bone, the nasopharynx via the sinuses, or the cranium via the foramina. Tumor cells that have invaded the optic nerve and leptomeninges and then disperse into the circulating subarachnoid fluid are characteristic of the second pattern of metastasis. This may occur even when there is no tumor detected at the cut end of the optic nerve. Via the circulating subarachnoid fluid, tumor cells can also reach the spinal cord, distant sites of brain, and the contralateral optic nerve. Tumor formation in these sites represents true metastasis rather than local invasion. The third pattern of metastasis is hematogenous dissemination that results in widespread metastasis to the lungs, bones, brain, and other viscera. Metastasis after orbital invasion, and to a lesser degree choroidal invasion, is often via this route. The fourth pattern of metastasis, characterized by lymphatic spread, occurs when tumor is located anteriorly or massive extraocular invasion has occurred. Only tumors with these characteristics can spread via the lymphatic system, because there are no lymphatic vessels in the eye or orbit. Only the conjunctiva and eyelids have lymphatic drainage. When tumor reaches regional lymph nodes, hematogenous spread can also occur.
Retinoblastoma is a uniquely human disease. Attempts to develop animal models have provided a number of model systems each with distinct advantages but also with limitations. The majority of the animal models are xenograft models that have been created by injecting human retinoblastoma tumor cells into either the anterior chamber or the subretinal space of the eyes of immunodeficient mice or rats. Injection of tumor cells into the anterior chamber is favored because of that site’s accessibility for both the injection and the follow-up observation.6 Anterior chamber injection of Y79 cells has previously been reported to result in the invasion of the optic nerve and brain.7 These studies did not attempt to clarify whether the tumor spread by nonspecific extension or by specific migration of the tumor through the optic nerve to the central nervous system as is characteristic of human retinoblastoma. Tumor involvement of the anterior chamber is a late occurrence in human disease and the physiological environment of the anterior chamber differs significantly from that of the vitreal cavity in which naturally occurring tumors form.8,9 The anterior chamber xenograft model therefore has limited utility for the study of metastatic behavior. Another animal model in which retinoblastoma cells are injected into the subretinal space produces tumors that more closely resemble human retinoblastoma in location.10,11 However, injections into the subretinal space disrupt the choroid and the retina; therefore, use of this model to study invasiveness and progression of retinoblastoma is limited.
Several transgenic models of retinoblastoma have been developed. These models have recently been reviewed.12 Each of the successful models developed used viral oncogene products, most notably the SV40-T antigen, to bind to and interrupt the function of the endogenous tumor suppressor proteins pRb or p53. Several of the viral oncogene constructs used to create the transgenic mice used eye-specific promoters (IRBP, opsin, crystallin),13-17 although the model most often used for therapeutic studies was created using the promoter for the human luteinizing protein ß subunit.18,19 The tumors formed in the eyes of these transgenic mice resemble human retinoblastoma in many histopathological characteristics, however, they often lack the photoreceptor phenotype seen in the naturally occurring human disease and there is a higher frequency of tumors developing in the brain and other extraocular locations. A transgenic model generated by inactivating both alleles of the retinoblastoma gene has not been established; the homozygous Rb negative genotype is incompatible with embryonic development. The heterozygous Rb genotype seen in children with inherited retinoblastoma does not result in intraocular tumor formation in the mouse.20-22
Animal models of retinoblastoma that contain the naturally occurring mutated Rb gene and that accurately reproduce the tumors’ biological behaviors are critical for the examination of new therapeutic approaches. This paper reports two murine models of intraocular retinoblastoma, one that effectively resembles the histopathological and biological behavior of an aggressively invasive human tumor with metastatic potential and the other that mimics nonmetastatic disease. Therefore, these models may also lead to a better understanding of the mechanisms of retinoblastoma metastasis and allow the development of innovative therapies for metastatic retinoblastoma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human retinoblastoma Y79 and WERI- Rb (ATCC HTB 18 and ATCC HTB 169, respectively), the human embryonic kidney HEK293 (ATCC CRL 1573), the rhesus monkey choroidal RF/6A (ATCC CRL 1780), and the rat C6 glioma (ATCC CCL 107) cell lines (all from American Type Culture Collection, Manassas, VA) were cultured in GVL modified Eagle’s minimum essential medium supplemented with fetal calf serum (5%; Hyclone), streptomycin (100 µg/ml; Irvine Scientific, Santa Ana, CA) and penicillin (100 units/ml; Irvine Scientific). Y79 and WERI-Rb cells were grown in suspension at a concentration of 105-106 cells/ml. Passages 2 through 8 were used for the in vivo studies. Adherent choroidal and glioma cells were propagated by diluting trypsin-treated cells 1:10 in medium when the cultures reached 80% confluence.
Development of Intraocular Tumors
Adult transgenic Rag-2 knockout immunodeficient mice23 were used for the study. Animals were handled at all times in accordance with the Association for Research in Vision and Ophthalmology Statement of the Use of Animals in Ophthalmology and Vision Research. The right eyes of mice were injected with either Y79 or WERI-Rb human retinoblastoma cells. Each animal was first anesthetized with an intraperitoneal injection of 20–30 µl of sodium pentobarbital (Nembutal) solution, 50 mg/ml. The pupil was then dilated with 2 to 3 drops of 2.5% phenylephrine hydrochloride solution, and a drop of topical anesthetic proparacaine hydrochloride (0.5%) was applied. Cellulose eye drops (2.5%) and a glass contact lens were applied to the cornea to aid visualization of the surgical procedures. Injections were performed using a surgical microscope. The conjunctiva of the temporal area of the eye was dissected and an incision was made at the scleral sulcus with a #11 disposable scalpel. Two microliters of sterile phosphate buffered saline containing 2 x 104 cells were injected into the vitreal cavity through the scleral sulcus using a Hamilton syringe with a 33 gauge cannula. Special care was taken to prevent lens damage or posterior retinal punctures. The animals were examined at 1 and 24 hours and then weekly after surgery. Clinical findings regarding the presence of tumor were recorded. After 2 weeks, animals from the Y79 and WERI-Rb groups were sacrificed weekly. Necropsies were performed on every animal. The brain, eyes, mediastinum, lungs, heart, and liver were examined microscopically. The brain was serially cross-sectioned to obtain coronal sections from frontal to occipital lobes and cross-sections of the cerebellum, medulla, and spinal cord.
Histopathology
After dissection, the organs were immediately fixed in 10% formalin. The tissues were processed and embedded in paraffin using conventional automated systems. The blocks were sectioned to obtain levels and serial sections 4 to 5 microns thick and stained with conventional hematoxylin-eosin (H&E). The slides were examined and scored by an unbiased pathologist.
Binding of Retinoblastoma Cell Lines to Choroidal and Glioma Cell Lines
Monkey choroid, rat C6 glioma, or human embryonic kidney cells (1 x 105) were plated in a six-well microtiter plate and allowed to adhere overnight. Y79 or WERI-Rb retinoblastoma cells (1 x 106) were washed in 2 ml PBS and pelleted by centrifugation. The cells were resuspended in 0.2 ml trypsin-EDTA solution (0.25% trypsin and 1 mmol/L EDTA, Gibco BRL, Rockville, MD) for times varying from 0 to 20 minutes. The reaction was terminated by the addition of 2 ml culture media. Cells (0.2 ml or 1 x 105 cells) were then layered over the adherent choroid, glioma, or embryonic kidney cell cultures and incubated at 37°C in humidified air containing 5% CO2 for 3 hours. The culture fluid was decanted and the culture washed twice by vigorous shaking with culture medium (0.2 ml) warmed to 37°C. The three aliquots were pooled and the cells counted using a hemacytometer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Y79 retinoblastoma cell line was derived from the tumor of a 21/2-year-old Caucasian female with a strong maternal history of retinoblastoma. The original tumor was mostly undifferentiated and, although intraocularly invasive, no extraocular extensions were evident.24 A total of 24 mice received Y79 retinoblastoma cells. Systematic in vivo examination of the mouse eyes using an operating microscope first revealed tumors in the vitreal cavity 2 weeks after injection of the cells. Small cortical cataracts were found in the lens of all mice in the study; however, the cataracts did not interfere with adequate evaluation of the vitreal tumors. By 5 weeks, the tumors had obliterated the anterior chamber with the appearance of a whitish, focally neovascularized cornea. After 7 weeks, some eyes also showed proptosis. Contralateral eyes were unremarkable.
Histopathological Findings
Eyes
Serial sections and levels of the eyes were obtained to evaluate
the extent of tumor involvement. Early retinoblastomas (2 weeks after
injection) showed vitreal tumors that were located in the posterior and
mid-equatorial regions of the cavity (Figure 1A
1). The tumors were composed of small
to medium size cells with scanty cytoplasm, hyperchromatic nuclei,
frequent mitoses, and rare necrotic cells (Figure 1A
2). No rosettes or
fleurettes were present. The observed features are similar to those of
a poorly differentiated human retinoblastoma. The site of injection was
identified as a fibroblastic scar in the sclera with attached
peripheral retina and an occasional small focal vitreal hemorrhage
(data not shown). As the time between the tumor cell injection and the
sacrifice of the mice lengthened, tumors were larger and invaded
various ocular structures (Table 1)
. By 3
weeks, the tumor showed subretinal and focal retinal invasion. By 3 to
4 weeks, focal subretinal and ciliary body invasion was observed
(Figure 1A
3). By 5 to 6 weeks, retinoblastoma had invaded the choroid,
retina, subretinal space, and optic nerve of most of the eyes studied
(Figures 1A
4, 1A5). At this time the vitreal tumor showed foci of
necrosis. The tumor was focally present in the anterior chamber by 7
weeks (Figure 1A
6). Between weeks 5 and 7, the tumors showed
involvement of the ocular coats with focal corneal ulceration and
invasion of the sclera. The sclera showed invasion by the tumor
primarily at the injection site and extraocular tumor was also seen at
this area. At 9 weeks, one mouse had evidence of tumor invading the
lens. Two of the contralateral eyes of animals with tumors involving
the optic nerve showed subarachnoid, perineural metastasis by
retinoblastoma (Figure 1A
7), however, no intraocular tumor was observed
in any of the contralateral eyes.
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Mice with early intraocular tumors without invasion of the
subretinal space or optic nerve had no metastases to the brain. After 5
weeks, eight mice had either tumor cells in the subarachnoid space
(Figure 1A
8) or focal brain parenchyma retinoblastoma invasion (Figure 1A
9). The corresponding ocular tumors had involvement of at least the
choroid and, in most cases, the optic nerve and extraocular structures
(Table 1)
.
Heart, Lung, Mediastinum, and Liver
There was no tumor involvement at these sites by microscopic examination. Some animals had mild chronic inflammatory infiltrates in the liver at the level of the portal triads with associated microvesicular steatosis. These changes were unrelated to the extent of tumor progression of ocular retinoblastoma.
WERI-Rb Retinoblastoma Tumors
The WERI-Rb cell line was derived from the tumor of a 1-year-old Caucasian female with no family history of retinoblastoma. This tumor was also essentially undifferentiated and, although it had invaded the optic nerve head, there was no evidence of tumor at any distal site.25 Twenty mice received intraocular injections of WERI-Rb cells and were followed in this arm of the study. Examination of mice showed small vitreal tumors, first noted at 2 weeks after injection and visible only under the operating microscope. The lens frequently had cataracts. There was tumor extension in the anterior chamber by 5 weeks. After this time point most of the eyes appeared to have extensive tumor involvement in the anterior segment.
Histopathological Findings
Eyes
Histological examination of early tumors (2–3 weeks)
showed scanty amounts of tumor cells present mostly in the posterior
vitreous (Table 2
, Figure 1B
1). The tumor
cells were small to medium in size with scanty cytoplasm and
hyperchromatic nuclei. The cells grew in a trabecular pattern. No
fleurettes or true rosettes were found (Figure 1B
2). There were no
areas of necrosis or calcification seen. By day 28, the tumor focally
invaded the retina but rarely the subretinal space (Figure 1B
3). In
most of the cases with subretinal involvement, tumors were contiguous
with the site of injection or were evident in advanced disease. In
striking contrast to the Y79 retinoblastoma cells, WERI-Rb
retinoblastoma cells were predisposed to invade the anterior uveal
tissues (anterior choroid, ciliary body, and iris), and lens (Figure 1
,
B4 and B5). Tumor invasion of optic nerve (Figure 1B
6) or brain (data
not shown) was lacking in the WERI-Rb-injected eyes. Extraocular
extension was seen in advanced tumors and almost always through the
injection site (data not shown).
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There were no tumor cells found in the meninges or brain parenchyma in the WERI-Rb-injected group.
Heart, Lung, Mediastinum, and Liver
No involvement of the heart, lung, mediastinum, or liver was observed. Inflammatory changes in the liver were similar to those seen in the Y79-injected group.
In Vitro Binding of Retinoblastoma Cell Lines to Choroidal and Glioma Cell Lines
One contributing characteristic to the development of metastases
is the specific trafficking of tumor cells to the target organ. To test
the hypothesis that Y79 cells might invade by specifically adhering to
choroidal or glial cells, suspensions of either Y79 or WERI-Rb cells
were incubated with the adherent RF/6A monkey choroidal cell line. The
RF/6A cell line was established from a spontaneously transformed
endothelial cell line derived from the choroid-retina of a rhesus
macaque fetus and exhibits endothelial cell
characteristics.26
After incubation for 1 hour,
non-adherent cells were removed by vigorous washing with media and the
plates were observed under the microscope (Figure 2A)
. Plates containing Y79 cells showed a
significant percentage of the retinoblastoma cells bound to the monkey
choroidal cell line, whereas plates containing WERI-Rb cells showed
little binding. Similar results were found when the two retinoblastoma
cell lines were incubated with the rat C6 glioma
cell line27
(Figure 2A)
. Neither retinoblastoma cell line
exhibited significant binding to the HEK293 human embryonic kidney cell
line (Figure 2A)
. Trypsin treatment of Y79 cells reduced the binding of
these cells to both the choroidal and glioma cell lines (Figure 2B)
,
whereas similar treatment with neuraminidase had no effect on binding
(data not shown). The trypsin treatment of Y79Rb cells reduced binding
to either the choroid cells or the glioma cells in a time-dependent
manner (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This manuscript aims to describe not only two models of retinoblastoma but also, and maybe more importantly, some of the striking differences between the two models. Although the procedures used to induce tumor formation are identical and tumors that closely resembled human retinoblastoma result from injection of either retinoblastoma cell line, the characteristics of the tumors formed are remarkably and reproducibly different. The WERI-Rb tumors exhibit characteristics of localized, nonmetastatic human retinoblastoma, whereas the Y79 tumors exhibit the histopathological characteristics of aggressive human retinoblastoma with invasion of the optic nerve and brain that would likely result in distant metastatic disease and metastasis to the contralateral optic nerve. Differences in the growth rates of these two cell lines cannot completely explain the histopathological differences exhibited by the tumors because, by the end of the experiments, animals in both groups had tumors that had completely replaced the normal ocular tissues in the affected eye. Only animals in the Y79 tumor group had tumors that appeared to mimic the aggressive, rapidly growing, metastatic retinoblastomas only occasionally observed in North America and Europe but more commonly seen in Mexico, Central and South America, Saudi Arabia, and India.29 The WERI-Rb animal model behaved like patients with nonmetastatic retinoblastoma more commonly seen in Europe and North America.
In vitro binding studies suggested that a specific difference in the membrane protein structure of the cells may, at least in part, play a role with respect to the observed difference in metastatic behavior of the two animal models. Adherent cell lines derived from monkey choroid or rat C6 glioma (derived from tissues that are similar in origin to the target tissues of invasive retinoblastoma), were found to rapidly bind Y79 cells. Y79 cells also bound to human Hs 683 glioma cells (data not shown). Specificity was suggested since Y79 cells did not bind to human embryonic kidney cells. Trypsin abolished this binding suggesting protein on the surface of Y79 cells is apparently involved in the cell-cell interaction. Treatment with neuraminidase had no effect. WERI-Rb cells did not appear to interact with choroidal, glioma, or embryonic kidney cell lines under the same experimental conditions. Understanding the mechanism of this binding may aid in understanding the biochemical events related to metastasis of retinoblastoma. Furthermore, this simple in vitro binding assay may help predict which patients with retinoblastoma might be prone to develop metastatic disease.
Distant metastases outside the central nervous system were not observed in these animal models. Metastases outside of the central nervous system in human disease is only rarely observed and is considered a very late event in the course of the disease. Distant metastases may be seeded via the vasculature, probably after choroidal or orbital invasion. Although choroidal invasion was eventually always observed in the Y79 cell animal model, the animals in these particular studies were not allowed to survive for longer than 9 weeks. If the animals were allowed to progress, it is possible that distant metastases outside of the central nervous system might have been observed.
All of the animal models described previously may have utility in particular research protocols, but none is perfect in all settings. The extent of choroidal, extraocular, or optic nerve invasion at the time of diagnosis1,30 most often determines the prognosis of children with retinoblastoma. The tumors formed in the models described in this paper closely mimic human disease. We have recently used these models of retinoblastoma in preclinical evaluations of the potential efficacy of suicide gene therapy28 and gene replacement therapy31 for retinoblastoma. These models provide an opportunity for research into the similarities and differences in the biological behavior and the response to treatment of metastatic and nonmetastatic retinoblastoma. These two models will also allow a detailed study of the molecular biological and physiological differences between metastatic and nonmetastatic retinoblastoma originating from cells with the naturally occurring mutations in the Rb genes.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Foundation for Research and the Retina Research Foundation (to R. L. H.), the Lions Eye Bank and the Moran Foundation (to P. C.-B.), the Knights Templar Eye Foundation, Inc. (to M. Y. H.), and the General Clinical Research Center no. M01RR00188 (to C. E. A.-C.).
Accepted for publication July 1, 2000.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). 1, Eye
at 2 weeks with a tumor in the vitreal cavity between a normal lens
(L) and a normal retina
(R). Original
magnification, x40. 2, Tumor in the vitreal space showing
small to medium size cells with scanty cytoplasm, hyperchromatic
nuclei, and frequent mitoses. Original magnification, x400.
3, Eye at 3 weeks showing normal lens
(L), retina
(R), tumor in the
vitreous and tumor invasion of the ciliary body
(
). Original
magnification, x40. 4, Eye at 6 weeks showing tumor in the
vitreal space (Vi) with
an area of necrosis
([trioro]) and tumor
invading the retina (R),
subretinal space (SR),
and choroid (C). Original
magnification, x40. 5, Eye at 6 weeks showing tumor in the
subretinal space (SR) and
tumor invading the optic nerve
(ON). Original
magnification, x40. 6, Eye at 7 weeks showing lens
(L) with tumor in the
anterior (AC) and
posterior (PC) chambers.
Original magnification, x40. 7, Contralateral eye at 8
weeks showing normal retina
(R) and tumor surrounding
the optic nerve (ON).
Original magnification, x40. 8, Brain at 6 weeks showing
tumor in the ventricular space
(V). Original
magnification, x40. 9, Optic chiasm
(OC) at 6 weeks showing
tumor invasion of the neural tissue. Original magnification, x100.
B: Histology of WERI-Rb retinoblastoma mouse model. WERI-Rb
cells (2 x
104) were injected into
the vitreal space of transgenic Rag-2 knockout immunodeficient mice.
Tissues were histologically examined at the indicated times
postinjection as described in Methods. Tumor is indicated by the
arrows in each panel
(