Human Retinoblastomas Have Similar Molecular Profiles
To assemble a molecular profile of human retinoblastoma, we carried out gene expression profiling of 52 fresh retinoblastomas from patients who underwent surgical enucleation prior to anti-cancer therapy (see Table S1
available online). For profile comparisons, we also included four retinoblastoma cell lines (Y79, Weri1, Rb-355, and Rb-13) (Griegel et al., 1990a
; Griegel et al., 1990b
; McFall et al., 1977
; Reid et al., 1974
) in this analysis. A principal component analysis (PCA) and hierarchical clustering revealed that the majority of tumor samples (46/52, 88.5%) cluster tightly around a central core molecular signature; no clear subtypes were detected (). Three tumors that had the same core molecular signature (SJRB-39, SJRB-41, and SJRB-42) were introduced into the vitreous of immunocompromised mice for future orthotopic xenograft studies. Transmission electron microscopy (TEM) analysis of these three primary tumors confirmed that they had many of the cellular features of retinoblastoma, including mitotic figures, abundant mitochondria, cellular processes, and extensive cell-cell junctions (Johnson et al., 2007
) (; Figure S1
Molecular Characterization of Human Retinoblastoma Cells
To gain insight into the differentiation features of human retinoblastomas, we analyzed the expression of 28 genes from the microarrays that are restricted to different retinal cell classes (). A mixture of genes that are normally expressed in retinal progenitor cells and differentiated retinal neurons (i.e., photoreceptors and amacrine cells) were expressed in human retinoblastomas (). Real-time RT-PCR was used to confirm these data (; data not shown). These results together suggested that human retinoblastomas are relatively homogenous and express a hybrid differentiation phenotype.
Mouse Retinoblastomas from Six Genotypes Show Striking Similarities
We have generated six mouse lines that develop retinoblastoma by using the Chx10-Cre
to conditionally inactivated tumor suppressor pathways in their developing retinae (Rowan and Cepko, 2004
) (). Chx10-Cre;RbLox/Lox;p107–/–
(p107s), and Chx10-Cre;RbLox/Lox;p107–/–; p130Lox/Lox
(RbTKO) develop retinoblastoma when different combinations of Rb family members are inactivated in retinal progenitor cells (). The other two strains developed retinoblastoma when Rb-pathway inactivation was combined with p53 pathway inactivation. The Chx10-Cre;RbLox/Lox
(p53TKO) mice lack p53, Rb, and p107, and the Chx10-Cre;RbLox/Lox;p107–/–;MDMXTg
(MDMX) mice conditionally overexpress the MDMX
gene (Xiong et al., 2010
) in retinal progenitor cells that lack Rb and p107 ().
Characterization of Mouse Retinoblastoma Cells from Six Different Strains
To characterize tumor incidence and progression in these mouse models of retinoblastoma, we monitored 509 mice weekly from 3 weeks of age until advanced tumor progression necessitated euthanasia (). Retinal camera images and fluorescein angiography of early-stage tumors () showed that in mice, retinoblastoma initiates in a manner similar to that seen in humans. Tumors originate in multiple foci across the retina and efficiently reorganize the retinal vasculature. The time from tumor diagnosis to moribund status reflected the ages at which the six strains developed advanced-stage retinoblastoma (). This finding suggests that the difference between strains was due to differences in the rate of tumor progression rather than the rate of initiation. Histopathologic analysis of five mice from each strain (60 eyes total) confirmed that optic nerve, anterior chamber, subretinal, and choroidal invasion were present in each strain (Figures S2A–S2F
). Anterior chamber and subretinal invasion was the most common (80%–100% of eyes examined), and optic nerve and choroidal invasion was the least common (30%–60% of eyes examined). Necropsy on 22 of the p53TKO mice revealed that 23% (5/22) of the mice had metastases. Four of the metastatic lesions were in the brain, and one was in a lymph node, which is consistent with the pattern seen in patients with metastatic retinoblastoma. All of the mouse retinoblastoma strains displayed abundant rosettes, except for the RbTKO strain, which had only a few areas with rosettes ( and data not shown).
We performed immunostaining for markers of proliferation (Ki67), apoptosis (caspase 3), neuronal differentiation (Tuj1, synaptophysin), astrocytes and activated Müller glia (GFAP), vascular endothelial cells (CD34), and macrophages (Mac2, F4/80) on paraffin-embedded sections from five mice per geno-type and scored the proportion of immunopositive cells (Figures S2G–S2I
). At the stage when these tumors were isolated (moribund status), there were no dramatic differences across strains in the expression of these markers by genotype. In addition, TEM analysis of another 30 tumors across the six genotypes indicated that there were no significant differences in cellular morphology, including differentiation features such as processes and junctions (Figure S2J
To develop a molecular profile of our retinoblastoma mice, we performed gene expression array analysis on 120 tumors (20 per strain). When combined on a PCA plot, the six retinoblastoma mouse strains exhibited extensive overlap and intermixing of their gene expression profiles (). The gene expression profiles were highly correlated (95%–99%) across all strains but the most distinct were p53 TKO and Rb TKO (Figures S2K and S2L
and Table S2
). The orthologs of the 28 genes used to characterize the retinal differentiation properties of human retinoblastomas () were also coexpressed in the mouse retinoblastomas (Figure S2M
). Cell type scoring and real time RT-PCR confirmed a hybrid molecular signature (Figures S2N and S2O
). Hierarchical cluster analysis () identified two clusters (j and k) that exhibited gene expression differences in photoreceptor and amacrine/horizontal neuron genes that were validated by real-time RT-PCR (Figures S2P–S2S
and Table S3
). However, even in the clusters with enriched gene expression of particular retinal differentiation pathways, multiple developmental programs were coexpressed (Figures S2P–S2S
Mouse and Human Retinoblastomas Share Molecular Profiles
To directly compare the molecular signatures of human and mouse retinoblastomas, we focused our analysis on the 15,646 unique ortholog-matched unigene pairs present on the human and mouse gene expression arrays. There are three potential sources of variance in comparing human and mouse array data: (1) differences in individual probe sets of the ortholog-matched unigenes; (2) general biological differences across species; and (3) biological differences between human and mouse tumors, which provide insight into the biology of retinoblastoma and how well the mouse models recapitulate the human disease.
To minimize the contribution of the first two sources of variance and highlight the major differences between the human and mouse retinoblastomas, we developed an algorithm for cross-species comparisons of array data (see Supplemental Experimental Procedures
). Using this method, we found that 12,267 of 15,646 (78%) ortholog-matched unigene pairs exhibited similar levels of expression (<1 quartile difference) across species. To establish the significance of this correlation, we randomized the probe sets in a series of 50,000 simulations per chip and performed the same analysis. These simulations demonstrated that any correlation greater than 3.5% is significant with a p value of at least 0.00002. The majority of the differences between human and mouse retinoblastoma unigene pairs (3094/3379; 92%) was a 1-quartile difference in expression; 280 of 3379 (8.3%) were a 2-quartile difference; and 5 of 3379 (0.1%) were a 3-quartile difference in expression. Among those unigene pairs that were most distinct across species (p = 2.3 × 10–17
), 269 differed across all mouse genotypes (Table S4
), and an additional 525 differed in subsets of the six genotypes (Table S4
PCA analysis demonstrated that most of the difference between the profiles of human and mouse retinoblastoma cells was in a single dimension (). A correlation analysis of all mouse genotypes and clusters to human tumors indicated that the RbTKO strain was the best match to human retinoblastomas by a small margin ().
Comparison of Gene Expression in Human and Mouse Retinoblastoma
Mouse and Human Retinoblastomas Express Multiple Differentiation Pathways
To perform an unbiased analysis of retinal cell type molecular signatures, we took advantage of a recent series of publications using gene expression array analysis of individually isolated retinal progenitor cells and differentiated retinal neurons and glia (Cherry et al., 2009
; Roesch et al., 2008
; Trimarchi et al., 2008
). Cone photoreceptors have not been analyzed using this single-cell technique, so we used in situ-validated microarray data from purified pools of cone photoreceptors (Akimoto et al., 2006
Cell type signatures of retinal progenitor cells, rod photoreceptors, cone photoreceptors, amacrine cells, ganglion cells, and Müller glia (Table S5
) were used to characterize the differentiation state of all 172 mouse and human retinoblastomas in our microarray study, as described previously (Cherry et al., 2009
; Roesch et al., 2008
; Trimarchi et al., 2008
). We also analyzed a group of previously characterized genes that regulate the G2/M phase of the cell cycle to compare the proliferation signature in the retinoblastomas to that of normal retinal progenitor cells. The most robust cell type-specific signatures for human retinoblastomas were those of progenitor cells, G2/M genes, and rod photoreceptors (; Figures S3A and S3B
). The rod signature was indistinguishable from normal differentiated rods, and the progenitor signature was indistinguishable from that of normal progenitor cells. More importantly, the human retinoblastomas also had robust signatures for amacrine cells and cone photoreceptors (). The strongest cell type-specific signature for mouse retinoblastomas was the amacrine cell score; these tumors also had a pronounced expression of genes that regulated the G2/M phase in progenitor cells (; Figures S3A and S3B
). Statistical analysis of the human and mouse retinal cell type signatures identified the amacrine cell signature and the rod cell signature as the most similar and the most distinct, respectively, across species (; Table S6 and Supplemental Experimental Procedures
Retinal Cell Type-Specific Gene Signatures for Human and Mouse Retinoblastomas
When signatures were compared, correlations became evident between rod and cone scores, G2/M and progenitor scores, and between amacrine and ganglion cell scores along a continuum for both mouse and human retinoblastomas (Figure S3B
). The cell type signatures that are normally incompatible (i.e., progenitor versus rod or amacrine versus rod) coexisted in the same tumors (; Figure S3B
). Immunostaining for cell type markers and scoring of dissociated cells provided additional evidence that individual cells express multiple retinal differentiation pathways (Figures S3C–S3F
; data not shown). These analyses suggest that human and mouse retinoblastomas represent a true hybrid of progenitor cells, photoreceptors, and amacrine interneurons.
To extend these observations and perform a broader, unbiased analysis of individual tumor cells, we isolated retinoblastoma cells from one of our orthotopic xenografts (SJ-RB39) and performed single-cell gene expression array analysis, as done previously (Cherry et al., 2009
; Roesch et al., 2008
; Trimarchi et al., 2008
). These data confirmed that individual retinoblastoma cells in humans or mice express a mixture of differentiation programs for photoreceptors, amacrine cells, and progenitor cells (; Figures S3G–S3I
Multiple Differentiation Pathways in Human Orthotopic Retinoblastoma Xenografts
The expression of multiple differentiation pathways in primary retinoblastomas may reflect contamination of normal retinal neurons in the tumors. To test this possibility, we established orthotopic xenografts of three human retinoblastomas (SJ-RB39, SJ-RB41, and SJ-RB42; see ) by injecting primary tumor cells into the vitreous of SCID mice. For comparison, we also obtained a previously described orthotopic xenograft (MSKCC176) (Xu et al., 2009
). If normal retinal cells contaminate primary tumors, then they would be rapidly overgrown in the orthotopic xenografts, and any contribution from the recipient mouse retina would not be detected because our probes and primers are specific for the human sequences. We carefully documented the morphology of the live cells and tissue specimens by TEM and intraocular histopathologic analyses (; Figure S4
). Tumor initiation, expansion, and progression were monitored using a digital retinal camera (), fluorescein angiography (), and weekly inspection for anterior chamber invasion (). The gene expression profile for the primary tumors used for the SJ-RB xenografts were representative of the overall population of human retinoblastomas in our study; minimal change occurred over the three passages in the vitreous of the eyes of the SCID mice for the SJ-RB xeno-grafts or the MSKCC176 xenograft (; Table S7
). The cell type signatures for these orthotopic xenografts were very similar to that of the primary human tumors ().
Orthotopic Xenografts of Human Retinoblastomas
Human retinoblastomas express high levels of the MDMX
gene and the MDMX protein (Laurie et al., 2006
). We confirmed that primary human retinoblastomas and the SJ-RB and MSKCC176 orthotopic xenografts express high levels of MDMX mRNA and protein ( and data not shown). In contrast, MDM2 levels were below the limit of detection in any of the human tumor xenografts or cell lines in our study, including the MSKCC176 xenograft (). These data suggest that the orthotopic xenografts provide an ideal model in which to study inhibitors of the MDMX-p53 interaction.
Neuroanatomical and Neurochemical Features of Amacrine Cells in Retinoblastoma
In addition to their molecular signatures, each retinal cell type exhibits distinct morphological and anatomical features that can be quantified on TEM images. We established a series of six morphometric features of cell bodies and ten neuroanatomical features of cellular processes that allowed us to unambiguously distinguish the different classes of retinal cell types (; Table S8
and Table S9
). On the basis of these morphometric and neuroanatomical measurements, the human and mouse retinoblastomas most closely resembled amacrine interneurons than photoreceptors, bipolar cells, or Müller glia (; Table S8
and Table S9
). One of the most striking features of the neuroanatomical analysis of plexus regions was the presence of dense core vesicles in human and mouse retinoblastomas (). These structures were present in the same tumors with connecting cilia and basal bodies (). Dense core vesicles are a hallmark of monoamine neurotransmitter secretion, and the only cell type in the retina that has dense core vesicles and produces monoamines are amacrine cells (Macneil et al., 1999
; MacNeil and Masland, 1998
). We used HPLC-ED to measure the levels of dopamine, serotonin, and their metabolites in normal retinae, mouse retinoblastoma, and human retinoblastoma xenografts (). The levels of these molecules in human and mouse retinoblastoma were similar to those found in normal retinae.
Morphometric, Neuroanatomical, and Neurochemical Analyses of Retinoblastoma Cells
Functional Significance of Neuronal Differentiation in Retinoblastoma
To begin to explore the functional significance of the features of neuronal differentiation in retinoblastoma, we searched our database of pediatric solid tumor cell line sensitivity to FDA-approved drugs and well-characterized cytotoxic agents (A.A. Shelat and R.K. Guy, unpublished data). This database includes dose-response data in triplicate for more than 600 compounds on 21 different solid tumor cell lines including retinoblastoma, osteosarcoma, rhabdomyosarcoma, and neuroblastoma. The Y79 and Weri1 retinoblastoma cell lines were found to be uniquely sensitive to a subset of antipsychotic agents. Cheminformatic analysis showed that the agents were all derivatives of phenothiazines, which are broad-acting monoamine receptor inhibitors, including dopamine, serotonin, histamine, and epinephrine receptors. These intriguing results prompted us to analyze neurotransmitter receptors, transporters, and regulators in detail and to study their functional significance for retinoblastoma growth and survival.
We first identified genes encoding all major neurotransmitter receptors and transporters in the retina that were represented on the gene expression microarrays (175 genes, 212 probe sets, Table S10
). For example, we reviewed the data for 29 genes (43 probe sets) involved in GABA neurotransmission. Among those 29 genes, only 6 had expression levels for either tumor or normal retina greater than Log2
= 8, and only 3 genes (GABBR1
, and GABRB3
) were expressed in primary human retinoblastomas or cell lines at levels similar to or greater than that in normal retina (). Those three genes were selected for further analysis (see below). Identical criteria were used to select genes involved in glutamate, glycine, melatonin, histamine, epinephrine, dopamine, serotonin, cholinergic/ muscarinic, and opioid signaling in neurons (). In the gene expression array analysis, the monoamine receptors and pathways (dopamine, histamine, epinephrine, and serotonin) were most abundant in retinoblastoma, but other pathways (GABA, glycine, and glutamate) were also expressed ().
Inhibition of Neurotransmitter Receptors Reduces Retinoblastoma Growth In Vitro and In Vivo
To validate the gene expression microarray data, we generated TaqMan real-time RT-PCR probes for each gene that was expressed in the human retinoblastomas and analyzed expression in the SJ39 xenograft, human retinoblastoma cell lines, and normal retinae. In total, 96 genes were analyzed in duplicate for each sample (complete data set available upon request). Several of the monoamine/catecholamine receptors including serotonin receptors (HTR3A, HTR1E), a dopamine receptor (DRD5), and a histamine receptor (HRH3) were expressed in retinoblastoma at levels equal to or greater than normal human retina (). In addition, several of the genes involved in the biosynthesis of catecholamines such as TPH1, which encodes the rate-limiting enzyme for serotonin synthesis, and PHOX2A, which regulates genes that are important for dopamine neurotransmission, were expressed at high levels in the human retinoblastomas (). All three alpha-2 adrenergic receptors (ADRA2A, ADRA2B, and ADRA2C) were expressed in the tumors (). These proteins regulate the catecholamine-mediated inhibition of adenylate cyclase through the action of G-proteins in neurons. Immunoblotting confirmed the expression of these proteins ( and data not shown).
Although most genes involved in neurotransmission that are upregulated in retinoblastomas are those found in amacrine cells and involved in catecholamine/monoamine signaling, there were some that are from other pathways in the retina including GABA, glycine, and glutamate. It is possible that proteins important in other neurotransmitter pathways are expressed in retinoblastoma and are functionally important but were expressed at levels below the limit of detection in our molecular studies. Therefore, to determine which of the major neurotransmitter pathways are functionally relevant, we performed a series of pharmacological experiments with retinoblastoma cell lines in culture. We tested 13 well-characterized pharmacological agents targeting each pathway in all 3 retinoblastoma cell lines (Weri1, Y79, and RB355) and a nonneuronal cell line (BJ) in triplicate dose-response experiments ( and data not shown). Only the broad-acting monoamine transporter inhibitors showed any activity in these experiments, and fluphenazine and chlorpromazine were the most active among those agents ( and ). We analyzed the receptor-binding profile of these two agents by using data from the PDSP Ki
). Using this approach, we identified another pharmacological agent with a similar profile (thioridazine) and tested it in dose response studies against all three retinoblastoma cell lines in triplicate. This compound gave results similar to fluphenazine and chlorpromazine (). None of the agents were active against the BJ cells (data not shown). In addition, more specific catecholamine pathway inhibitors such as the selective serotonin reuptake inhibitors (SSRIs) had no effect on retinoblastoma cell lines (data not shown). These data suggest that broad inhibition of multiple classes of monoamine receptors is required to affect retinoblastoma cells in culture.
Inhibition of Neurotransmitter Receptors in Retinoblastoma
To test whether the inhibition of monoamine receptors affected retinoblastoma cell proliferation and/or cell viability, we exposed the three retinoblastoma cell lines to different concentrations of fluphenazine and chlorpromazine for 24 hr and then labeled the cells in S-phase with a 1 hr BrdU pulse. We performed immunostaining for BrdU and phospho histone H3 (pH3) to monitor proliferation and activated caspase-3 to monitor cell death (). Overall, the retinoblastoma cells exhibited reduced BrdU and pH3 labeling with increasing concentration of fluphenazine or chlorpromazine (). There was no significant effect on the proportion of activated caspase-3+ cells ().
To extend our observations to a more relevant in vivo model, we tested the effect of these monoamine transporter inhibitors on the orthotopic xenograft SJ-39. Twenty immunocompromised mice were injected intravitreally with the SJ-39 retinoblastoma xenograft, and after 3 weeks, engraftment was confirmed with retinal camera and fluorescein angiography. The 20 animals were divided into four groups, and each group received a subconjunctival injection of vehicle, topotecan/carboplatin, fluphenazine, or chropromazine once weekly for 3 consecutive weeks in a double-blind study design. The tumor volume was then measured in each of the 40 eyes by using MRI (), and histological analysis was performed (). Fluphenazine and chlorpromazine injections appeared to significantly reduce tumor volume in vivo (), and this effect was confirmed by histopathologic analysis ().