Histochem Cell Biol DOI 10.1007/s00418-014-1285-z
ORIGINAL PAPER
Re-characterization of established human retinoblastoma cell lines Maike Busch · Claudia Philippeit · Andreas Weise · Nicole Dünker
Accepted: 29 September 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell Electronic supplementary material The online version of this article (doi:10.1007/s00418-014-1285-z) contains supplementary material, which is available to authorized users. M. Busch · C. Philippeit · N. Dünker (*) Department of Neuroanatomy, Medical Faculty, Institute of Anatomy, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany e-mail: [email protected] A. Weise Institute of Biology II/Cell Biology, University of Freiburg, Freiburg, Germany
lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro. Keywords Retinoblastoma · RB cell lines · Soft agar assay · Colony formation · CAM assay · Invasiveness · Dissemination · Tumor formation
Introduction Retinoblastoma (RB) is the most common malignant eye tumor of childhood occurring in infants as both, a heritable (usually bilateral) and non-heritable (unilateral) form. The most important factor for its prognosis is the extension of invasion and dissemination of the retinoblastoma cells and the formation of metastasis. There are different patterns of invasion: (i) invasion along the optic nerve to the brain, (ii) dispersion into the circulating subarachnoid fluid toward the spinal cord and distant sites of the brain, (iii) hematogenous dissemination with formation of metastasis in lungs, bones and brain and (iv) lymphatic spread (McLean et al. 1994). Retinoblastoma is a uniquely human disease and all animal models like injections in the subretinal space or the anterior chamber of eyes of immunosuppressed or deficient rats and mice (Gallie et al. 1977; del Cerro et al. 1993), which have been developed so far, had limitation. The application of tissue and cell culture techniques permits detailed studies of the biology of the tumor, focussing on growth and metastatic potential of RB cells. In addition, the chick chorioallantoic membrane (CAM) is an established, naturally immunodeficient model system
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for in vivo studies of tumor cell dissemination (Deryugina and Quigley 2008). Different steps in the metastatic tumor cascade can be addressed using this model: (i) spontaneous metastasis after tumor cell grafting onto the CAM and (ii) experimental metastasis after intravenous inoculation of tumor cells. In the spontaneous metastasis setting, aggressive tumor cells escape from the primary grafted cells and intravasate into the chick vasculature, providing a model for the intravasation step of the metastatic cascade. In the second setting, tumor cells extravasate from the CAM capillary system and colonize. Even after years of research, still little is known about the functional properties of RB cells. In 1974, Reid et al. established the first human retinoblastoma cell line called Y-79 and characterized it morphologically, cytogenetically and with respect to cell growth and biochemistry. Later, McFall and her group published the characterization of a second established RB cell line: WERI-Rb1 (McFall et al. 1977). The growth of RB cells in culture for a prolonged time remained difficult till in the eighties, two groups described methods of establishing cell lines from RB tumors (Gallie et al. 1982; Bogenmann and Mark 1983). Jiang et al. (1984) demonstrated that WERI-Rb1 and Y-79, which grew in loose clusters and attached to coverslips, pre-coated with poly-l-ornithine solution, both exhibit neuronal and glial properties. In the nineties, Griegel et al. (1990a, b) established and characterized seven new RB cell lines: RBL-13, RBL-14, RBL-18 and RBL-30, derived from unilateral retinoblastomas and RBL-7, RBL-15 and RBL-20, derived from bilateral retinoblastomas. The authors investigated colony formation properties in soft agar as well as the invasive potential of these cell lines and reported (i) that none of them was able to form anchorage-independent colonies from a single cell suspended in semisolid agar and (ii) that they were noninvasive when tested for invasion into chick heart fragments (Griegel et al. 1990a). Very different growth rates and morphologies have been described for Y-79, RB 355 and WERI-Rb27 cells (Madreperla et al. 1991). Previous studies addressing the genetic characteristics of RB cell lines showed a partial deletion and a total loss of the RB1 gene in Y-79 and WERI-RB1 cells, respectively (Bookstein et al. 1988; Choi et al. 1993). Griegel et al. (1990a) observed loss of heterozygosity of the RB1 gene in RBL-30 cells, but not in RBL-13 and RBL-15 cells. Evidence for a point mutation in RB1 was observed in the RB 30 tumor, where the RBL-30 cell line is derived from (Griegel et al. 1990a). Forty years after their first description and thus 40 years after establishment and propagation in vitro, in the study presented, we set out to re-characterize the retinoblastoma cell lines RBL-13, RBL-15, RBL-30, RB 355, RB 383,
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Histochem Cell Biol
Y-79 and WERI-Rb1 with regard to (i) their RB1 mutation status (ii) their growth kinetics, (iii) endogenous apoptosis levels, (iv) colony formation potential and (v) invasiveness and metastasis capacity. Our study updates current knowledge on established RB cell lines and provides valuable new data for future in vitro approaches in retinoblastoma therapy-related research with regard to main tumor cell properties like growth behavior, anchorage-independent growth as well as intravasation and dissemination potential.
Materials and methods Cell culture Seven human retinoblastoma (RB) cell lines RBL-13, RBL15, RBL-30, RB 355, RB 383, Y-79 and WERI-Rb1 (generously provided by H. Stephan; Reid et al. 1974; McFall et al. 1977; Griegel et al. 1990a; Madreperla et al. 1991) were cultivated as suspension cultures in Dulbecco’s modified Eagle’s medium (DMEM; PAN-Biotech) with 10 % fetal calf serum (FCS; PAN-Biotech), 100 U penicillin/ml and 100 µg streptomycin/ml (Invitrogen), 4 mM l-glutamine (Sigma), 50 µM β-mercaptoethanol (Roth) and 10 µg insulin/ml (Sigma) at 37 °C, 10 % CO2 and 95 % humidity. Human embryonic kidney cells (HEK293T; generously provided by B. Royer-Pokora) were grown as adherent cultures in DMEM (PAN-Biotech) with 10 % FCS (PANBiotech), 100U penicillin/ml and 100 µg streptomycin/ml (Invitrogen) at 37 °C, 5 % CO2 and 95 % humidity. Growth kinetic Growth kinetics of the different retinoblastoma cell lines were determined by serial dilution of the cells in 96-well plates as described in Weise and Dünker (2013). In brief, serial dilutions followed this scheme: any of the wells of a 96-well plate was filled with 100 µl growth medium. Hundred microliters cell suspensions from a culture with 0.3–0.5 × 106 cells/ml was pipetted into well A1 and subsequently horizontally diluted 1:2 up to the last well (A12). Afterward, vertical dilutions (1:2) of the entire row A were carried out up to the last row (H) of the 96-well plate. Wells with single cells were examined every 24 h, and cells were counted. Cells which did not at least reduplicate were designated as dead and were not included into the evaluations. In order to compare the growth kinetic of RB cells growing in aggregates to that starting from single cells, we plated a fix concentration (1.5 × 105 cells/ml) of all RB cells lines in 24-well plates. Wells with cell aggregates were examined, and living cells were counted in a Neubauer chamber after trypan blue staining every 24 h.
Histochem Cell Biol
Determination of apoptosis To determine apoptosis and visualize the morphology of the RB cell lines by 4′,6-diamidino-2-phenylindole (DAPI)counter staining, 1 × 105 cells were seeded on poly-d-lysine (Sigma) coated coverslips and grown in 1 ml medium in 24-well plates. The number of DAPI-stained pycnotic nuclei was determined as described before (Haubold et al. 2010). For each cell line, five coverslips were evaluated. A total of at least 1,000 cells per coverslip were counted, and the percentage of apoptotic, clearly pycnotic nuclei (at least 10) was calculated.
(10 µg/ml, H9268, Sigma). Additional 2 ml of DMEM medium with supplements (as described under “cell culture”) was added after another 24 h. After another 48 h, the medium was completely changed. Invasiveness of RB cells: CAM assay For our metastasis model, we mainly followed the protocol published by Zijlstra et al. (2002) and most recently visualized by Palmer et al. (2011). 1. Spontaneous metastasis model: grafting of GFP-labeled RB cells
Colony formation assay For growth in soft agarose 5 × 104, RB cells were suspended in 2 ml DMEM/F12 medium (Sigma) containing 10 % fetal calf serum, 100 U penicillin/ml and 100 µg streptomycin/ml, 4 mM l-glutamine, 50 µM β-mercaptoethanol, 10 µg insulin/ml and 0.35 % agarose (Roth). The cell suspension was layered on 2 ml 0.5 % agarose, containing DMEM/F12 medium with the same supplements as indicated above and cultured in 6-well plates at 37 °C, 10 % CO2 and 95 % humidity. As positive control 5 × 104 HEK293T cells were suspended in DMEM/F12 medium with 10 % fetal calf serum and 100U penicillin/ml and 100 µg streptomycin/ml containing 0.35 or 0.5 % agarose as described. Cells were fed repeatedly after 4–5 days and documented weekly over a time period of 4 weeks. Colony formation, starting from single cells seeded in soft agarose, was quantified after 3 weeks of incubation. Plating was repeated three times. The colony formation efficiency (%) per visual field was determined by counting the colonies and single cells in three visual fields (10×) per cell line in triplicates. Lentiviral GFP labeling of RB cell lines For virus production, HEK293T cells were transfected with 6 µg of each of the following plasmid DNA: packaging vector pczVSV-G (Hartmann et al. 2010), pCD NL-BH (Hartmann et al. 2010) and a GFP expressing vector (pCL7EGwo, kindly provided by Dr. H. Hanenberg) in the presence of 45 µg polyethyleneimine (PEI, branched, Aldrich). The medium was changed after one day to Iscove’s Modified Dulbecco’s medium (IMDM, Gibco) with 10 % FCS and 1 % penicillin/streptomycin and 48 h after transfection viral supernatants were harvested, filtered (0.45 µm) and cryoconserved. For stable GFP labeling, RB cells were seeded in a 6-well plate (6 × 105 cells/well). After 24 h, cells were infected with GFP lentiviruses in the presence of polybrene
Fertilized eggs were incubated in a humidified rotary incubator at 38 °C and 50 % humidity for 10 days. On embryonic (E) day 10, the eggs were candled by shining light into the eggshell at the blunt end of the egg. The chorioallantoic vein was located, and a 1 cm square was marked with a pencil approximately 1 cm away from the veins branching point. A hole was drilled through the blunt end of the egg into the air sac and within the drawn square, and the “upper CAM” (see Fig. 1 in Kim et al. 1998) was dropped by gentle suction, creating an artificial air sac. The CAM was gently abraded by the use of a cotton-tipped applicator. Immediately after damaging the CAM, 50 μl of the cell suspension (1 × 106 cells in PBS) was placed onto the traumatized CAM area. The window in the egg was sealed tightly with tape and returned to the incubator. 2. Experimental metastasis model: intravenous injection of GFP-labeled RB cells Fertilized eggs were incubated for 12 days. On developmental day E12, the eggs were candled by shining light into the eggshell at the blunt end of the egg. The chorioallantoic vein was located, and a 1 cm by 0.5 cm rectangle was marked with a pencil approximately directly above the vein. A small window was created in the eggshell, and the eggshell membrane was rendered transparent with a drop of mineral oil. Fifty microliters of cell suspension (1.5 × 105 cells in DMEM) was injected into the allantoic vein of each embryo. The window in the egg was sealed tightly with tape and returned to the incubator. Injections were performed under the dissection microscope in order to monitor if the injected cells are carried by the blood flow (=correct direction of the injection). In each experiment, 10–15 eggs were injected and if the cells were wrongly injected against the blood flow (=wrong direction), we excluded the eggs from our experiments. We likewise only used eggs with minimal or no blood leakage after injection to make sure that the complete amount of cell suspension was injected.
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Table 1 Mutation status of RB cell lines
Histochem Cell Biol RB cell line
References
Y-79 WERI-Rb1
Reid et al. (1974) McFall et al. (1977)
RB 355
Madreperla et al. (1991)
Unilateral
no RB1 deletion
RB 383
Griegel et al. (1990a, b)
Unilateral
RBL-13 RBL-15
Griegel et al. (1990a, b) Griegel et al. (1990a, b)
Unilateral Bilateral
Genomic rearrangements of the RB1 locus c.958 C>T/c.2390 T>G
RBL-30
Griegel et al. (1990a, b)
Unilateral
Harvesting of tissue For each RB cell line, 10 eggs were grafted and intravenously injected, respectively. The duration of the CAM assay is limited to a 7–9 day window available before the chick hatches. Thus, after distinct time points (7 days after inoculation (E10-17) and 6 days after injection (E12-E18)), six standardized (1.4 cm internal diameter) tissue punches of each ventral “lower CAM” (see Fig. 1 in Kim et al. 1998) were collected and transferred into a 24-well plate filled with PBS. Each of the six lower CAM punches was scanned for GFP-positive RB cells by fluorescence microscopy, combined and cryoconserved for RNA extraction. In order to prevent cross-contamination of the samples, the dissection tools were sterilized by sequential rinsing in: (1) 10 % NaOH, (2) 70 % ethanol, (3) double distilled water, and (4) again double distilled water, following the protocol published by Palmer et al. (2011). RNA isolation and quantitative real-time PCR analysis For RNA isolation, the CAM tissue punches were mechanically pulverized on dry ice without defrosting. RNA isolations from RB cell lines and CAM tissue were performed using the NucleoSpin RNA II kit (Macherey and Nagel), and cDNA was synthesized with the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s protocol. Quantitative real-time PCR analyses were performed using a 7300 Real-Time PCR System (Applied Biosystems). The following human Taqman Gene Expression Assays (Applied Biosystems) were used: GAPDH (Hs99999905_m1) and 18S (Hs99999901_s1). The latter was used as an endogenous control for human and chicken samples. Real-time PCRs were performed in duplicate, and a total volume of 20 µl was applied to the following program: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Mutation analysis Mutation analysis was performed as described previously (Rushlow et al. 2013). In brief, a MLPA Kit
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Type of tumor
RB1 mutation del exon 2–6/IVS20+1G>A del RB1/del RB1 (including/TM2B to DLEU)
del RB1/del RB1 (from RB1 to DLEU) g59695 C>T/g59695 C>T
(MRC-Holland, Amsterdam, Salsa MLPA Kit P047 RB1) was used and reactions were performed according to the manufacturer’s instructions. Additional sequencing of the RB1 gene was performed for all retinoblastoma cells lines.
Results Mutation status of RB cell lines We analyzed the RB1 mutation status of seven RB cell lines in order to verify already published data and reinvestigate mutations in the RB1 gene. A description of the RB1 mutation status of all RB cell lines investigated is summarized in Table 1. A previous genetic characterization of the commercially available RB cell line Y-79 revealed a partial deletion of the RB1 gene (Bookstein et al. 1988). Our re-investigation of Y-79 cells uncovered a heterozygous deletion of exon 2–6 and an intronic point mutation (IVS20+1 G>A) on the other allele. A former study reported on a total loss of the RB1 gene in the likewise commercially available RB cell line WERI-Rb1 (Choi et al. 1993). Our mutation analysis confirmed a complete deletion of RB1 in WERI-Rb1 cells and additionally revealed that this deletion ranges from ITM2B up to DLEU, resulting in an absent RB1 protein. In our MPLA analysis, the RB cell line RB 355 showed no deletions in the RB1 gene and other alterations in the RB1 gene (e.g., point mutations in one of the 27 exons) have not been determined yet for these cells. RB 383 cells exhibited extensive genomic alterations in our MPLA analysis, indicating large rearrangements of the RB1 locus. RBL-13 cells showed two heterozygous point mutations in the RB1 gene: c.958 C>T and c.2390 T>G. RBL-15 cells, derived from a bilateral tumor, displayed a complete deletion of RB1 reaching from RB1 up to DLEU, causing an absent RB1 protein. Griegel et al. reported on evidence for a point mutation in RB1 in the primary retinoblastoma tumor, the RB cell line RBL-30 originally derives from (Griegel et al. 1990a). Our analysis revealed a homozygous g59695 C>T point mutation in the RB1 gene of RBL-30 cells.
Histochem Cell Biol
RB cell line morphology and growth Six of the seven established human RB cell lines RBL-13, RBL-15, RBL-30, RB 355, RB 383, Y-79 and WERI-Rb1 investigated grew in suspension and were predominantly small epithelioid cells with little cytoplasm and large nucleus (Fig. 1). RB 355 cells, by contrast, partly attached to the flask and grew as adherent spindle-shaped cells with a larger cytoplasm (Fig. 1c4). All RB cell lines investigated were able to attach to coverslips pre-treated with poly-d-lysine. They mainly grew in cell clusters and form aggregates and clumps from ten to hundreds of cells (Fig. 1). A chainlike structure could frequently be observed in WERI-Rb1 suspension cell cultures (Fig. 1a2–c2), but also occurred in Y-79 cultures (Fig. 1a1–c1). After attachment to polyd-lysine-coated coverslips, Y-79 more frequently tends to form chain-like structures and WERI-Rb1 cells mainly grew as long chains, sometimes also forming chain loops, which, however, did not resemble Flexner-Wintersteiner rosettes. Besides normal aggregates, RBL-13 cells formed three-dimensional “hollow cell balls” in different sizes (Fig. 1a5–c5). In general, RBL-30 cells were the smallest in size, whereas the adherent RB 355 cell line comprised the largest RB cells. Population doubling times and endogenous apoptosis rates Under our culture conditions, we observed different growth rates for different retinoblastoma cell lines, which are summarized in Fig. 2b, d. We counted growing RB cells (a) starting from single cells (Fig. 2a) and (b) starting from cell aggregates (Fig. 2c), naturally formed by these cells, in order to compare potential differences in cell growth rates. In both approaches, RBL-30, RBL-13 and RBL 383 cells grew very slowly and hardly doubled within 1 week. These RB cell lines displayed drastically reduced growth rates with doubling times of 69 up to 220 h, depending on the culture method used. By contrast, Y-79 cells were one of the fastest growing cell lines under both culture conditions with calculated doubling times between 45 and 61 h. All other cell lines were slightly variable in their growth kinetic depending on the method used (Fig. 2). It was not possible to determine a growth curve for RBL-13 cells, when these cell lines were grown starting from cell aggregate due to very strong aggregate formation, which leads to inaccuracy in cell number counts. The endogenous apoptosis levels of the different RB cell lines were quantified by counting DAPI-stained pycnotic nuclei after 24 h culture on poly-d-lysine pre-treated coverslips (Fig. 3). The apoptosis rates differed from two to nearly eight percent with the highest apoptosis in RB 383 and RB 355 and the lowest in WERI-RB1 and RBL-15.
Anchorage-independent growth: capacity of different RB cell lines to form colonies in soft agarose For determination of anchorage-independent growth in soft agarose, RB cell lines and HEK293T cells as positive control were seeded in 0.35 % soft agarose and bedded on a layer of 0.5 % agarose in 6-well plates. Visible colonies were photographed weekly and counted after 2 weeks of incubation by the use of an inverse microscope (10×). Six of the seven RB cell lines analyzed were able to form colonies in soft agarose upon single cell seeding after 3 weeks of incubation (Fig. 4). RB 355 did not form detectable colonies even after 4 weeks of incubation. The average colony size of all RB cell lines investigated was around 76 µm, except in WERI-Rb1 cells, which displayed the largest colonies with a size of 200 µm (Fig. 4). WERI-Rb1 likewise displayed the highest colony formation efficiency with 66.1 %, followed by Y-79 (54.4 %) and RBL-15 (51.5 %) cells. The frequency of colony formation of the other four cell lines ranged from 16.2 to 5 % and is slightly variable (Table 2). CAM Assay: invasiveness and metastasis capacity The chick chorioallantoic membrane (CAM) is an established model system for in vivo studies of tumor cell dissemination (Deryugina and Quigley 2008). Fluorescent GFP-tagging of all RB cell lines enabled us to localize the human tumor cells within the chick embryo CAM tissue. In our spontaneous metastasis setting, GFP-labeled RB cells were grafted onto the upper, dorsal CAM and 10 days after inoculation, tumor cells could be identified in distant portions of the lower, ventral CAM (Fig. 5). Intravasated cells could be visualized and appeared extravasating and scattering in the lower CAM. A total of 60 CAM punches were analyzed for each RB cell line, and the average percentage of GFP-positive lower CAM tissue punches was estimated. Four out of seven RB cell lines (Y-79, RBL-13, RBL-15 and RBL30) displayed GFP-positive cells in the lower CAMs after grafting, whereby RBL-13, RBL-15 and Y-79 showed the highest percentage with 16–18 % positive CAMs (Table 3). One RB cell line (RBL-13) caused tumor development on the upper CAM upon grafting (Fig. 5a; Table 3). Second, in experimental metastasis setting, intravenously injected GFP-labeled RB cells extravasated from the CAM capillary system and colonized near the CAM vessels (Fig. 5b). All RB cell lines had the potential to extravasate from the capillary system after injection. The calculated average of GFP-positive lower CAMs ranged from 6–80 %, whereby WERI-Rb1 (80 %) and RB 383 (72 %) displayed the highest rates of positive CAMs (Table 3). Even in this experimental setting, tumor formation on the upper CAM was detected in the two RB cell lines Y-79 and RBL-13 (Fig. 5a).
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Fig. 1 Morphology of RB cell lines in suspension culture (a1–7) and adherent on polyd-lysine-coated coverslips after DAPI staining (b1–7 and c1–7). The arrow heads (c1–c7) indicate the cytoplasmic rim of the RB cells. Cell nuclei are counter-stained with DAPI. (A1–7) phase contrast microscopy photographs (20×); (b1–7) merged DAPI-stained phase contrast photographs, (c1–7) zoom in of RB cell clusters to visualize the relationship of large nucleus to small cytoplasmic rim. Bar 20 µm applies to b1–7
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Histochem Cell Biol
Histochem Cell Biol
Fig. 2 Growth curves of different RB cell lines with doubling times and doubling time ranges (95 % confidence interval). Doubling times were calculated based on best-fitted exponential growth curves using the GraphPad Prism4 program. Indicated doubling times and time
Fig. 3 Endogenous apoptosis rates of RB cell lines. Cells were seeded on poly-d-lysine-coated coverslips and cultivated for 24 h. Pycnotic cell counts from DAPI stains were performed to determine apoptosis rates. Values are mean ± SD from three independent experiments, each with five different coverslips
Quantification of human RNA in chicken tissue by realtime PCR analyses Levels of intravasated and disseminated human RB tumor cells into the chick CAM and embryo tissue can be
ranges represent means of 6–16 counts in the case of seeded single cells and duplicates in the case of seeded aggregates. RB cells were seeded as single cells (a) or aggregates with a starting concentration of 1.5 × 105 cells/ml (c), and doubling times were calculated (b, d)
determined by quantitative real-time PCR analysis (e.g., Zijlstra et al. 2002; Palmer et al. 2011). Previous studies made use of the amplification of species-specific Alu DNA repeats characteristic of the primate genome (Schmid and Jelinek 1982; Deryugina et al. 2005; Deryugina and Quigley 2008; Kim et al. 1998; Zijlstra et al. 2002; Mira et al. 2002; Palmer et al. 2011). Human Alu repeat sequences represent approximately 5 % of the human genomic DNA and can easily be amplified, but this fact often represents the reason for contaminations (Mira et al. 2002). The method works basically well under complete sterile conditions, but is not readily applicable to the CAM model. It is technically very difficult to open the egg shell under a sterile bench without injuring the CAM and injections into the CAM blood vessels need to be done under a dissection microscope. Under these conditions, the contamination risk with human genomic DNA (gDNA) is very high. Contamination with gDNA is sequentially visible in Alu DNA repeat signals in negative controls, and these background levels detected in CAMs of control chick embryos that did not receive any human tumor cells were routinely subtracted from the curves (Deryugina et al. 2005; Mira et al. 2002). To circumvent this problem, we decided to establish a gene expression-based method for the quantification
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Histochem Cell Biol
Fig. 4 Colony formation of RB cell lines and HEK293T cells in soft agarose after 1, 2 and 3 weeks in culture. 5 × 104 RB or HEK293T cells were suspended in medium. The cell suspension was layered on 0.5 % agarose containing medium and cultured in 6-well plates. The outermost left row of figures shows the cells under the microscope
(10× magnification) at day 0 (day of seeding) and depicts that colony formation started from single cells seeded into the soft agarose. The 4× phase contrast photographs after 2 weeks of cultivation serves as an overview to judge the average colony formation
of the amount of human tissue in the chicken CAMs. For this purpose, we provided evidence that human GAPDH (hGAPDH) expression is completely undetectable in chicken CAM tissue and 18S is suitable as endogenous control for both human and chick tissue (Fig. S1). To determine the range and sensitivity of this method and to create a standard curve, increasing human RNA concentrations of RB cells were mixed with chick RNA from CAM tissue at proportions of 0.01, 0.05, 0.1, 1, 5, 10
and 20 %. One hundred percent human RB cell RNA and 100 % chick CAM tissue RNA were used as positive and negative control, respectively, in every experimental setting. Human GAPDH expression was detectable from very low concentrations of 100 pg (0.01 %) up to 1 µg (100 %) human RNA displayed by the standard curves of the RB cell lines (Fig. 6). In order to correlate the hGAPDH signal—and thereby the amount of human tumor cell extravasation—with the amount of amplified cDNA, 18S RNA,
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Histochem Cell Biol
equally expressed in human and chick tissue (Fig. S1) was used as an internal control (Fig. S2). To quantify the intravasation of the RB cells into the chick tissue, we pooled the standardized chick CAM punches from each embryo, isolated total RNA, and synthetized cDNA from 1 µg RNA. For each RB cell line, eight chick embryos were analyzed: four after CAM grafting and four after intravenous injection of RB cells. Each analysis included a RB cell
Table 2 Colony formation capacity (%) from single cells seeded in soft agarose and colony formation time of RB cell lines RB cell lines
Colony formation efficiency (%)
Colony formation time (weeks)
WERI-Rb1 Y-79 RBL-15 RBL-30 RB 383 RBL-13
66.1 54.4 51.5 12 5.7 5
3 3 3 3 3 3
RB 355
NCD
3
Colony formation capacity was determined by the mean number of colonies relative to the seeded cells of three different visual field counting’s (10×) in triplicate. NCD no colonies detected
line-specific standard curve. The hGAPDH threshold cycle (CthGAPDH) was normalized against the 18S internal control (Ct18S/CthGAPDH), to calculate a relative amount of human RNA to be compared with the RB cell line-specific standard curve in order to determine the amount (ng) of intravasated RB cells. We were able to amplify hGAPDH from GFP-positive lower CAMs after intravenous CAM injection of different human RB cell lines with decreasing concentration of human RNA (inferred from hGAPDH amplification) in the following order: WERI-Rb1 > RB 383 > RBL-15 > RBL30 > Y-79 (Table 4). No hGAPDH expression was detectable after inoculation as it turned out that—depending on the cell concentration per CAM punch—four out of six GFPpositive CAM punches are required to amplify hGAPDH successfully, indicating the detection limit of our newly established method. WERI-Rb1 displayed the highest potential to extravasate and migrate into the CAM tissue resulting in high human RNA concentration in the lower CAM upon injection correlating with a high number of GFP-labeled cells visualized by fluorescence microscopy. All lower CAM punches were GFP-positive with high concentration of WERI-Rb1 cells, reflected by up to 0.5 ng human RNA in the lower CAM tissue as revealed by hGAPDH amplification (Table
Fig. 5 Localization of RB tumor cells after intravenously injection and inoculation on the chicken CAM. Tumor formation on the upper chicken CAM was detectable 6–7 days after inoculation and injection of RBL-13 cell (a). Arrows tumors of RBL-13 cells; bold arrow place of injection. Six day after intravenous injection, green-fluorescent, GFP-labeled RB 383 and WERI-RB1 cells extravasated from the CAM capillary system and colonized near the vessels of the lower chicken CAM (b)
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Histochem Cell Biol
Table 3 Overview of the average percentage of GFP-positive lower CAM tissue punches and tumor formation on the upper CAM after intravenously injection and inoculation of RB cell lines RB cell lines
Graft
Injection
∅ GFP + lower CAM punch (%)
Tumor formation upper CAM ∅ GFP + lower CAM punch (%)
Y-79_GFP WERI-Rb1_GFP RB 383_GFP RBL-13_GFP RBL-15_GFP
16.6 – – 18.5 18.3
RBL-30_GFP
8.3
− − − + −
−
18.3 80 71.6 18.3 25 20
Tumor formation upper CAM + − − + −
−
The average percentage of GFP-positive tissue punches of lower CAMs was calculated from 10 eggs per RB cell line and six punches per egg
Fig. 6 Human GAPDH standard curves of the 6 RB cell lines investigated in the CAM experiments. Quantitative real-time PCR was performed on cDNA synthetized from RNA extracted from the chicken CAM and mixed with different concentrations of human RNA from different RB cells (100 pg–1,000 ng)
4). By contrast, after inoculation of WERI-Rb1 cells onto the upper CAM no GFP-positive CAM could be detected and also tumor formation was absent. In comparison, GFPlabeled Y-79 cells reached the lower CAMs after inoculation and injection and also formed tumors after injection into the chorioallantoic vein (Table 4), indicating a highly invasive and tumorigenic phenotype although the amount of cells per tissue punch was lower compared to WERI-Rb1.
Discussion Growth rate When RB cell lines were established from different retinoblastoma tumors in the past, highly different proliferation rates and population doubling times were observed (Reid et al. 1974; McFall et al. 1977). For Y-79, Reid et al. (1974)
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determined a doubling time of 52 h. McFall et al. (1977) stated an average population doubling time of 33 h for Y-79 and 96 h for WERI-Rb1. Other investigators observed that growth rates of Y-79, WERI-Rb27 and RB 355 are obviously different in the order RB 355 > WERI-Rb27 > Y79 (Madreperla et al. 1991). Under our culture conditions, Y-79 was one of the fastest growing cell lines with a doubling time between 47 and 61 h, resembling the data published by Reid et al. (1974). RB 383, RBL-13 and RBL-30 are slow-growing cells with an average doubling time between 94 and 140 h. In general, one can state that the doubling times of the RB cell lines differ between cells that were seeded as single cells and those grown from aggregates. One should keep this fact in mind planning cell culture experiments with RB cell lines. Morphology It has been described that Y-79, WERI-Rb27, RB 355, RB 383, RBL-13 and RBL-15 cell lines grow in suspension cultures, whereas RB 355 cells are substrate-bound and require trypsinization to collect them from the culture plate (Griegel et al. 1990a). Under our culture conditions, RB 355 cells likewise grow semi-adherent but with additional suspension cells. A former study showed that cultured RB cells exhibit characteristic aggregate formation and some Rb cell lines (RBL7, RBL15 and RBL18) are able to form Flexner-Wintersteiner rosettes and retained this ability for more than 2 year in culture (Griegel et al. 1990a). Other RB lines, however, lost the ability to form rosettes after prolonged cultivation (Griegel et al. 1990a). With continued culture, Y-79 cells, which have been described to form FlexnerWintersteiner rosettes (Campbell and Chader 1988; Reid et al. 1974), likewise lack these (Reid et al. 1974). Thus, after nearly 25–40 years of propagation in vitro, the RB cell lines described here obviously lost the ability to form FlexnerWintersteiner rosettes as we never observed rosettes in any
Histochem Cell Biol
of our cultures independent of the culture condition (suspension or monolayer on coated coverslips) investigated. Comparing the morphology of Y-79 and WERI-Rb1 RB cells, investigators found these cell lines to be similar as both have small, round cells with little cytoplasm and large nuclei, grew as suspension cultures in loose grape-like clusters and both attach on coverslips pre-treated with a polyl-ornithine solution (McFall et al. 1977; Jiang et al. 1984; Campbell and Chader 1988). Besides, both RB lines exhibited growth of cells in rosettes and chain formation (McFall et al. 1977). In attachment cultures with pre-treatment of the culture dishes with poly-d-lysine, Y-79 cells grow in a monolayer, in clusters, or in chains with occasional rosettes (Sasabe and Inana 1991). Reid et al. (1974) reported that with continued culture Y-79 cells frequently arrange in columns. In our cultures, formation of long chains—especially in monolayer cultures—is a main characteristic of WERIRb1 cells, frequently described to grow in grape-like clusters and is only sometimes seen in Y-79 cells, mainly after attachment to poly-d-lysine-coated coverslips. Colony formation It has been reported that retinoblastoma cells only insufficiently form colonies in the standard semisolid medium (McFall et al. 1977). Y-79 could be cloned in soft agar, while WERI-Rb1 could not (McFall et al. 1977; Griegel et al. 1990a). Y-79 RB cells formed rapidly growing, visible colonies in soft agar at a rate of 15 % (Sasabe and Inana 1991). Colony formation was obtained not only with Y-79, but also with RB 247 C3, RB 355, RB 383, RBL-13 and RBL-15, but only when small aggregates of 2–10 cells were seeded into soft agar. In contrast to our results, Griegel et al. (1990a) stated that colony formation was never observed after single cells from each of the RB lines investigated had been seeded into soft agar medium. Y-79 displayed high frequency of colony formation after 4 weeks, whereas RB 247 C3, RB 355, RB 383, RBL-13 and RBL-15 cells exhibited low frequencies after 6 weeks of culturing in soft agar medium (Griegel et al. 1990a). RB13 and RB15 cells likewise displayed very low colony formation frequencies (Griegel et al. 1990a). Slightly modifying the original human tumor clonogenic assay (HTCA) established by Hamburger and Salmon (1977), the group around Inomata and Kaneko improved colony formation of the cultured RB cell lines Y-79 and WERI-Rb1 (Inomata et al. 1986; Inomata and Kaneko 1987). In this setting, WERI-Rb1 cells formed colonies, which remained in the same “chain form” seen in monolayer culture (Inomata et al. 1986; McFall et al. 1977). In our soft agarose assay—cells were seeded in 0.35 % agarose on a layer of 0.5 % agarose—all RB cell lines except RB 355 cells were able to form colonies after single
cell seeding. After 2 weeks of incubation, colony formation was visible, with the highest anchorage-independent growth capacity and colony size detected in WERI-Rb1 cells followed by Y-79 cells. Metastasis and invasiveness The CAM of the chick embryo is rich in blood vessels and capillaries, allowing survival of tumor cells placed on the dropped CAM (Deryugina et al. 2005; Kim et al. 1998). The CAM is an impenetrable barrier to invasive cells unless it has been traumatized by removing the upper peridermal part of the double epithelia layer, leaving the basal layer intact (Armstrong et al. 1982; Martinez-Madrid et al. 2009). Under these conditions, tumor cells can enter the tissue regardless of their invasive potential (Ossowski 1988), but only cells capable of penetrating blood vessel walls will be able to circulate and arrest in chick embryonic vessels and tissues. The ventral, so-called “lower CAM” (Kim et al. 1998) is a compartment physically connected to the so-called “upper CAM”, resembling the site of inoculation, only through blood and lymphatic vessels. Tumor cell intravasation, describing the process of cancer cells entering the vasculature, is one of the rate-limiting, but leaststudied steps in the metastatic cascade. Contradictory to a former study reporting that the RB cell lines RBL-13, RBL-30 and RBL-15 are noninvasive at least when tested for invasion into chick heart fragments (Griegel et al. 1990a), in the study presented nearly all RB cell lines investigated migrated into the lower CAM, although the rate of GFP-positive cells within the lower CAM punches analyzed was highly variable, indicating different intravasation (spontaneous metastasis model) and migration and/or extravasation (experimental metastasis model) capacities of the six RB cell lines investigated. In general, the GFP-positivity reflecting the number of RB cells reaching the lower CAM, was lower after grafting (spontaneous metastasis model), whereas the tumor formation on the upper CAM was slightly higher after inoculation. There are two possible explanations for higher tumor formation after grafting: (i) the tumor cell concentration was higher in the spontaneous metastasis model (grafting of 1 × 106 cells) compared to the experimental metastasis model (injection of 1.5 × 105 cells) and (ii) the cells were inoculated onto the upper CAM, representing the tumor formation site. In our spontaneous and experimental metastasis model, GFP-labeled RB cells, which intravasated into and migrated in the CAM vasculature after inoculation onto the upper CAM or intravenous injection could be visualized spreading in the lower CAM and extravasating from the CAM capillary system, resembling the results of former studies, in which the authors inoculated variants of
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HT-1080 fibrosarcoma cells onto the CAM (Deryugina et al. 2005; Deryugina and Quigley 2008). Our results support data of other groups suggesting that Y-79 cells represent invasive and metastatic RB tumor cells, whereas WERI-Rb1 cells resemble a non-metastatic phenotype (McFall et al. 1977; Gallie et al. 1977; Chévez-Barrios et al. 2000). In this context, Chévez-Barrios et al. (2000) showed that after injection into the vitreal cavity of immunodeficient mice, Y-79 cells not only formed intraocular tumors in the vitreal cavity, but invaded the retina, the optic nerve head and the anterior chamber of the eye and form metastases in the contralateral optic nerve. After intravitreal injection of WERI-Rb1 cells, by contrast, tumors were confined to the eye with only anterior choroidal invasion (Chévez-Barrios et al. 2000). McFall et al. (1977) likewise reported that WERIRb1 cells were weakly tumorigenic at either inoculum, whereas Y-79 cells were strongly tumorigenic at an inoculum as low as 106. Further along this line, Gallie et al. (1977) reported that RB cells isolated from primary tumors proliferate after intra-ocular implantation into the anterior eye chamber of nu/nu (T−) mice, but only Y-79 invade the orbit, the optic nerve and the brain and produce rapidly growing tumors in nu/nu (T−) mice after subcutaneous injection of high cell numbers, thus displaying a much more invasive phenotype. Thus, our data confirm the notion that even after years of propagation human Y-79 RB cells retain their high tumorigenic potential (del Cerro et al. 1993). The expression-based quantification method of human cells in the CAM model, implemented in our study, is a good alternative to the established Alu DNA repeat-based quantification system (Kim et al. 1998; Zijlstra et al. 2002; Mira et al. 2002; Palmer et al. 2011). There are, however, certain shortcomings of our quantitative CAM method. Most retinoblastomas have metastatic potential through invasion of the optic nerve, a characteristic that cannot be addressed using the CAM assay. As other patterns of RB invasion like hematogenous dissemination with formation of metastasis in lungs, bones and brain and lymphatic spread (McLean et al. 1994) have been described, the CAM, nevertheless, provides a useful in vivo-like model system to address the metastatic potential of retinoblastoma cells. Besides, it turned out that a certain amount of GFPpositive lower CAM punches (4 out of 6) are required to successfully amplify hGAPDH by real-time PCR, indicating the detection limit of our quantification method. Thus, future iterations will have to further optimize the correlation between the amount of human cells per lower CAM punch and the quantification by hGAPDH expression and lower the detection limit, in order to refine the determination of human cell content per chick CAM. The detection of gene expression instead of analysis of genomic DNA, which leads to frequent contaminations in the negative controls, is critical as GAPDH expression
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Histochem Cell Biol
might change if human Rb cells are inoculated into a chick tissue environment. To minimize the risk of false positive or false negative GAPDH detection, we, however, employed a control step by evaluating the chick CAM punches for GFPpositive human cells under the fluorescence microscope and always found the level of hGAPDH expression in the lower CAM punches detected by our real-time PCR analyses to correlate with the number of GFP-positive CAMs. Additionally, in the study presented, we never observed any hGAPDH amplification in our negative controls.
Summary All retinoblastomas have mutations of both RB alleles, but they always display additional non-RB mutations (Squire et al. 1985; Chen et al. 2001, 2002). The RB1 mutation status of the RB cell lines investigated in this study indicates that the cell lines originate from RB tumor tissue. It is reasonable to assume that mutations in additional genes contribute to promotion and progression of this tumor (Gallie et al. 1999). Against the background that primary retinoblastoma material from patients is not readily available, RB cell lines provide an alternative to investigate the effects of (i) overexpression or knockdown of genes believed to be associated with retinoblastoma dissemination and progression and (ii) newly established, prospective pharmacological therapeutics for RB. If a new gene or reagent is believed to decrease RB tumor cell growth, a fast-growing RB cell line like Y-79 would be the ideal test system to proof this hypothesis. Intending to analyze induction of apoptosis, one would depict a RB cell line with low endogenous cell death levels like RBL-15 or WERI-Rb1. The capability to grow anchorage independent and form colonies in soft agarose represents one feature of a tumor cell and loss of this ability upon overexpression or knockdown of a certain gene or application of a reagent would indicate an effect on tumorigenicity. Last but not least, the CAM assay provides a helpful model system to test for effects on tumor cell intra- and extravasation, dissemination, migration and new tumor formation (metastasis). In contrast to Matrigel™ invasion or three-dimensional collagen assays, reported to be useful tools in metastasis research, but not necessarily reflecting the physiological events facilitating the dissemination of tumor cells, this in vivo-like system has a long history as an efficient model to study complex physiological processes such as tumor metastasis (Gordon and Quigley 1986; Scher et al. 1976). Summarizing one can state that our data display no predictable correlation between the growth capacity or apoptosis rates of the RB cell lines investigated and their anchorage-independent growth or between the ability to form colonies in soft agarose and to intravasate into the chick CAM tissue or to develop tumors on the CAM (Table S1).
Histochem Cell Biol Table 4 Correlation of the number (#) of GFP-positive lower CAM tissue punches and real-time PCR-based quantification of human RNA in lower chicken CAMs 6 days after intravenous injection of different RB cell lines RB cell lines
GFP + tissue punches of CAM (#)
Human RNA in CAM (pg)
Y-79_GFP
1 of 6 4 of 6 2 of 6 None 6 of 6 6 of 6 6 of 6 4 of 6 6 of 6
nd
ORIGINAL PAPER
Re-characterization of established human retinoblastoma cell lines Maike Busch · Claudia Philippeit · Andreas Weise · Nicole Dünker
Accepted: 29 September 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell Electronic supplementary material The online version of this article (doi:10.1007/s00418-014-1285-z) contains supplementary material, which is available to authorized users. M. Busch · C. Philippeit · N. Dünker (*) Department of Neuroanatomy, Medical Faculty, Institute of Anatomy, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany e-mail: [email protected] A. Weise Institute of Biology II/Cell Biology, University of Freiburg, Freiburg, Germany
lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro. Keywords Retinoblastoma · RB cell lines · Soft agar assay · Colony formation · CAM assay · Invasiveness · Dissemination · Tumor formation
Introduction Retinoblastoma (RB) is the most common malignant eye tumor of childhood occurring in infants as both, a heritable (usually bilateral) and non-heritable (unilateral) form. The most important factor for its prognosis is the extension of invasion and dissemination of the retinoblastoma cells and the formation of metastasis. There are different patterns of invasion: (i) invasion along the optic nerve to the brain, (ii) dispersion into the circulating subarachnoid fluid toward the spinal cord and distant sites of the brain, (iii) hematogenous dissemination with formation of metastasis in lungs, bones and brain and (iv) lymphatic spread (McLean et al. 1994). Retinoblastoma is a uniquely human disease and all animal models like injections in the subretinal space or the anterior chamber of eyes of immunosuppressed or deficient rats and mice (Gallie et al. 1977; del Cerro et al. 1993), which have been developed so far, had limitation. The application of tissue and cell culture techniques permits detailed studies of the biology of the tumor, focussing on growth and metastatic potential of RB cells. In addition, the chick chorioallantoic membrane (CAM) is an established, naturally immunodeficient model system
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for in vivo studies of tumor cell dissemination (Deryugina and Quigley 2008). Different steps in the metastatic tumor cascade can be addressed using this model: (i) spontaneous metastasis after tumor cell grafting onto the CAM and (ii) experimental metastasis after intravenous inoculation of tumor cells. In the spontaneous metastasis setting, aggressive tumor cells escape from the primary grafted cells and intravasate into the chick vasculature, providing a model for the intravasation step of the metastatic cascade. In the second setting, tumor cells extravasate from the CAM capillary system and colonize. Even after years of research, still little is known about the functional properties of RB cells. In 1974, Reid et al. established the first human retinoblastoma cell line called Y-79 and characterized it morphologically, cytogenetically and with respect to cell growth and biochemistry. Later, McFall and her group published the characterization of a second established RB cell line: WERI-Rb1 (McFall et al. 1977). The growth of RB cells in culture for a prolonged time remained difficult till in the eighties, two groups described methods of establishing cell lines from RB tumors (Gallie et al. 1982; Bogenmann and Mark 1983). Jiang et al. (1984) demonstrated that WERI-Rb1 and Y-79, which grew in loose clusters and attached to coverslips, pre-coated with poly-l-ornithine solution, both exhibit neuronal and glial properties. In the nineties, Griegel et al. (1990a, b) established and characterized seven new RB cell lines: RBL-13, RBL-14, RBL-18 and RBL-30, derived from unilateral retinoblastomas and RBL-7, RBL-15 and RBL-20, derived from bilateral retinoblastomas. The authors investigated colony formation properties in soft agar as well as the invasive potential of these cell lines and reported (i) that none of them was able to form anchorage-independent colonies from a single cell suspended in semisolid agar and (ii) that they were noninvasive when tested for invasion into chick heart fragments (Griegel et al. 1990a). Very different growth rates and morphologies have been described for Y-79, RB 355 and WERI-Rb27 cells (Madreperla et al. 1991). Previous studies addressing the genetic characteristics of RB cell lines showed a partial deletion and a total loss of the RB1 gene in Y-79 and WERI-RB1 cells, respectively (Bookstein et al. 1988; Choi et al. 1993). Griegel et al. (1990a) observed loss of heterozygosity of the RB1 gene in RBL-30 cells, but not in RBL-13 and RBL-15 cells. Evidence for a point mutation in RB1 was observed in the RB 30 tumor, where the RBL-30 cell line is derived from (Griegel et al. 1990a). Forty years after their first description and thus 40 years after establishment and propagation in vitro, in the study presented, we set out to re-characterize the retinoblastoma cell lines RBL-13, RBL-15, RBL-30, RB 355, RB 383,
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Histochem Cell Biol
Y-79 and WERI-Rb1 with regard to (i) their RB1 mutation status (ii) their growth kinetics, (iii) endogenous apoptosis levels, (iv) colony formation potential and (v) invasiveness and metastasis capacity. Our study updates current knowledge on established RB cell lines and provides valuable new data for future in vitro approaches in retinoblastoma therapy-related research with regard to main tumor cell properties like growth behavior, anchorage-independent growth as well as intravasation and dissemination potential.
Materials and methods Cell culture Seven human retinoblastoma (RB) cell lines RBL-13, RBL15, RBL-30, RB 355, RB 383, Y-79 and WERI-Rb1 (generously provided by H. Stephan; Reid et al. 1974; McFall et al. 1977; Griegel et al. 1990a; Madreperla et al. 1991) were cultivated as suspension cultures in Dulbecco’s modified Eagle’s medium (DMEM; PAN-Biotech) with 10 % fetal calf serum (FCS; PAN-Biotech), 100 U penicillin/ml and 100 µg streptomycin/ml (Invitrogen), 4 mM l-glutamine (Sigma), 50 µM β-mercaptoethanol (Roth) and 10 µg insulin/ml (Sigma) at 37 °C, 10 % CO2 and 95 % humidity. Human embryonic kidney cells (HEK293T; generously provided by B. Royer-Pokora) were grown as adherent cultures in DMEM (PAN-Biotech) with 10 % FCS (PANBiotech), 100U penicillin/ml and 100 µg streptomycin/ml (Invitrogen) at 37 °C, 5 % CO2 and 95 % humidity. Growth kinetic Growth kinetics of the different retinoblastoma cell lines were determined by serial dilution of the cells in 96-well plates as described in Weise and Dünker (2013). In brief, serial dilutions followed this scheme: any of the wells of a 96-well plate was filled with 100 µl growth medium. Hundred microliters cell suspensions from a culture with 0.3–0.5 × 106 cells/ml was pipetted into well A1 and subsequently horizontally diluted 1:2 up to the last well (A12). Afterward, vertical dilutions (1:2) of the entire row A were carried out up to the last row (H) of the 96-well plate. Wells with single cells were examined every 24 h, and cells were counted. Cells which did not at least reduplicate were designated as dead and were not included into the evaluations. In order to compare the growth kinetic of RB cells growing in aggregates to that starting from single cells, we plated a fix concentration (1.5 × 105 cells/ml) of all RB cells lines in 24-well plates. Wells with cell aggregates were examined, and living cells were counted in a Neubauer chamber after trypan blue staining every 24 h.
Histochem Cell Biol
Determination of apoptosis To determine apoptosis and visualize the morphology of the RB cell lines by 4′,6-diamidino-2-phenylindole (DAPI)counter staining, 1 × 105 cells were seeded on poly-d-lysine (Sigma) coated coverslips and grown in 1 ml medium in 24-well plates. The number of DAPI-stained pycnotic nuclei was determined as described before (Haubold et al. 2010). For each cell line, five coverslips were evaluated. A total of at least 1,000 cells per coverslip were counted, and the percentage of apoptotic, clearly pycnotic nuclei (at least 10) was calculated.
(10 µg/ml, H9268, Sigma). Additional 2 ml of DMEM medium with supplements (as described under “cell culture”) was added after another 24 h. After another 48 h, the medium was completely changed. Invasiveness of RB cells: CAM assay For our metastasis model, we mainly followed the protocol published by Zijlstra et al. (2002) and most recently visualized by Palmer et al. (2011). 1. Spontaneous metastasis model: grafting of GFP-labeled RB cells
Colony formation assay For growth in soft agarose 5 × 104, RB cells were suspended in 2 ml DMEM/F12 medium (Sigma) containing 10 % fetal calf serum, 100 U penicillin/ml and 100 µg streptomycin/ml, 4 mM l-glutamine, 50 µM β-mercaptoethanol, 10 µg insulin/ml and 0.35 % agarose (Roth). The cell suspension was layered on 2 ml 0.5 % agarose, containing DMEM/F12 medium with the same supplements as indicated above and cultured in 6-well plates at 37 °C, 10 % CO2 and 95 % humidity. As positive control 5 × 104 HEK293T cells were suspended in DMEM/F12 medium with 10 % fetal calf serum and 100U penicillin/ml and 100 µg streptomycin/ml containing 0.35 or 0.5 % agarose as described. Cells were fed repeatedly after 4–5 days and documented weekly over a time period of 4 weeks. Colony formation, starting from single cells seeded in soft agarose, was quantified after 3 weeks of incubation. Plating was repeated three times. The colony formation efficiency (%) per visual field was determined by counting the colonies and single cells in three visual fields (10×) per cell line in triplicates. Lentiviral GFP labeling of RB cell lines For virus production, HEK293T cells were transfected with 6 µg of each of the following plasmid DNA: packaging vector pczVSV-G (Hartmann et al. 2010), pCD NL-BH (Hartmann et al. 2010) and a GFP expressing vector (pCL7EGwo, kindly provided by Dr. H. Hanenberg) in the presence of 45 µg polyethyleneimine (PEI, branched, Aldrich). The medium was changed after one day to Iscove’s Modified Dulbecco’s medium (IMDM, Gibco) with 10 % FCS and 1 % penicillin/streptomycin and 48 h after transfection viral supernatants were harvested, filtered (0.45 µm) and cryoconserved. For stable GFP labeling, RB cells were seeded in a 6-well plate (6 × 105 cells/well). After 24 h, cells were infected with GFP lentiviruses in the presence of polybrene
Fertilized eggs were incubated in a humidified rotary incubator at 38 °C and 50 % humidity for 10 days. On embryonic (E) day 10, the eggs were candled by shining light into the eggshell at the blunt end of the egg. The chorioallantoic vein was located, and a 1 cm square was marked with a pencil approximately 1 cm away from the veins branching point. A hole was drilled through the blunt end of the egg into the air sac and within the drawn square, and the “upper CAM” (see Fig. 1 in Kim et al. 1998) was dropped by gentle suction, creating an artificial air sac. The CAM was gently abraded by the use of a cotton-tipped applicator. Immediately after damaging the CAM, 50 μl of the cell suspension (1 × 106 cells in PBS) was placed onto the traumatized CAM area. The window in the egg was sealed tightly with tape and returned to the incubator. 2. Experimental metastasis model: intravenous injection of GFP-labeled RB cells Fertilized eggs were incubated for 12 days. On developmental day E12, the eggs were candled by shining light into the eggshell at the blunt end of the egg. The chorioallantoic vein was located, and a 1 cm by 0.5 cm rectangle was marked with a pencil approximately directly above the vein. A small window was created in the eggshell, and the eggshell membrane was rendered transparent with a drop of mineral oil. Fifty microliters of cell suspension (1.5 × 105 cells in DMEM) was injected into the allantoic vein of each embryo. The window in the egg was sealed tightly with tape and returned to the incubator. Injections were performed under the dissection microscope in order to monitor if the injected cells are carried by the blood flow (=correct direction of the injection). In each experiment, 10–15 eggs were injected and if the cells were wrongly injected against the blood flow (=wrong direction), we excluded the eggs from our experiments. We likewise only used eggs with minimal or no blood leakage after injection to make sure that the complete amount of cell suspension was injected.
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Table 1 Mutation status of RB cell lines
Histochem Cell Biol RB cell line
References
Y-79 WERI-Rb1
Reid et al. (1974) McFall et al. (1977)
RB 355
Madreperla et al. (1991)
Unilateral
no RB1 deletion
RB 383
Griegel et al. (1990a, b)
Unilateral
RBL-13 RBL-15
Griegel et al. (1990a, b) Griegel et al. (1990a, b)
Unilateral Bilateral
Genomic rearrangements of the RB1 locus c.958 C>T/c.2390 T>G
RBL-30
Griegel et al. (1990a, b)
Unilateral
Harvesting of tissue For each RB cell line, 10 eggs were grafted and intravenously injected, respectively. The duration of the CAM assay is limited to a 7–9 day window available before the chick hatches. Thus, after distinct time points (7 days after inoculation (E10-17) and 6 days after injection (E12-E18)), six standardized (1.4 cm internal diameter) tissue punches of each ventral “lower CAM” (see Fig. 1 in Kim et al. 1998) were collected and transferred into a 24-well plate filled with PBS. Each of the six lower CAM punches was scanned for GFP-positive RB cells by fluorescence microscopy, combined and cryoconserved for RNA extraction. In order to prevent cross-contamination of the samples, the dissection tools were sterilized by sequential rinsing in: (1) 10 % NaOH, (2) 70 % ethanol, (3) double distilled water, and (4) again double distilled water, following the protocol published by Palmer et al. (2011). RNA isolation and quantitative real-time PCR analysis For RNA isolation, the CAM tissue punches were mechanically pulverized on dry ice without defrosting. RNA isolations from RB cell lines and CAM tissue were performed using the NucleoSpin RNA II kit (Macherey and Nagel), and cDNA was synthesized with the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s protocol. Quantitative real-time PCR analyses were performed using a 7300 Real-Time PCR System (Applied Biosystems). The following human Taqman Gene Expression Assays (Applied Biosystems) were used: GAPDH (Hs99999905_m1) and 18S (Hs99999901_s1). The latter was used as an endogenous control for human and chicken samples. Real-time PCRs were performed in duplicate, and a total volume of 20 µl was applied to the following program: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Mutation analysis Mutation analysis was performed as described previously (Rushlow et al. 2013). In brief, a MLPA Kit
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Type of tumor
RB1 mutation del exon 2–6/IVS20+1G>A del RB1/del RB1 (including/TM2B to DLEU)
del RB1/del RB1 (from RB1 to DLEU) g59695 C>T/g59695 C>T
(MRC-Holland, Amsterdam, Salsa MLPA Kit P047 RB1) was used and reactions were performed according to the manufacturer’s instructions. Additional sequencing of the RB1 gene was performed for all retinoblastoma cells lines.
Results Mutation status of RB cell lines We analyzed the RB1 mutation status of seven RB cell lines in order to verify already published data and reinvestigate mutations in the RB1 gene. A description of the RB1 mutation status of all RB cell lines investigated is summarized in Table 1. A previous genetic characterization of the commercially available RB cell line Y-79 revealed a partial deletion of the RB1 gene (Bookstein et al. 1988). Our re-investigation of Y-79 cells uncovered a heterozygous deletion of exon 2–6 and an intronic point mutation (IVS20+1 G>A) on the other allele. A former study reported on a total loss of the RB1 gene in the likewise commercially available RB cell line WERI-Rb1 (Choi et al. 1993). Our mutation analysis confirmed a complete deletion of RB1 in WERI-Rb1 cells and additionally revealed that this deletion ranges from ITM2B up to DLEU, resulting in an absent RB1 protein. In our MPLA analysis, the RB cell line RB 355 showed no deletions in the RB1 gene and other alterations in the RB1 gene (e.g., point mutations in one of the 27 exons) have not been determined yet for these cells. RB 383 cells exhibited extensive genomic alterations in our MPLA analysis, indicating large rearrangements of the RB1 locus. RBL-13 cells showed two heterozygous point mutations in the RB1 gene: c.958 C>T and c.2390 T>G. RBL-15 cells, derived from a bilateral tumor, displayed a complete deletion of RB1 reaching from RB1 up to DLEU, causing an absent RB1 protein. Griegel et al. reported on evidence for a point mutation in RB1 in the primary retinoblastoma tumor, the RB cell line RBL-30 originally derives from (Griegel et al. 1990a). Our analysis revealed a homozygous g59695 C>T point mutation in the RB1 gene of RBL-30 cells.
Histochem Cell Biol
RB cell line morphology and growth Six of the seven established human RB cell lines RBL-13, RBL-15, RBL-30, RB 355, RB 383, Y-79 and WERI-Rb1 investigated grew in suspension and were predominantly small epithelioid cells with little cytoplasm and large nucleus (Fig. 1). RB 355 cells, by contrast, partly attached to the flask and grew as adherent spindle-shaped cells with a larger cytoplasm (Fig. 1c4). All RB cell lines investigated were able to attach to coverslips pre-treated with poly-d-lysine. They mainly grew in cell clusters and form aggregates and clumps from ten to hundreds of cells (Fig. 1). A chainlike structure could frequently be observed in WERI-Rb1 suspension cell cultures (Fig. 1a2–c2), but also occurred in Y-79 cultures (Fig. 1a1–c1). After attachment to polyd-lysine-coated coverslips, Y-79 more frequently tends to form chain-like structures and WERI-Rb1 cells mainly grew as long chains, sometimes also forming chain loops, which, however, did not resemble Flexner-Wintersteiner rosettes. Besides normal aggregates, RBL-13 cells formed three-dimensional “hollow cell balls” in different sizes (Fig. 1a5–c5). In general, RBL-30 cells were the smallest in size, whereas the adherent RB 355 cell line comprised the largest RB cells. Population doubling times and endogenous apoptosis rates Under our culture conditions, we observed different growth rates for different retinoblastoma cell lines, which are summarized in Fig. 2b, d. We counted growing RB cells (a) starting from single cells (Fig. 2a) and (b) starting from cell aggregates (Fig. 2c), naturally formed by these cells, in order to compare potential differences in cell growth rates. In both approaches, RBL-30, RBL-13 and RBL 383 cells grew very slowly and hardly doubled within 1 week. These RB cell lines displayed drastically reduced growth rates with doubling times of 69 up to 220 h, depending on the culture method used. By contrast, Y-79 cells were one of the fastest growing cell lines under both culture conditions with calculated doubling times between 45 and 61 h. All other cell lines were slightly variable in their growth kinetic depending on the method used (Fig. 2). It was not possible to determine a growth curve for RBL-13 cells, when these cell lines were grown starting from cell aggregate due to very strong aggregate formation, which leads to inaccuracy in cell number counts. The endogenous apoptosis levels of the different RB cell lines were quantified by counting DAPI-stained pycnotic nuclei after 24 h culture on poly-d-lysine pre-treated coverslips (Fig. 3). The apoptosis rates differed from two to nearly eight percent with the highest apoptosis in RB 383 and RB 355 and the lowest in WERI-RB1 and RBL-15.
Anchorage-independent growth: capacity of different RB cell lines to form colonies in soft agarose For determination of anchorage-independent growth in soft agarose, RB cell lines and HEK293T cells as positive control were seeded in 0.35 % soft agarose and bedded on a layer of 0.5 % agarose in 6-well plates. Visible colonies were photographed weekly and counted after 2 weeks of incubation by the use of an inverse microscope (10×). Six of the seven RB cell lines analyzed were able to form colonies in soft agarose upon single cell seeding after 3 weeks of incubation (Fig. 4). RB 355 did not form detectable colonies even after 4 weeks of incubation. The average colony size of all RB cell lines investigated was around 76 µm, except in WERI-Rb1 cells, which displayed the largest colonies with a size of 200 µm (Fig. 4). WERI-Rb1 likewise displayed the highest colony formation efficiency with 66.1 %, followed by Y-79 (54.4 %) and RBL-15 (51.5 %) cells. The frequency of colony formation of the other four cell lines ranged from 16.2 to 5 % and is slightly variable (Table 2). CAM Assay: invasiveness and metastasis capacity The chick chorioallantoic membrane (CAM) is an established model system for in vivo studies of tumor cell dissemination (Deryugina and Quigley 2008). Fluorescent GFP-tagging of all RB cell lines enabled us to localize the human tumor cells within the chick embryo CAM tissue. In our spontaneous metastasis setting, GFP-labeled RB cells were grafted onto the upper, dorsal CAM and 10 days after inoculation, tumor cells could be identified in distant portions of the lower, ventral CAM (Fig. 5). Intravasated cells could be visualized and appeared extravasating and scattering in the lower CAM. A total of 60 CAM punches were analyzed for each RB cell line, and the average percentage of GFP-positive lower CAM tissue punches was estimated. Four out of seven RB cell lines (Y-79, RBL-13, RBL-15 and RBL30) displayed GFP-positive cells in the lower CAMs after grafting, whereby RBL-13, RBL-15 and Y-79 showed the highest percentage with 16–18 % positive CAMs (Table 3). One RB cell line (RBL-13) caused tumor development on the upper CAM upon grafting (Fig. 5a; Table 3). Second, in experimental metastasis setting, intravenously injected GFP-labeled RB cells extravasated from the CAM capillary system and colonized near the CAM vessels (Fig. 5b). All RB cell lines had the potential to extravasate from the capillary system after injection. The calculated average of GFP-positive lower CAMs ranged from 6–80 %, whereby WERI-Rb1 (80 %) and RB 383 (72 %) displayed the highest rates of positive CAMs (Table 3). Even in this experimental setting, tumor formation on the upper CAM was detected in the two RB cell lines Y-79 and RBL-13 (Fig. 5a).
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Fig. 1 Morphology of RB cell lines in suspension culture (a1–7) and adherent on polyd-lysine-coated coverslips after DAPI staining (b1–7 and c1–7). The arrow heads (c1–c7) indicate the cytoplasmic rim of the RB cells. Cell nuclei are counter-stained with DAPI. (A1–7) phase contrast microscopy photographs (20×); (b1–7) merged DAPI-stained phase contrast photographs, (c1–7) zoom in of RB cell clusters to visualize the relationship of large nucleus to small cytoplasmic rim. Bar 20 µm applies to b1–7
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Histochem Cell Biol
Histochem Cell Biol
Fig. 2 Growth curves of different RB cell lines with doubling times and doubling time ranges (95 % confidence interval). Doubling times were calculated based on best-fitted exponential growth curves using the GraphPad Prism4 program. Indicated doubling times and time
Fig. 3 Endogenous apoptosis rates of RB cell lines. Cells were seeded on poly-d-lysine-coated coverslips and cultivated for 24 h. Pycnotic cell counts from DAPI stains were performed to determine apoptosis rates. Values are mean ± SD from three independent experiments, each with five different coverslips
Quantification of human RNA in chicken tissue by realtime PCR analyses Levels of intravasated and disseminated human RB tumor cells into the chick CAM and embryo tissue can be
ranges represent means of 6–16 counts in the case of seeded single cells and duplicates in the case of seeded aggregates. RB cells were seeded as single cells (a) or aggregates with a starting concentration of 1.5 × 105 cells/ml (c), and doubling times were calculated (b, d)
determined by quantitative real-time PCR analysis (e.g., Zijlstra et al. 2002; Palmer et al. 2011). Previous studies made use of the amplification of species-specific Alu DNA repeats characteristic of the primate genome (Schmid and Jelinek 1982; Deryugina et al. 2005; Deryugina and Quigley 2008; Kim et al. 1998; Zijlstra et al. 2002; Mira et al. 2002; Palmer et al. 2011). Human Alu repeat sequences represent approximately 5 % of the human genomic DNA and can easily be amplified, but this fact often represents the reason for contaminations (Mira et al. 2002). The method works basically well under complete sterile conditions, but is not readily applicable to the CAM model. It is technically very difficult to open the egg shell under a sterile bench without injuring the CAM and injections into the CAM blood vessels need to be done under a dissection microscope. Under these conditions, the contamination risk with human genomic DNA (gDNA) is very high. Contamination with gDNA is sequentially visible in Alu DNA repeat signals in negative controls, and these background levels detected in CAMs of control chick embryos that did not receive any human tumor cells were routinely subtracted from the curves (Deryugina et al. 2005; Mira et al. 2002). To circumvent this problem, we decided to establish a gene expression-based method for the quantification
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Histochem Cell Biol
Fig. 4 Colony formation of RB cell lines and HEK293T cells in soft agarose after 1, 2 and 3 weeks in culture. 5 × 104 RB or HEK293T cells were suspended in medium. The cell suspension was layered on 0.5 % agarose containing medium and cultured in 6-well plates. The outermost left row of figures shows the cells under the microscope
(10× magnification) at day 0 (day of seeding) and depicts that colony formation started from single cells seeded into the soft agarose. The 4× phase contrast photographs after 2 weeks of cultivation serves as an overview to judge the average colony formation
of the amount of human tissue in the chicken CAMs. For this purpose, we provided evidence that human GAPDH (hGAPDH) expression is completely undetectable in chicken CAM tissue and 18S is suitable as endogenous control for both human and chick tissue (Fig. S1). To determine the range and sensitivity of this method and to create a standard curve, increasing human RNA concentrations of RB cells were mixed with chick RNA from CAM tissue at proportions of 0.01, 0.05, 0.1, 1, 5, 10
and 20 %. One hundred percent human RB cell RNA and 100 % chick CAM tissue RNA were used as positive and negative control, respectively, in every experimental setting. Human GAPDH expression was detectable from very low concentrations of 100 pg (0.01 %) up to 1 µg (100 %) human RNA displayed by the standard curves of the RB cell lines (Fig. 6). In order to correlate the hGAPDH signal—and thereby the amount of human tumor cell extravasation—with the amount of amplified cDNA, 18S RNA,
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equally expressed in human and chick tissue (Fig. S1) was used as an internal control (Fig. S2). To quantify the intravasation of the RB cells into the chick tissue, we pooled the standardized chick CAM punches from each embryo, isolated total RNA, and synthetized cDNA from 1 µg RNA. For each RB cell line, eight chick embryos were analyzed: four after CAM grafting and four after intravenous injection of RB cells. Each analysis included a RB cell
Table 2 Colony formation capacity (%) from single cells seeded in soft agarose and colony formation time of RB cell lines RB cell lines
Colony formation efficiency (%)
Colony formation time (weeks)
WERI-Rb1 Y-79 RBL-15 RBL-30 RB 383 RBL-13
66.1 54.4 51.5 12 5.7 5
3 3 3 3 3 3
RB 355
NCD
3
Colony formation capacity was determined by the mean number of colonies relative to the seeded cells of three different visual field counting’s (10×) in triplicate. NCD no colonies detected
line-specific standard curve. The hGAPDH threshold cycle (CthGAPDH) was normalized against the 18S internal control (Ct18S/CthGAPDH), to calculate a relative amount of human RNA to be compared with the RB cell line-specific standard curve in order to determine the amount (ng) of intravasated RB cells. We were able to amplify hGAPDH from GFP-positive lower CAMs after intravenous CAM injection of different human RB cell lines with decreasing concentration of human RNA (inferred from hGAPDH amplification) in the following order: WERI-Rb1 > RB 383 > RBL-15 > RBL30 > Y-79 (Table 4). No hGAPDH expression was detectable after inoculation as it turned out that—depending on the cell concentration per CAM punch—four out of six GFPpositive CAM punches are required to amplify hGAPDH successfully, indicating the detection limit of our newly established method. WERI-Rb1 displayed the highest potential to extravasate and migrate into the CAM tissue resulting in high human RNA concentration in the lower CAM upon injection correlating with a high number of GFP-labeled cells visualized by fluorescence microscopy. All lower CAM punches were GFP-positive with high concentration of WERI-Rb1 cells, reflected by up to 0.5 ng human RNA in the lower CAM tissue as revealed by hGAPDH amplification (Table
Fig. 5 Localization of RB tumor cells after intravenously injection and inoculation on the chicken CAM. Tumor formation on the upper chicken CAM was detectable 6–7 days after inoculation and injection of RBL-13 cell (a). Arrows tumors of RBL-13 cells; bold arrow place of injection. Six day after intravenous injection, green-fluorescent, GFP-labeled RB 383 and WERI-RB1 cells extravasated from the CAM capillary system and colonized near the vessels of the lower chicken CAM (b)
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Histochem Cell Biol
Table 3 Overview of the average percentage of GFP-positive lower CAM tissue punches and tumor formation on the upper CAM after intravenously injection and inoculation of RB cell lines RB cell lines
Graft
Injection
∅ GFP + lower CAM punch (%)
Tumor formation upper CAM ∅ GFP + lower CAM punch (%)
Y-79_GFP WERI-Rb1_GFP RB 383_GFP RBL-13_GFP RBL-15_GFP
16.6 – – 18.5 18.3
RBL-30_GFP
8.3
− − − + −
−
18.3 80 71.6 18.3 25 20
Tumor formation upper CAM + − − + −
−
The average percentage of GFP-positive tissue punches of lower CAMs was calculated from 10 eggs per RB cell line and six punches per egg
Fig. 6 Human GAPDH standard curves of the 6 RB cell lines investigated in the CAM experiments. Quantitative real-time PCR was performed on cDNA synthetized from RNA extracted from the chicken CAM and mixed with different concentrations of human RNA from different RB cells (100 pg–1,000 ng)
4). By contrast, after inoculation of WERI-Rb1 cells onto the upper CAM no GFP-positive CAM could be detected and also tumor formation was absent. In comparison, GFPlabeled Y-79 cells reached the lower CAMs after inoculation and injection and also formed tumors after injection into the chorioallantoic vein (Table 4), indicating a highly invasive and tumorigenic phenotype although the amount of cells per tissue punch was lower compared to WERI-Rb1.
Discussion Growth rate When RB cell lines were established from different retinoblastoma tumors in the past, highly different proliferation rates and population doubling times were observed (Reid et al. 1974; McFall et al. 1977). For Y-79, Reid et al. (1974)
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determined a doubling time of 52 h. McFall et al. (1977) stated an average population doubling time of 33 h for Y-79 and 96 h for WERI-Rb1. Other investigators observed that growth rates of Y-79, WERI-Rb27 and RB 355 are obviously different in the order RB 355 > WERI-Rb27 > Y79 (Madreperla et al. 1991). Under our culture conditions, Y-79 was one of the fastest growing cell lines with a doubling time between 47 and 61 h, resembling the data published by Reid et al. (1974). RB 383, RBL-13 and RBL-30 are slow-growing cells with an average doubling time between 94 and 140 h. In general, one can state that the doubling times of the RB cell lines differ between cells that were seeded as single cells and those grown from aggregates. One should keep this fact in mind planning cell culture experiments with RB cell lines. Morphology It has been described that Y-79, WERI-Rb27, RB 355, RB 383, RBL-13 and RBL-15 cell lines grow in suspension cultures, whereas RB 355 cells are substrate-bound and require trypsinization to collect them from the culture plate (Griegel et al. 1990a). Under our culture conditions, RB 355 cells likewise grow semi-adherent but with additional suspension cells. A former study showed that cultured RB cells exhibit characteristic aggregate formation and some Rb cell lines (RBL7, RBL15 and RBL18) are able to form Flexner-Wintersteiner rosettes and retained this ability for more than 2 year in culture (Griegel et al. 1990a). Other RB lines, however, lost the ability to form rosettes after prolonged cultivation (Griegel et al. 1990a). With continued culture, Y-79 cells, which have been described to form FlexnerWintersteiner rosettes (Campbell and Chader 1988; Reid et al. 1974), likewise lack these (Reid et al. 1974). Thus, after nearly 25–40 years of propagation in vitro, the RB cell lines described here obviously lost the ability to form FlexnerWintersteiner rosettes as we never observed rosettes in any
Histochem Cell Biol
of our cultures independent of the culture condition (suspension or monolayer on coated coverslips) investigated. Comparing the morphology of Y-79 and WERI-Rb1 RB cells, investigators found these cell lines to be similar as both have small, round cells with little cytoplasm and large nuclei, grew as suspension cultures in loose grape-like clusters and both attach on coverslips pre-treated with a polyl-ornithine solution (McFall et al. 1977; Jiang et al. 1984; Campbell and Chader 1988). Besides, both RB lines exhibited growth of cells in rosettes and chain formation (McFall et al. 1977). In attachment cultures with pre-treatment of the culture dishes with poly-d-lysine, Y-79 cells grow in a monolayer, in clusters, or in chains with occasional rosettes (Sasabe and Inana 1991). Reid et al. (1974) reported that with continued culture Y-79 cells frequently arrange in columns. In our cultures, formation of long chains—especially in monolayer cultures—is a main characteristic of WERIRb1 cells, frequently described to grow in grape-like clusters and is only sometimes seen in Y-79 cells, mainly after attachment to poly-d-lysine-coated coverslips. Colony formation It has been reported that retinoblastoma cells only insufficiently form colonies in the standard semisolid medium (McFall et al. 1977). Y-79 could be cloned in soft agar, while WERI-Rb1 could not (McFall et al. 1977; Griegel et al. 1990a). Y-79 RB cells formed rapidly growing, visible colonies in soft agar at a rate of 15 % (Sasabe and Inana 1991). Colony formation was obtained not only with Y-79, but also with RB 247 C3, RB 355, RB 383, RBL-13 and RBL-15, but only when small aggregates of 2–10 cells were seeded into soft agar. In contrast to our results, Griegel et al. (1990a) stated that colony formation was never observed after single cells from each of the RB lines investigated had been seeded into soft agar medium. Y-79 displayed high frequency of colony formation after 4 weeks, whereas RB 247 C3, RB 355, RB 383, RBL-13 and RBL-15 cells exhibited low frequencies after 6 weeks of culturing in soft agar medium (Griegel et al. 1990a). RB13 and RB15 cells likewise displayed very low colony formation frequencies (Griegel et al. 1990a). Slightly modifying the original human tumor clonogenic assay (HTCA) established by Hamburger and Salmon (1977), the group around Inomata and Kaneko improved colony formation of the cultured RB cell lines Y-79 and WERI-Rb1 (Inomata et al. 1986; Inomata and Kaneko 1987). In this setting, WERI-Rb1 cells formed colonies, which remained in the same “chain form” seen in monolayer culture (Inomata et al. 1986; McFall et al. 1977). In our soft agarose assay—cells were seeded in 0.35 % agarose on a layer of 0.5 % agarose—all RB cell lines except RB 355 cells were able to form colonies after single
cell seeding. After 2 weeks of incubation, colony formation was visible, with the highest anchorage-independent growth capacity and colony size detected in WERI-Rb1 cells followed by Y-79 cells. Metastasis and invasiveness The CAM of the chick embryo is rich in blood vessels and capillaries, allowing survival of tumor cells placed on the dropped CAM (Deryugina et al. 2005; Kim et al. 1998). The CAM is an impenetrable barrier to invasive cells unless it has been traumatized by removing the upper peridermal part of the double epithelia layer, leaving the basal layer intact (Armstrong et al. 1982; Martinez-Madrid et al. 2009). Under these conditions, tumor cells can enter the tissue regardless of their invasive potential (Ossowski 1988), but only cells capable of penetrating blood vessel walls will be able to circulate and arrest in chick embryonic vessels and tissues. The ventral, so-called “lower CAM” (Kim et al. 1998) is a compartment physically connected to the so-called “upper CAM”, resembling the site of inoculation, only through blood and lymphatic vessels. Tumor cell intravasation, describing the process of cancer cells entering the vasculature, is one of the rate-limiting, but leaststudied steps in the metastatic cascade. Contradictory to a former study reporting that the RB cell lines RBL-13, RBL-30 and RBL-15 are noninvasive at least when tested for invasion into chick heart fragments (Griegel et al. 1990a), in the study presented nearly all RB cell lines investigated migrated into the lower CAM, although the rate of GFP-positive cells within the lower CAM punches analyzed was highly variable, indicating different intravasation (spontaneous metastasis model) and migration and/or extravasation (experimental metastasis model) capacities of the six RB cell lines investigated. In general, the GFP-positivity reflecting the number of RB cells reaching the lower CAM, was lower after grafting (spontaneous metastasis model), whereas the tumor formation on the upper CAM was slightly higher after inoculation. There are two possible explanations for higher tumor formation after grafting: (i) the tumor cell concentration was higher in the spontaneous metastasis model (grafting of 1 × 106 cells) compared to the experimental metastasis model (injection of 1.5 × 105 cells) and (ii) the cells were inoculated onto the upper CAM, representing the tumor formation site. In our spontaneous and experimental metastasis model, GFP-labeled RB cells, which intravasated into and migrated in the CAM vasculature after inoculation onto the upper CAM or intravenous injection could be visualized spreading in the lower CAM and extravasating from the CAM capillary system, resembling the results of former studies, in which the authors inoculated variants of
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HT-1080 fibrosarcoma cells onto the CAM (Deryugina et al. 2005; Deryugina and Quigley 2008). Our results support data of other groups suggesting that Y-79 cells represent invasive and metastatic RB tumor cells, whereas WERI-Rb1 cells resemble a non-metastatic phenotype (McFall et al. 1977; Gallie et al. 1977; Chévez-Barrios et al. 2000). In this context, Chévez-Barrios et al. (2000) showed that after injection into the vitreal cavity of immunodeficient mice, Y-79 cells not only formed intraocular tumors in the vitreal cavity, but invaded the retina, the optic nerve head and the anterior chamber of the eye and form metastases in the contralateral optic nerve. After intravitreal injection of WERI-Rb1 cells, by contrast, tumors were confined to the eye with only anterior choroidal invasion (Chévez-Barrios et al. 2000). McFall et al. (1977) likewise reported that WERIRb1 cells were weakly tumorigenic at either inoculum, whereas Y-79 cells were strongly tumorigenic at an inoculum as low as 106. Further along this line, Gallie et al. (1977) reported that RB cells isolated from primary tumors proliferate after intra-ocular implantation into the anterior eye chamber of nu/nu (T−) mice, but only Y-79 invade the orbit, the optic nerve and the brain and produce rapidly growing tumors in nu/nu (T−) mice after subcutaneous injection of high cell numbers, thus displaying a much more invasive phenotype. Thus, our data confirm the notion that even after years of propagation human Y-79 RB cells retain their high tumorigenic potential (del Cerro et al. 1993). The expression-based quantification method of human cells in the CAM model, implemented in our study, is a good alternative to the established Alu DNA repeat-based quantification system (Kim et al. 1998; Zijlstra et al. 2002; Mira et al. 2002; Palmer et al. 2011). There are, however, certain shortcomings of our quantitative CAM method. Most retinoblastomas have metastatic potential through invasion of the optic nerve, a characteristic that cannot be addressed using the CAM assay. As other patterns of RB invasion like hematogenous dissemination with formation of metastasis in lungs, bones and brain and lymphatic spread (McLean et al. 1994) have been described, the CAM, nevertheless, provides a useful in vivo-like model system to address the metastatic potential of retinoblastoma cells. Besides, it turned out that a certain amount of GFPpositive lower CAM punches (4 out of 6) are required to successfully amplify hGAPDH by real-time PCR, indicating the detection limit of our quantification method. Thus, future iterations will have to further optimize the correlation between the amount of human cells per lower CAM punch and the quantification by hGAPDH expression and lower the detection limit, in order to refine the determination of human cell content per chick CAM. The detection of gene expression instead of analysis of genomic DNA, which leads to frequent contaminations in the negative controls, is critical as GAPDH expression
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might change if human Rb cells are inoculated into a chick tissue environment. To minimize the risk of false positive or false negative GAPDH detection, we, however, employed a control step by evaluating the chick CAM punches for GFPpositive human cells under the fluorescence microscope and always found the level of hGAPDH expression in the lower CAM punches detected by our real-time PCR analyses to correlate with the number of GFP-positive CAMs. Additionally, in the study presented, we never observed any hGAPDH amplification in our negative controls.
Summary All retinoblastomas have mutations of both RB alleles, but they always display additional non-RB mutations (Squire et al. 1985; Chen et al. 2001, 2002). The RB1 mutation status of the RB cell lines investigated in this study indicates that the cell lines originate from RB tumor tissue. It is reasonable to assume that mutations in additional genes contribute to promotion and progression of this tumor (Gallie et al. 1999). Against the background that primary retinoblastoma material from patients is not readily available, RB cell lines provide an alternative to investigate the effects of (i) overexpression or knockdown of genes believed to be associated with retinoblastoma dissemination and progression and (ii) newly established, prospective pharmacological therapeutics for RB. If a new gene or reagent is believed to decrease RB tumor cell growth, a fast-growing RB cell line like Y-79 would be the ideal test system to proof this hypothesis. Intending to analyze induction of apoptosis, one would depict a RB cell line with low endogenous cell death levels like RBL-15 or WERI-Rb1. The capability to grow anchorage independent and form colonies in soft agarose represents one feature of a tumor cell and loss of this ability upon overexpression or knockdown of a certain gene or application of a reagent would indicate an effect on tumorigenicity. Last but not least, the CAM assay provides a helpful model system to test for effects on tumor cell intra- and extravasation, dissemination, migration and new tumor formation (metastasis). In contrast to Matrigel™ invasion or three-dimensional collagen assays, reported to be useful tools in metastasis research, but not necessarily reflecting the physiological events facilitating the dissemination of tumor cells, this in vivo-like system has a long history as an efficient model to study complex physiological processes such as tumor metastasis (Gordon and Quigley 1986; Scher et al. 1976). Summarizing one can state that our data display no predictable correlation between the growth capacity or apoptosis rates of the RB cell lines investigated and their anchorage-independent growth or between the ability to form colonies in soft agarose and to intravasate into the chick CAM tissue or to develop tumors on the CAM (Table S1).
Histochem Cell Biol Table 4 Correlation of the number (#) of GFP-positive lower CAM tissue punches and real-time PCR-based quantification of human RNA in lower chicken CAMs 6 days after intravenous injection of different RB cell lines RB cell lines
GFP + tissue punches of CAM (#)
Human RNA in CAM (pg)
Y-79_GFP
1 of 6 4 of 6 2 of 6 None 6 of 6 6 of 6 6 of 6 4 of 6 6 of 6
nd
