Adhesion G protein-coupled receptor, ELTD1, is a potential therapeutic target for retinoblastoma migration and invasion

Background Prognosis for pediatric metastatic Retinoblastoma (Rb) is poor and current therapies are limited by high systemic toxicity rates and insufficient therapeutic efficacy for metastatic Rb. Tumor dissemination to the brain is promoted by the heterogeneous adhesive and invasive properties of Rb cells within the tumor. In this study we evaluate, for the first time, the expression, and roles of the ELTD1 and GPR125 adhesion G protein-coupled receptors (GPCRs) in Rb cell migration, viability and invasion. Methods We characterized the RNA expression of adhesion-GPCRs in 64 Rb tumors compared to 11 fetal retinas using the database from the Childhood Solid Tumor Network from St Jude Children’s Research Hospital. The role of ELTD1 and GPR125 in Rb were investigated ex vivo by microarray analysis, in vitro by cell viability, Western blot and migration assays, in addition to imaging of the subcellular localization of the GPCRs. To elucidate their role in vivo we utilized siRNA technology in an established Rb orthotopic xenograft murine model. Results Our investigation demonstrates, for the first time, that ELTD1 but not GPR125, is significantly increased in Rb tumors compared to fetal retinas. We utilized established the Rb cell lines Y79 and Weri-Rb-1, which represent an aggressive, metastatic, and non-metastatic phenotype, respectively, for the in vitro analyses. The studies demonstrated that ELTD1 is enriched in Weri-Rb-1 cells, while GPR125 is enriched in Y79 cells. The measured differences extended to their subcellular localization as ELTD1 labeling displayed punctate clusters in cell-to-cell adhesion sites of Weri-Rb-1 cells, while GPR125 displayed a polarized distribution in Y79 cells. Lastly, we demonstrated the lack of both adhesion receptors does not affect Rb cell viability, yet inhibition of ELTD1 decreases Y79 cell migration in vitro and invasion in vivo. Conclusion Taken together, our data suggest that ELTD1, is a potential target to prevent extraocular Rb. The results within establish ELTD1 as a potential therapeutic target for metastatic Rb. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-020-07768-3.


Background
Retinoblastoma (Rb) is the most common intraocular tumor that threatens pediatric survival rates if not diagnosed and treated early. Upon mutation of both alleles of the tumor suppressor RB1 gene, cells from the retina abnormally proliferate to create a tumor mass that disrupts intraocular structures. The dissemination of the disease leads to central nervous system (CNS) metastasis, increasing mortality rates. Current therapies aim to limit tumor proliferation and growth. The administration of these therapies remains challenging due to several clinical factors, including patient's age, sex, laterality, vascularity, staging at diagnosis [2,6,11,12,32], and tumor heterogeneity [8,17]. Recent work has shown Rb cell heterogeneity influences resistance to therapies as some tumor cells display different degrees of invasiveness and aggressiveness within the same tumor mass [35]. Therefore, this highlights the importance of identifying novel therapeutic modalities that target Rb cells with varied metastatic potential.
Adhesion G protein-coupled receptors (adhesion-GPCRs) have been at the forefront of cancer research for their roles in [3,20,40], regulating cellular adhesion, polarity, migration and angiogenesis [21,27]. To date, 33 receptors have been identified, with at least 15 of these being dysregulated in distinct tumor types [3]. To our knowledge, no prior study has elucidated the role of adhesion-GPCRs in Rb. We focused our investigation on two of these adhesion-GPCRs in Rb tumors, the epidermal growth factor, latrophilin and seven transmembrane domain containing 1 (ELTD1/ ADGRL4) and the G-protein receptor 125 (GPR125/ADR-GRA3). While ELTD1 is associated with glioblastoma, colorectal cancer, cardiac and renal function, [1,14,26,33,34,39,42], GPR125 plays vital roles during embryonic development, cell adhesion, signaling and planar cell polarity [19]. Overexpression of GPR125 has also been reported after brain injury and myeloid sarcoma formation, while its downregulation during colorectal cancer is a predictive biomarker of poor prognosis and higher probability of recurrence [13,29,38]. Our aim was to investigate the role of these two adhesion-GPCRs in Rb, as both ELTD1 and GPR125 influence multiple aspects of cancer during disease development and progression. To accomplish this, we utilized a combination of in vivo work, ex vivo tumors and the established and well-characterized Rb cell lines, Y79 and Weri-Rb-1 cells, representing the metastatic and nonmetastatic phenotype. Our results demonstrate that ELT-D1is a potential target to prevent Rb cell migration and optic nerve dissemination, while GPR125 may play a role in Rb cell adhesion.

MicroArray
Adhesion-GPCR microarray expression data were obtained from the CSTN at SJCRH [36]. This dataset of genes was analyzed from 64 human Rb tumors compared to 11 fetal retinas using the JMP v12.2 Software (www.jmp.com/en_us/software/jmp.html) to conduct a hierarchical cluster analysis (HCA). Ward's method, in which clustering is based on the within-cluster sum of square, was applied for the HCA and dendrogram, and supervised heat maps were presented. Boxplots were generated to graphically examine the distribution of log base 2 transformed data of expression per select gene per group (normal retina vs. retinoblastoma) and a series of ANOVA tests were conducted to determine the group difference of expression level per select gene using SAS 9.3 (www.sas.com/en_us/software/analytics/stat.html).

RT-PCR RNA isolation and cDNA synthesis
Sections of paraffin-embedded enucleated eyes from Rb patients were cut approximately 8 μm thick before being placed in a sterile RNase free microcentrifuge tube (Mid-Sci, St. Louis, MO). Each sample was incubated with deparaffinization solution (Qiagen, Valencia, CA) and incubated at 56°C for 3 mins followed by Proteinase K incubation. RNA isolation was performed using the Qiagen® miRNease Mini Kit (Qiagen) using manufacturer's specifications. RNA concentration was assessed using a Nanodrop Spectrophotometer (Nanodrop, Wilmington DE). A total of 50 ng of RNA was used to synthesize cDNA using the SuperScript® VILO™ cDNA Synthesis Kit (Life Technologies, Carlsbad, CA).

cDNA pre-amplification
Material was pre-amplified using cDNA, TaqMan® Pre-Amp Master Mix and a cocktail of the pooled primers mix listed in Table 1. Enzyme activation was carried out at 95°C for 10 mins, followed by 14 cycles of amplification (95°C for 15 s, followed by 60°C for 4 mins). Pre-amplified material was diluted 1:10 in Tris EDTA buffer and kept at − 20°C until ready for use. Gene targets were previously tested using an amplification efficiency test.

qPCR reaction
Each reaction was prepared in a 10uL final volume, containing the TaqMan Universal Master Mix, the diluted cDNA, primers, and nuclease free water, as previously described [9,10]. Samples were analyzed using the Roche LightCycler® 480 using the following conditions: hold step of 50°C for 2 mins followed by 95°C for 10 mins. Thermal cycling was programmed to run 40 cycles of 95°C for 15 s followed by 60°C for 1 min. All measurements were done in replicates of 4. Relative quantitation was performed using the Comparative C T Method using the values of the endogenous or housekeeping gene (HPRT1) and target genes in each sample ( Table 1). The relative fold change was calculated using the following equation: R q = 2 −ΔC T , where ΔC T = C T target gene − C T reference gene. Data are presented as mean ± SEM. Differences were assessed by Prism, Graph Pad (Graph Pad, La Jolla, CA).

Fetal retina and retinoblastoma human samples
Human fetal retina was purchased from Advanced Bioscience Resources (ABR, Alameda, California). Upon arrival, retina tissue was dissected out of the eyecup, cut into small pieces, placed in sterile Eppendorf tubes and flash frozen in dry ice prior to their storage at a − 80°C until ready to use for protein extraction and Western blot assays. Rb tumor samples were obtained from the CSTN at SJCRH. All studies received approval from the University of Puerto Rico Medical Science Campus Institutional Review Board (IRB) (Approval #B0580215).

Indirect immunofluorescence assay
Y79 and Weri-Rb-1 cells were cultured into 35 mm petri dishes that contained 10% Poly-L Lysine (Sigma P4832) coated glass coverslips. After seeding cells at a density of 1.0 × 10 6 /mL in coated plates for 30 min, they were fixed

Cell viability assay
Transfected cells and respective non-transfected controls were assayed for viability using Trypan Blue. Cells were seeded in 12-well plates containing RPMI with 10% FBS and incubated at 37°C for 24 h. After cell collection, these were diluted in Trypan Blue Dye (1:1 dilution) and quantified for viability with a hemocytometer. Viable cells remain unlabeled while non-viable cells were labeled blue due to compromising of the cellular membrane. Percentage of cell viability was determined using a Nexcelom Cellometer 2000 Cell Counter System.

Transwell assay
Cell migration was conducted by using Boyden chamber assays to examine Rb cell migration. These contained 8 μm-pore-size polyethylene terephthalate basement membranes (BM) with a Falcon cell-culture insert (BD Biosciences, Bedford, MA). Y79 and Weri-Rb-1 cells (2 × 10 5 cells/well) were seeded in 100uL serum-starved media (RPMI without serum) into the upper chamber, while 500uL of RPMI supplemented media with 10% FBS was added to the lower chamber. Seeding conditions of Rb cells (Y79 and Weri-Rb-1) were as follows: non-transfected cells, negative control (non-targeted RNA), and either siELTD1 or siGPR125. Once the cells were seeded, the membrane was inserted into the lower chamber and cells were incubated for 24 h at 37°C. Following the incubation period, the nonmigrating cells were removed from the upper chamber by gently scraping with a cotton swab moistened with 1x PBS 5 times. Fixation and staining of cells located on the lower side of the insert membrane was achieved using cold methanol (10-15 min) and 1:10,000 Hoechst dye (Sigma, B2261) at room temperature for 30 min. Cells were mounted using Richard-Allan Scientific™ Cytoseal™ 60 mounting media (ThermoScientific, 8319-16). Images of four different fields were taken using a 20x objective and cells were counted to calculate the average number of cells that migrated through the membrane.
To ensure that quantified Rb cells corresponded to migrating Hoechst-labeled cells, confocal Z stack images were taken for 3D reconstruction and quantification as validated by a maximum intensity projection image of each membrane (20x; Nikon Inverted Microscope Model TI, with Confocal, along with a Nikon Imaging Software-Confocal).
Orthotopic xenograft Rb mouse model Y79 and Weri-Rb-1 cell lines are well established and were previously demonstrated to follow metastatic and non-metastatic patterns in vivo, respectively [8]. We utilized these cell lines to do the orthotopic xenograft Rb murine model [17,25]. Prior to eye injections or intraocular pressure measurements, mice were anesthesized with 2% isoflurane via gas (air) delivery of 2 L/min. Our in vivo conditions included a total of 5 control mice (inoculated with untransfected Y79 cells) as well as a total of 7 experimental mice (inoculated with Y79 cells that were previously transfected with siELTD1). Cells were inoculated into the vitreous of ICR-SCID mice (Taconic Laboratories, ICRSC-M, IcrTac:ICR-Prkdc scid ). Approximately 50,000 cells were injected in a 5 μL final volume per eye into anesthetized ICR-SCID (IcrTac:ICR-Prkdc scid) mice. The intraocular pressure (IOP) of inoculated mice was measured using a tonometer on a weekly basis as a signature of tumor growth. Mice with IOP ≥ 15 were euthanized in a CO2 chamber, followed by cervical dislocation and lack of withdraw reflex to assure animal death. Eyes were carefully dissected to preserve the attached optic nerve (ON). Dissected eyes were fixed in 4% PFA overnight at 4°C and subsequently embedded in paraffin. Rb tumor invasion to the ON was analyzed with histological sections of inoculated eyes and extensive invasion was defined when tumor was detected2 00-300 μm beyond the ON head. Eyes embedded in paraffin were sectioned (5 μm) in the sagittal plane through the ON and stained with H&E. Tumor invasion to the anterior chamber, cornea, ciliary epithelium, retina and optic nerves were compared across transfected and non-transfected eyes. These animal studies were conducted in accordance with the University of Puerto Rico (UPR) Institutional Animal Care and Use committee (IACUC) guidelines (IACUC #A190115).

Microscopy
Membranes of the transwell assays as well as ELTD1 and GPR125 protein distribution in Rb cells were imaged using a Nikon Inverted Microscope Model TI, with Confocal, along with a Nikon Imaging Software-Confocal. A 20x objective (Numeric Aperture: 0.5, Camera type Andor DU-897) was used to visualize and quantify migrating Hoechst-labeled Rb cells, while 20x and 40x objectives were used to examine ELTD1 and GPR125 protein distribution. In vivo H&E labeling was imaged using a Fluorescent Nikon Eclipse Ts2 microscope using 4x and 10x objectives. Imaging settings were the same across evaluations, according to each respective assay.

Statistical analysis
All data analysis was done using GraphPad PRISM 8. Results were examined by Ordinary One-Way ANOVA data analysis of variance followed by a Tukey's multiple comparison test or by a student's paired T test. Significance of these tests were considered when *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Results
RB tumors expressed high levels of ELTD1, but not GPR125, RNA An RNA profile comparing adhesion GPCRs was generated from a cohort of 64 human pediatric retinoblastoma tumor tissues and 11 human control fetal retinas. As shown in Fig. 1a, the heatmap of 31 adhesion-GPCRs showed distinct RNA expression patterns of some, but not all receptors in Rb tumors compared to fetal retina controls (Additional file 1: Supplementary Table 1). Cluster analyses reflected three distinct groups of adhesion-GPCRs expressed in the Rb tumors relative to fetal retina (Fig. 1b). Quantitative statistical analysis of the RNA expression signatures of these receptors showed that while ELTD1, EMR1, EMR2, CD97 and GPR97 were upregulated in Rb tumors, LPHN2, LPHN3, GPR124, CELRS2 and GPR56 were downregulated (Additional file 1: Supplementary Table 1). LPHN1, EMR2, GPR125, GPR110, GPR111, GPR115, BAI1, BAI2, BAI3, GPR64, GPR112, GPR114, GPR128, CELSR3 and GPR144 were not found to display any difference between tumor and normal fetal retina. For the current study we focused on two adhesion-GPCRs, ELTD1 and GPR125, both which influence different aspects of cancer development and progression [1,13,26,38]. As shown in Fig. 1c-d, ELTD1, but not GPR125 RNA expression, was significantly increased in Rb tumors compared to fetal retina controls (ELTD1: p < 0.0001 versus GPR125: p = 0.1731).

ELTD1 protein was upregulated in RB cells and tumors
Next, we investigated the protein levels of ELTD1 and GPR125 in Rb tumors and Rb cell lines by immunoblotting (Fig. 2). The ELTD1 antibody specificity was tested using human brain lysates as negative controls and HeLa lysates as positive controls ( Fig. 2a; ****p < 0.0001; N = 10). Given our RNA microarray results, we hypothesized ELTD1, but not GPR125, to be increased in Rb cells and tumors. As illustrated in Fig. 2b, ELTD1 showed high levels in Weri-Rb-1 cells and Rb tumors compared to fetal retina (Fetal retina versus Weri-Rb-1 cells: **p < 0.01; Fetal retina versus Rb tumors: ***p < 0.001; N = 8), but this significant difference did not extend to the aggressive Y79 cells. The GPR125 antibody specificity was tested using Jurkat cell lysates as negative controls and HeLa lysates as positive controls ( Fig. 2c; *p < 0.05; N = 6). We hypothesized there would be no difference in GPR125 protein expression based on the RNA microarray results which compared fetal retinas with human Rb tumors (Fig. 1). As expected, the GPR125 protein expression was not different between fetal retina and Rb tumors. However, Weri-Rb-1 and Y79 cells displayed a significant increase in GPR125 protein expression when compared to fetal retina samples Fig. 2d; (Fetal retina versus Weri-Rb-1: **p < 0.01 and Fetal retina versus Y79: ****p < 0.0001; N = 6).
ELTD1 was enriched in cell-to-cell adhesion sites of the non-metastatic, Weri-Rb-1, cells, while GPR125 was distributed in a polarized manner in Y79, cells To address whether ELTD1 and GPR125 mRNA are differently expressed in the Y79 and Weri-Rb-1 cells, we conducted RT-PCR analysis, as shown in Fig. 3A-B. The results demonstrated a differential expression of the tested adhesion-GPCRs in the Rb cell lines. The differential expression was both at the transcriptional (Fig. 2b) and protein (Fig. 2d) levels. Next, we investigated the subcellular distribution of these adhesion-GPCRs (Fig. 3C  -R1). The intensity of ELTD1 labeling was more abundant at cell-to-cell adhesion sites in Weri-Rb-1 cells (Fig. 3C and C1; white arrows) compared to Y79 (Fig. 3G  and G1), where ELTD1 displayed a punctate labeling pattern in the cytoplasmic and nuclear regions. The labeling profile of GPR125 was dispersed, not punctuated. Weri-Rb-1 cells showed a diffused profile (Fig. 3K, K1), while Y79 displayed a polarized GPR125 labeling (Fig. 3O  and O1; white arrowheads).
Silencing of ELTD1 decreased migration, but not viability of the metastatic, Y79, cells To determine the functional effects of ELTD1 in Rb, we modulated gene expression by RNA silencing. Given the RNA upregulation of ELTD1 in Rb tumors (Fig. 1) we hypothesized that silencing of ELTD1 would reduce Rb cell migration. In addition, we hypothesized that while the ELTD1 protein upregulation in Weri-Rb-1 cells could contribute to the adhesive properties that characterize these less invasive Rb cells, the ELTD1 protein expression and distribution in the Y79 cells could contribute to the invasive ability of these cells as they display lower adhesive characteristics. Therefore, after silencing of ELTD1 in these Rb cells, we measured their migration using the Boyden Chamber system. The cells were added to the upper chamber with serum-free RPMI media; to stimulate migration of the cells through basement membrane, the lower chamber contained complete media to more closely mimic in vivo physiology. As a control, untransfected cells were also evaluated to ensure proper quantification of migrating Rb cells (Fig. 4, right inserts; white arrowheads). Our results demonstrated a significant reduction in migration in Y79 cells compared to Weri-Rb-1 cells (Fig. 4a-c, g-i; m; controls, untransfected: Y79 versus Weri-Rb-1: ****p < 0.0001; N = 4). This result extended to Y79 cells that were treated with the siELTD1, illustrating a significant decrease in migration when compared to untreated Y79 cells (Fig. 4a-c; d-f; m; Y79 controls, untransfected versus Y79 siELTD1: ***p < 0.001; N = 4). Quantitative analysis demonstrated that there was no statistical difference between siELTD1 transfected Weri-Rb-1 cells and untransfected Weri-Rb-1 cells (Fig. 4g-i; j-l; m; N = 4). Next, we investigated the role of ELTD1 in cellular viability. As shown in Fig. 4n, there was no significant difference in cellular viability between treated and non-treated cells ( Fig. 4n; N = 3). These findings support a role for ELTD1 in Rb tumor cell migration, but not cellular viability, in vitro.

Silencing of GPR125 did not decrease migration or viability of Rb cells
To investigate the role of the adhesion GPR125 receptor in Rb, we performed similar experiments to those conducted with ELTD1 as described in the section above. Briefly, Rb cell lines were transfected with siGPR125. We hypothesized that, given the lack of GPR125 RNA and protein expression difference between Rb tumors and fetal retinas (Fig. 1d and Fig. 2d, respectively), yet its protein overexpression in both, Weri-Rb-1 and Y79 cells, siGPR125 could affect Rb cell migration or viability. As expected, there were differences in migratory activity between the Y79 and the Weri-Rb-1 cells (Fig. 5 a-c, g-i; m; controls, untransfected: Y79 versus Weri-Rb-1: **p < Fig. 2 Western blot analysis show overexpression of ELTD1, but not GPR125, in Rb human tumors compared to fetal retina samples. a, c Immunoblot illustrate specificity of ELTD1 (77 kDa) and GPR125 (146 kDa) antibodies, respectively. For ELTD1 antibody specificity, human brain and HeLa cell lysates were used as negative and positive controls, respectively. For GPR125 antibody specificity, Jurkat and HeLa cell lysates were used as negative and positive controls, respectively. b Quantitative analysis show a significantly higher ELTD1 protein expression in Rb cells as well as in Rb tumors compared to fetal retina controls. d Quantitative analysis show a significantly higher GPR125 protein expression in Rb cells, Weri-Rb-1 and Y79, but not in Rb tumors compared to fetal retina controls. GAPDH (37 kDa) antibody was utilized as a loading control. Images shown are cropped from full-length blots presented in Additional File 2: Supplementary Figure 1, to improve the clarity and conciseness of the data presented. Relative protein expression in blot images was analyzed with Image Studio Lite Version software (version 5.2.5). Two-tailed Student's t test (A and C) and Ordinary One-Way ANOVA followed by Tukey's multiple comparison test (B and D) were used to compare each column with their control counterpart. Error bars, SEM. Significance was expressed as: * p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 0.01; N = 6). However, knockdown of GPR125 did not have any effect on the migration (Fig. 5m; N = 6) of Y79 and Weri-Rb-1 cells when compared to their silenced counterparts. Cellular viability was not affected by GPR125 gene knockdown in any of the Rb cell lines ( Fig. 5n; N = 6).

In vivo silencing of ELTD1 reduced optic nerve invasion in an orthotopic xenograft RB mouse model
To test the hypothesis that ELTD1 influences Rb invasion from intraocular regions to the optic nerve, we developed an orthotopic xenograft Rb mouse model [5,25,41]. As demonstrated in Fig. 6A, anesthetized control Merged images which were equally adjusted to improve representation. Scale bar for panels C-J and K-R is 10 μm and for panels C1-J1 and K1-R1 is 5 μm mice (N = 5; inoculated with untransfected Y79 cells) as well as experimental mice (N = 7; inoculated with Y79 cells that were previously transfected with siELTD1), had their intraocular pressure (IOP) and weight measured prior to inoculation, and weekly, after cellular engraftment. A total of 50,000 cells in a 5 μL final volume were inoculated intravitreously in each eye of 8-weeks old male ICR-SCID mice. Tumors were visible after 2 weeks, as signs of anterior chamber invasion became obvious. Tumor growth was monitored for 2 months, after which orthotopic xenografts were euthanized and eyes were dissected for histopathological analysis, as before [5]. All eyes injected with untransfected Y79 cells demonstrated rapid tumor growth (n = 5 animals), while the siELTD1-Y79 (n = 7) inoculated eyes did not (data not shown). As expected, due to its less aggressive phenotype, untransfected Weri-Rb-1 (n = 5) and siELTD1-Weri-Rb-1 (n = 7) inoculated eyes, did not show rapid tumor growth. Hematoxylin and eosin (H&E) staining of the eyes demonstrated the dissemination of Y79 cells to other areas including the ON (Fig. 5d, D1-3), while Were-Rb-1 cells only invaded intraocular tissues (Fig. 5b, B1-3). Consistent with previous studies from members of our team and others [4,17], Y79 cells exhibited aggressive invasion to the anterior chamber, the retina, the subretinal space, the choroid, and the optic nerve and Weri-Rb-1 limited their invasive capacity to intraocular regions [8,17]. Silencing of ELTD1 significantly reduced Y79 invasion to the optic nerve ( Fig. 5e and E1-E2), while not displaying any change in the transfected non-metastatic Weri-Rb-1 cell (Fig. 5c and C1-C2).

Discussion
Adhesion-GPCR are orphan receptors that play critical physiological and pathological roles. In cancer, their aberrant expression strongly contributes to disease development and progression. However, the expression profile, cellular and molecular roles of these adhesion GPCRs have not been investigated within the context of Rb. The work presented herein provides the first report of the RNA expression signature of adhesion-GPCRs, with a focus on ELTD1 and GPR125 functions in this intraocular pediatric malignancy.
ELTD1 and GPR125 are adhesion-GPCRs expressed in different tumors and have been linked to tumor progression [3,13,42]. Consistent with other tumors, Rb expresses ELTD1 and GPR125; however, ELTD1 RNA and protein were upregulated in Rb tumors when compared to fetal retina controls (Figs. 1 and 2). At the cellular level, ELTD1 was enriched in the less invasive Weri-Rb-1 cells where it highly distributed as punctate clusters in regions of cell-to-cell contact. In the more invasive Y79 . m Quantitative analysis show a significant reduction in migration of siELTD1 cells compared to UT cells. This effect was specific to Y79 cells, as Weri-Rb-1 cell migration was unaffected. n Silencing of ELTD1 did not have an effect in Rb cell viability. Ordinary One-Way ANOVA followed by Tukey's multiple comparison test (M and N) was used to compare between columns. Scale bar for panels A-L is 10 μm. Error bars, SEM. Significance was expressed as ***p < 0.001 and ****p < 0.0001 Fig. 6 Inhibition of ELTD1 reduced optic nerve invasion in the aggressive Rb phenotypic cell line. A Schematic images illustrate procedure prior to Rb cell inoculation. Co-immunocompromised mice were weighted and intraocular pressure (IOP) was measured prior to and after Rb cell inoculation (controls and Y79 transfected with siELTD1). After mice reached an IOP ≥15 mmHg, closely near 2 months' post-inoculation, mice were enucleated, and eyes for removed for histological analysis. B, B1-B3 Controls untransfected Weri-Rb-1 cells show intraocular invasion, without extraocular involvement. C, C1-C2 Eyes inoculated with transfected siELTD1 Weri-Rb-1 cells displayed similar invasion patterns as their counterpart control conditions (B). D, D1-D3 Controls untransfected Y79 cells show intraocular invasion, with extraocular optic nerve involvement. E, E1-E3 Eyes inoculated with transfected siELTD1 Y79 cells displayed substantial reduction in optic nerve invasion. Objective 4x was used for images B, B1, C, D and E while 10x was used for images B2-B3, C1-C2, D1-D3 and E1-E2. AC: Anterior Chamber; ON: Optic Nerve; VC: Vitreous Cavity Migrating cells were fixed and stained with Hoechst dye for nuclear stain to ensure that quantification of blue migrating cells overlapped with the membrane's pores (white arrowhead). m Quantitative analysis show a significant increase in migration of UT Y79 cells compared to UT Weri-Rb-1 cells, as expected. However, no reduction in migration was observed in the Rb cells after GPR125 silencing. n. Silencing of GPR125 did not have an effect in Rb cell viability. Ordinary One-Way ANOVA followed by Tukey's multiple comparison test (M and N) was used to compare between columns. Scale bar for panels A-L is 10 μm. Error bars, SEM. Significance was expressed as **p < 0.01 cells, ELTD1 punctuate clusters are less pronounced and are distributed in cytoplasmic and nuclear regions. GPR125 was less intense and more diffused in nucleocytoplasmic regions of Weri-Rb-1 cells while in the Y79 cells, the protein distributed in a more polarized manner. The nucleo-cytoplasmic ELTD1 and GPR125 distribution pattern observed in Rb cells has been described for other proteins that shuttle between the nucleus and cytoplasm relaying information between these two cellular sites and zones of cell adhesion to extracellular matrix [15,28]. Considering the unique structure of adhesion-GPCRs (e.g., long N-terminal with domains vital for adhesion), our results suggest that ELTD1 and GPR125 are distributed in this characteristic manner within Rb cells, and, thus, may represent a possible mechanism for transcellular coordination and communication, as well as for cell-to-cell and cell to matrix adhesion [15,28].
ELTD1 has been associated with the development and progression of tumors given its role in cancer progression, including angiogenesis [3,22]. Recent work from Kann and colleagues showed that silencing of ELTD1 dramatically reduced invasiveness of hepatocellular carcinoma cells [16], confirming prior studies connecting ELTD1 to metastasis [42]. Furthermore, ELTD1 has been found to associate with poor prognosis in colorectal cancer [1]. Meanwhile, the role(s) of GPR125 in cancer remains poorly understood. Fu and colleagues suggested an oncogenic role in myeloid sarcoma [13], while others showed it works as a tumor suppressor in colorectal cancer by activation of the Wnt/β-catenin signaling pathway [38].
Our results showed that silencing of ELTD1 decreased in vitro migration and in vivo invasion of the Y79 cells without affecting viability. Considering that ELTD1 protein was not upregulated in the Y79 cells, it is possible that these effects are affecting ELTD1 targets, which influence cell proliferation, differentiation, migration and apoptosis, and have been found to be altered in Rb [7,18,30].
Silencing of GPR125 in Weri-Rb-1 or Y79 cells did not affect cell migration or viability. From its upregulation in Rb cells and polarized protein distribution in the Y79 cells, we hypothesized its knockdown would affect Rb cell migration. However, our results suggest that GPR125 could be contributing to other tumor-related processes that were beyond the scope of this manuscript, such as in Rb cell proliferation, cell death resistance, replicative potential, among others.
The variability in protein expression profile between Rb cell lines and primary tumors suggests different tumor microenvironmental conditions that may or may not be captured in vitro. The level of heterogeneity found in our studies highlights the importance of using distinct cell line models that reflect the complexity and heterogeneity characteristic of Rb tumors [8,17].
Remaining questions that should be further examined include: (1) an investigation of the association between ELTD1 and GPR125 expression from the primary tumor throughout Rb development and progression as tumor metastasizes; (2) identification of ELTD1-associated mechanisms contributing to regulating Rb migration and invasion and, (3) examination of ELTD1's role in Rb angiogenesis. Taken together, our in vitro, ex vivo and in vivo study reveal a novel biological platform that must be further explored as a potential personalized targeted therapy to better manage and treat metastatic Rb.

Conclusion
This work reports differences in the expression and functional roles of two adhesion-GPCRs, ELTD1 and GPR125. We identified ELTD1, but not GPR125, to be overexpressed in primary Rb tumors and demonstrated that ELTD1 disruption reduces Y79 cell migration in vitro and metastasis in vivo without affecting cell viability. These results confirm a specific role for ELTD1 in migration and invasion and represent the first step towards a better comprehension of the expression profile and biological roles of adhesion-GPCRs in Rb with the hope of investigating and identifying new therapeutic avenues against metastatic Rb.
Additional file 1: Supplementary Table 1. RNA expression microarray illustrates expression profile of adhesion-GPCRs that were significantly upor downregulated in Rb pediatric tumors (N = 64) compared to fetal retina controls (N = 11), and those that did not display significant differences.
Additional file 2: Supplementary Figure 1. Full-length Western blot Shown in Fig. 2. A. hela cell, not human brain, lysates show immunopositivity against the anti-ELTD1 antibody shown by the 77kDA band. GAPD H, a 37kDA protein, was used as loading control. B. Full length image measuring immunopositivity against ELTD1 in fetal retina (Fet Ret), Rb cells and Rb tumors. GAPDH was used as loading control. C. HeLa cell, not Jurkat cells, lysates show immunopositivity against the anti-GPR125 antibody shown by the 146kDA band. GAPDH was used as loading control. D. Full length image measuring immunopositivity against GPR125 in Fet Ret, Rb cells and Rb tumors. GAPDH was used as loading control. For all full-length blots, dotted line boxes around sample lanes indicate the area of the membrane that was cropped in Fig. 2.

Acknowledgements
We would like to acknowledge Dr. Jae E. Lee for his assistance with the microarray biostatistical analysis. Our thanks to Dr. Zachary K. Goldsmith for technical assistance with the RT-PCR studies and professional editing of the manuscript. We also thank Dr. Guillermo A. Yudowski, Dr. Cristina Velázquez, Dr. Luis Montaner and Dr. Gregory Quirk for their insightful feedback throughout the progress of this study. Finally, we acknowledge the Institute of Neurobiology-UPR for facilitation of the Nikon Confocal Microscope (COBRE-NIEF 5P20-GM103642). Author's information JG is a second-year osteopathic medical student at the Debusk College of Osteopathic Medicine at Lincoln Memorial University. NBT is a Pharmacology PhD candidate at the University of Michigan. DDL is a medical student at the Central University of the Caribbean of Puerto Rico. JCMS is medical student at University of Medicine and Health Sciences. RHC is a 4th year medical student at Ponce Health Sciences University. CMVR is a graduate student at the Department of Anatomy and Neurobiology at the university of Puerto Rico-Medical Sciences campus developing expertise in Retinoblastoma and the Endocannabinoid System. VMT is an Assistant Professor at the Department of Ophthalmology, Hamilton Eye Institute and the Department of Microbiology, Immunology and Biochemistry and leads the Translational Immunology and Ocular Oncology Research Unit at the University of Tennessee Health Science Center. She is a tumor immunologist whose laboratory research focuses on the primary intraocular malignancies of Retinoblastoma and Uveal Melanoma. Currently, VMT is a Principal Scientist at AbbVie Bioresearch Center, Worcester, MA. JFO is a tenured Associate Professor at the Department of Anatomy and Neurobiology at University of Puerto Rico-Medical Sciences Campus with expertise in Neuro-Oncology, Receptor Signaling and pre-clinical models of cancer. She serves as a Principal Investigator at the Institute of Neurobiology-UPR and the UPR-Comprehensive Cancer Center.

Availability of data and materials
The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request. Information on Y79 and Weri-Rb-1 Rb cells are available in www.ncbi.nlm.nih.gov/biosample and in www. atcc.org.
Ethics approval and consent to participate For the use of Rb tumors kindly provided by the public resource facility of the CSTN at STJCRH [36], we obtained IRB approval (Protocol #B0580215) by the institutional IRB committee and for the in vivo studies institutional IACUC approval was obtained (IACUC #A190115). Institutional IRB approval also included the use of human cell lines for this study (Protocol #B0580215).

Consent for publication
Not applicable.