Molecular Vision 2017; 23:680-694
Received 10 April 2017 | Accepted 01 October 2017 | Published 03 October 2017
The last two authors co-senior authors for this study
1Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo, Norway; 2Norwegian Center for Stem Cell Research, Oslo University Hospital, Norway; 3Department of Pathology, Oslo University Hospital, Norway; 4Department of Medical Biochemistry, Oslo University Hospital, Norway
Correspondence to: Agate Noer, Center for Eye Research, Department of Ophthalmology, Oslo University Hospital, Postboks 4956 Nydalen 0424 Oslo, Norway; Phone: +47 23 01 61 98; FAX: +47 22 11 80 00; email: email@example.com
Purpose: Uveal melanoma (UM) has a high propensity for metastatic spread, and approximately 40–50% of patients die of metastatic disease. Metastases can be found at the time of diagnosis but also several years after the primary tumor has been removed. The survival of disseminated cancer cells is known to be linked to anchorage independence, anoikis resistance, and an adaptive cellular metabolism. The cultivation of cancer cells as multicellular tumor spheroids (MCTS) by anchorage-independent growth enriches for a more aggressive phenotype. The present study examines the differential gene expression of adherent cell cultures, non-adherent MCTS cultures, and uncultured tumor biopsies from three patients with UM. We elucidate the biochemical differences between the culture conditions to find whether the culture of UM as non-adherent MCTS could be linked to an anchorage-independent and more aggressive phenotype, thus unravelling potential targets for treatment of UM dissemination.
Methods: The various culture conditions were evaluated with microarray analysis, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), RNAscope, immunohistochemistry (IHC), and transmission electron microscopy (TEM) followed by gene expression bioinformatics.
Results: The MCTS cultures displayed traits associated with anoikis resistance demonstrated by ANGPTL4 upregulation, and a shift toward a lipogenic profile by upregulation of ACOT1 (lipid metabolism), FADS1 (biosynthesis of unsaturated fatty acids), SC4MOL, DHCR7, LSS (cholesterol biosynthesis), OSBPL9 (intracellular lipid receptor), and PLIN2 (lipid storage). Additionally, the present study shows marked upregulation of synovial sarcoma X breakpoint proteins (SSXs), transcriptional repressors related to the Polycomb group (PcG) proteins that modulate epigenetic silencing of genes.
Conclusions: The MCTS cultures displayed traits associated with anoikis resistance, a metabolic shift toward a lipogenic profile, and upregulation of SSXs, related to the PcG proteins.
Uveal melanoma (UM) is the most common primary intraocular malignancy in adults with an incidence of approximately 5.1 per million per year in the United States , while in Europe, the incidence varies from less than 2 per million per year in Spain and southern Italy to more than 8 per million per year in Scandinavia [2,3]. Despite advances in the diagnosis and treatment of the disease, the prognosis has remained largely unchanged [1,4]. UM has a high propensity for metastatic spread. Relapse can be seen several years after treatment, and 40–50% of patients will eventually die of metastatic disease [4-7]. Dissemination of cancer cells from the primary tumor is believed to be an early event in UM. Circulating malignant cells (CMCs) have been detected in up to 88% of patients with UM and can be found at the time of diagnosis but also years after the primary tumor has been removed . Micrometastatic cells have also been found in the bone marrow of patients with UM in 29% of cases . Intriguingly, the presence of disseminated cells in bone marrow and the bloodstream does not correlate with overall survival [8,10]. Cancer cells disseminating from the primary tumor have to adapt to a changing micromilieu to generate metastatic disease. The various tissues of the metastatic route provide a different nutritional supply, pH, and oxygen concentration; thus, the malignant cells have to exhibit metabolic flexibility to sustain growth and survival [11-13].
Anchorage-independent growth and resistance to anoikis (cell death induced by loss of extracellular matrix attachment as in circulating metastatic cells) are essential features of disseminated cancer cells and metastatic progression [14-16]. The generation of multicellular tumor spheroids (MCTS) by anchorage-independent growth is associated with enrichment of an aggressive phenotype characterized by chemoresistance, invasiveness, and expression of undifferentiated markers [17-21]. The present study aims to compare the differential gene expression of MCTS of UM to primary tumor tissue and adherent cultures, with a special emphasis on unravelling the pathways and survival mechanisms pathognomonic for disseminated and circulating cancer cells.
All experiments were conducted in accordance with the Declaration of Helsinki (2013), and all tissue harvesting was approved by the Local Committees for Medical Research Ethics (REK Ref. 2009/1973 and REK Ref. 2013/803–1). The study is adhered to the ARVO statement on human subjects. Informed written consent was obtained from patients before tissue harvesting. All reagents used in the present study were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
UM biopsies from patients undergoing enucleation of the eye were included in this study. After enucleation, the ophthalmic pathologist excised fresh tumor tissue for use in research before formalin fixation for routine histopathological examination. The UM of the three donors (D1, D2, and D3) was classified as mixed (D1) and epithelioid (D2 and D3) types with a routine histopathological examination. Retrospectively, donors D1, D2, and D3 all had confirmed liver metastases. A fourth supplementary donor was added to the study after data were obtained. The UM of this donor, D(S), was classified as epithelioid, and the donor tissue underwent the same culture conditions as the tissue from donors D1, D2, and D3.
A fraction of the tissue was snap-frozen and stored at −80 °C. The remaining sample was minced with scissors in collagenase I and IV (1 mg/ml), before being incubated for 1 h at 37 °C. After dissociation, the tissue was cultured adherently for 7 days in RPMI 1640 (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/ml, P4333), and amphotericin B (2.5 µg/ml, A2942) in addition to gentamycin (75 µg/ml; Sanofi-Aventis, Gentilly, France) to ensure the removal of fibroblasts . After 7 days of adherent culturing, the cells were trypsinized using Trypsin-EDTA (0.25%, T4049) and pelleted into three fractions of 100,000 cells. The first fraction of the cells was collected for RNA analyses, the second fraction for further adherent growth, and the third for non-adherent growth as MCTS. The term MCTS is used for this non-adherent culture of tumor cells, in accordance with the nomenclature, and is considered aggregation and compaction of tumor cells . The cell fraction for MCTS culture was plated at a density of 500–1,000 cells per well on Corning Costar ultra-low attachment, polystyrene, round-bottom 96-well plates (CLS7007) in melanoma stem cell medium (MSCM) (1) and (2): (1) 30% human embryonic stem cell medium (hESC); (78% KnockOut DMEM/F12 (Cat. no. 12660–012, Thermo Fisher Scientific Inc., Waltham, MA), 20% KnockOut serum replacer (Cat. no. 10828–028, Thermo Fisher Scientific Inc.), 1% MEM non-essential amino acids (Cat. no. 11140–050, Thermo Fisher Scientific Inc.), 4 ng/ml basic fibroblast growth factor (b-FGF; Cat. no. 13,256-029, Thermo Fisher Scientific Inc.), 1% GlutaMAX (35,050-061, Thermo Fisher Scientific Inc.), and 1.4‰ 2-mercaptoethanol (M7522) and (2) 70% mouse embryonic fibroblast (MEF) conditioned medium (AR005, R&D Systems/Bio-Techne, Minneapolis, MN)  with penicillin/streptomycin (100 U/ml) and amphotericin B (2.5 µg/ml). The cells were collected after 12 days of cell culture and further embedded in paraffin for immunohistochemistry (IHC) or pelleted and stored at −80 °C for RNA analyses.
RNA from fresh frozen primary tumors (D1, D2, and D3) was isolated using the Qiagen RNeasy kit (Qiagen, Hilden, Germany). Briefly, the tissue was placed in a 4.5 ml cryotube, and 500 µl of QIAzol (Qiagen) was added before the sample was disrupted using Qiagen TissueRuptor (Qiagen), according to the manufacturer’s recommendations. The sample was centrifuged at 18 400 ×g for 10 min to remove insoluble material before being processed with the Qiagen RNeasy kit with DNase. Samples were purified using the Zymo PCR inhibitor removal kit (Zymo, Irvine, CA). RNA from the pelleted samples (adherent and cultured spheres from D1, D2, and D3) was isolated as described above, except the disruption step using the Qiagen TissueRuptor. RNA concentration and purity were determined using NanoDrop (Wilmington, DE) and Bioanalyzer (Agilent 2100, Agilent, Santa Clara, CA). All nine samples had RNA integrity number (RIN) values above 8 before being analyzed with microarray and PCR .
The growth media in the 96-well plates was diluted by gently adding Hanks’ Balanced Salt solution (Thermo Fisher Scientific Inc.). Then the MCTS were allowed to make sediment before the media was carefully removed. A mixture of human plasma and thrombin (Sigma–Aldrich) was used to clot the MCTS together before fixation in 4% paraformaldehyde (PFA) and embedment in paraffin. Then 3.5 μm sections were cut and stained . Ki-67 staining was performed using the Envision + Dual Link HRP (K4065, Dako, Glostrup, Denmark) and AEC + Substrate chromogen ready-to-use (k3461, Dako). Briefly, the K4065 kit protocol was followed until the addition of 3,3′-diaminobenzidine (DAB). After polymer horseradish peroxidase (HRP), 3-amino-9-ethylcarbazole (AEC) chromogen from the kit k3461 was added, and the sections were washed and counterstained with hematoxylin according to the k3461 protocol. Negative controls without primary antibody were included for all stainings. The following primary antibodies and dilutions were used (rabbit:rb, mouse:ms): Ki-67 (rb, 1:200; Thermo Fisher Scientific Inc.), SSX4 (rb, 1;50; Acris), anti-melanoma (a-melanoma) [HMB45 + MART1 (DT101 + BC199) + tyrosinase (T311)] (ms, 1:50; ab733, Abcam, Cambridge, UK), Perilipin (rb, 1:100; Santa Cruz Biotechnology Inc., Dallas, TX), and ANGPTL4 (rb, 1:500; Abcam). The secondary antibodies had the fluorescent marker Alexa Fluor 488 (1:500; Invitrogen). Hoechst (1:500; Invitrogen) was used for nuclear staining. The sections were analyzed using a Zeiss Axio Observer.Z1 fluorescence microscope (Zeiss, Oberkochen, Germany). Sections were also stained with hematoxylin and eosin (H&E) for morphological examination.
Microarray analysis was performed at the Genomics Core Facility, Oslo University Hospital and Helse Sør-Øst. HumanHT-12 v4 Expression BeadChip (Illumina, San Diego, CA) was used for the analysis. It targets more than 31,000 annotated genes with 47,000 probes mainly derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009). For each sample, 440 ng of total RNA was amplified and labeled using the Illumina TotalPrep-96 RNA Amplification Kit protocol. The quantity of labeled copy RNA (cRNA) was measured using the NanoDrop spectrophotometer (Wilmington, DE). The quality and size distribution of the labeled cRNA were assessed using the 2100 Bioanalyzer. This was done to be able to hybridize equal amounts of successfully labeled cRNA to the arrays. For each sample, 750 ng of biotin-labeled cRNA was hybridized to the Illumina HumanHT-12 v4 Expression BeadChip. J-Express and rank product (RP) analysis were used to further identify differently expressed genes with ≥2 fold up- or downregulation and q values ≤0.05 between the different groups. One thousand permutations (1,000*) were run for each RP analysis .
RNA concentration and purity were measured using NanoDrop. Reverse transcription (RT) was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Abingdon, UK) with 50 ng total RNA per 20 μl RT reaction. Copy DNA (cDNA) was diluted to a volume of 50 µl (1 ng/µl) after cDNA synthesis. Quantitative PCR (qPCR) was performed using the StepOnePlus RT–PCR system (Applied Biosystems) and Taqman Gene Expression assays following the manufacturer’s protocols (Applied Biosystems). The TaqMan Gene Expression Assays used include ANGPTL4 (Hs01101127_m1) and 18S (Hs03003631_g1). The thermal cycling conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All samples were run in duplicate (each reaction: 2.5 μl/2.5 ng cDNA in a total volume of 12.5 μl). The data were analyzed using the 2−ΔΔCt method to find the relative changes in gene expression as a fold change between the samples. The uncultured tumor sample was chosen as the calibrator and equaled one, while the other samples had fold changes related to the uncultured tumor calibrator sample. The 18S probe, primers, and assay (Hs03003631_g1) were used as a loading control to quantify the differences in cDNA input between the samples.
RNA in situ hybridization was performed using the RNAscope® 2.5 High Definition (HD)- Red assay (Advanced Cell Diagnostics, Hayward, CA) according to the manufacturer’s instructions using the standard pretreatment protocol. Sections were mounted using Prolong Gold with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI). RNAscope permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution . RNAscope Probe-Hs-SSX4–01 (Cat. no. 468,641, Advanced Cell Diagnostics) was used. Hybridization signals were detected with chromogenic reactions using Fast Red. Fast Red produces red fluorescence in addition to the red reaction product, thus providing a greater level of sensitivity . The RNA staining signal was identified as red punctate dots. Each sample was quality controlled for RNA integrity with a probe specific to peptidyl-prolyl cis-trans isomerase B (PPIB) mRNA. Negative control background staining was evaluated using a probe (Cat.no. 3100439, Advanced Cell Diagnostics, Newark, NJ) specific to the bacterial dihydrodipicolinate reductase (DapB) gene (Gene ID EF191515). The sections were analyzed with a Zeiss Axio Observer.Z1 fluorescence microscope.
Data from the microarray analysis were imported into Ingenuity Pathway Analysis (IPA) software in the search for biologic pathways and Gene Ontology to identify potential networks. Principal component analysis (PCA) and unsupervised hierarchical clustering were performed using the Partek Genomics Suite software (Partek, Inc., Chesterfield, MO).
Primary tissue from uncultured tumor D1 and the donor D(S) cultured as MCTS were fixed at 4 °C overnight in glutaraldehyde (0.1 M). The tissue was washed four times in cacodylate buffer (0.2 M) before post-fixation in a mixture of 1% osmium tetroxide and cacodylate buffer (0.2 M) for 60 min. The tissue was further rinsed in cacodylate buffer (0.2 M) before being dehydrated through a graded series of ethanol up to 100%. The tissue was then immersed in propylene oxide for 2 ×5 min and a mixture of Epon and propylene oxide before embedment in Epon. Ultrathin sections (60–70 nm thick) were cut on a Leica Ultracut Ultramicrotome UCT (Leica, Wetzlar, Germany), stained with uranyl acetate and lead citrate, and examined using a Tecnai12 transmission electron microscope (Phillips, Amsterdam, the Netherlands).
The cells cultured as MCTS grew as large aggregations involving the majority of the cells in the well (Figure 1A and insets). The MCTS were mitotically active, as seen with the positive Ki67 staining with a score of 1%, 2%, and 4% for donors D1, D2, and D3, respectively (Figure 1C). The melanoma profile of the MCTS was verified by staining for a-melanoma, a marker that recognizes HMB-45, MART-1, and tyrosinase. More than 90% of the cells in the MCTS-derived paraffin sections stained positive for this marker (Figure 1D).
The gene expression profiles of the UMs (D1, D2, and D3), uncultured, cultured as MCTS, or cultured as adherent primary cells, were comprehensively analyzed with microarray analysis. PCA was performed on raw data from the microarray with a false discovery rate (FDR) of 10%. This type of analysis clusters the samples and represents them on a three-dimensional space based on the differential relative gene expression. The PCA plot shows that the clustering was mainly determined by the culture conditions (Figure 2A).
The relative gene expression of UMs (uncultured, cultured as MCTS, or cultured as adherent primary tumor cells) was further investigated by performing an unsupervised hierarchical clustering with an FDR of 10% presented as a heat map (Figure 2B). The heat map shows significant downregulation of the surface markers in the cultured cells compared to the uncultured primary tumor biopsy. These markers reflect the cellular heterogeneity of the primary tumor and the loss of macrophages (CD68), endothelial cells (von Willebrand factor), and T-cells (CD3D, CD8A, and CD2) in the cell cultures (Table 1). Additionally, there is a marked downregulation of human leukocyte antigen (HLA) expression in MCTS (Table 1) and in the adherent primary tumor cells (Appendix 1). This finding is in accordance with the work of van Essen et al. who showed downregulation of HLA expression upon loss of tumor-infiltrating leukocytes .
The genes found to be upregulated in the unsupervised hierarchical clustering (Figure 2B) were in concordance with many of the genes found in the RP analysis (Table 1 and supplementary data). The RP analysis (q≤0.05) resulted in 206 genes ≥2 fold upregulated and 373 genes ≥2 fold downregulated in MCTS versus uncultured tumor biopsies. Two hundred eighteen genes were found to be ≥2 fold upregulated, and 552 genes were ≥2 fold downregulated in adherent cell cultures versus the uncultured tumor biopsies. Sixty-four genes were found to be ≥2 fold upregulated, and 71 genes were ≥2 fold downregulated in adherent cell cultures versus the MCTS.
The genes from the RP analysis were further analyzed with Ingenuity IPA software. The differences in molecular and cellular functions between the various culture conditions are shown in Figure 3.
There was a noticeable increase in the cellular strain in the MCTS compared to the uncultured tumor biopsies, indicated by increased free radical scavenging, enhanced drug metabolism, and the increase in lipid metabolism in the MCTS versus adherent cells and uncultured tumor biopsies. Associated pathways and molecules in lipid metabolism in the MCTS versus uncultured tumor biopsies are shown in Figure 3. Alterations in the lipid metabolism include seven networks: synthesis of lipids, steroid metabolism, metabolism of cholesterol, metabolism of lipid membrane derivatives, synthesis of cholesterol, and conversion of lipid and fatty acid metabolism.
Anoikis is a form of apoptosis induced by loss or inappropriate cell adhesion . The process of epithelial-to-mesenchymal-transition (EMT) is considered an important feature of anoikis . Rank product data revealed 3.5-fold upregulation of snail family transcriptional repressor 2 (SNAI2; Gene ID: 6591, OMIM 602150) and 0.6-fold downregulation of cadherin 1 (CDH1; Gene ID: 999, OMIM 192090; E-cadherin) in MCTS (Table 1). Anoikis resistance is also supported by the upregulation of pyruvate dehydrogenase kinase 4 (PDK4; Gene ID: 5166, OMIM 602527), an enzyme that inactivates pyruvate dehydrogenase (PDH), which is required for the conversion of pyruvate to acetyl-CoA. PDK4 is upregulated in response to loss of adherence (LOA) and reduces reactive oxygen species (ROS) strain . Noticeably, there was strong upregulation of angiopoietin like 4 (ANGPTL4; Gene ID: 51129, OMIM 605910) in the MCTS (Table 1, Figure 4). ANGPTL4 has recently been shown to be associated with an angiogenic phenotype of UM, and thus being involved in metastatic spread . ANGPTL4 is thought to contribute to anoikis resistance by inducing conformational changes that enable resistance to inducers of apoptosis [34,35]. ANGPTL4 is further known to stimulate intracellular lipolysis, thus supplying substrate for fatty acid oxidation (FAO) . Upregulation of FAO in MCTS is indicated by upregulation of enoyl-CoA hydratase 1 (ECH1; Gene ID: 1891, OMIM 600696), peroxisomal D3,D2-enoyl-CoA isomerase (PECI; Gene ID: 10455, OMIM 608024), and acyl-CoA thioesterase 1(ACOT1, Gene ID: 641371, OMIM 614313; Table 1). FAO has been proven to be an advantageous metabolic trait for cancer cells and is linked to anoikis resistance .
Microarray results indicated a metabolic shift toward a lipogenic profile in the MCTS. A high content of lipid droplets (LDs) and stored-cholesterol ester is strongly associated with tumor aggressiveness [37,38]. As shown in Table 1, ACOT1 (lipid metabolism), fatty acid desaturase 1 (FADS1, Gene ID: 3992, OMIM 606148; biosynthesis of unsaturated fatty acids), sterol-C4-mehtyl oxidase-like (SC4MOL, Gene ID: 6307, OMIM 607545), 7-dehydrocholesterol reductase (DHCR7, Gene ID: 1717, OMIM 602858), lanosterol synthase (LSS, Gene ID: 4047, OMIM 600909; cholesterol biosynthesis), and oxysterol binding protein like 9 (OSBPL9, Gene ID: 114883, OMIM 606737; intracellular lipid receptor) all showed marked upregulation in the MCTS cultures compared to primary tumors. SC4MOL, LSS, and FAD1 were also found to be upregulated in the MCTS cultures compared to the adherent cultures. The microarray results also demonstrated increased lipid storage by upregulation of perilipin 2 (PLIN2, Gene ID: 123, OMIM 103195). PLIN2 belongs to the perilipin family, members of which coat intracellular lipid storage droplets . The presence of PLIN2 was verified with IHC of donors D1, D2, and D3 (Figure 4). Morphological examination with transmission electron microscopy (TEM) revealed numerous lipid droplets in the supplementary donor D(S) cultured as MCTS (Figure 5). The TEM images also showed numerous mitochondria.
The synovial sarcoma X breakpoint (SSX, Gene ID: 6759, OMIM 300326) gene family consists of nine highly homologous members (SSX1–9) . SSX expression is confined to the testis, placenta, at low levels in the thyroid, and in a wide range of tumors (including synovial sarcoma), thus making them interesting targets for cancer therapy . SSXs have been linked to EMT and anoikis resistance .
The microarray results showed an increase in the expression of SSX4 in MCTS versus primary tumors and adherent cultures (Table 1). This presence of SSX4 mRNA was verified with RNAscope, while the SSX4 protein was verified with IHC staining. The proportion of cells expressing SSX4 in primary tumors and MCTS was found (Figure 6). Noticeably, the SSX protein was minimally expressed in the tumor biopsies.
By comparing the UM MCTS to biopsies and adherent cell cultures, the present study revealed a metabolic shift in the MCTS. The latter display traits associated with anoikis resistance, including a shift toward a lipogenic profile, as well as marked upregulation of SSXs, transcriptional repressors capable of humoral and cellular immune responses in cancer patients and putative targets for immunotherapy in cancers.
To disseminate, cancer cells have to undergo loss of adherence. Loss of adherence inhibits uptake of glucose and glycolysis which results in diminished levels of ATP and NADPH leading to metabolic stress and generation of ROS that induces anoikis . The induction of FAO restores ATP production and increases NADPH, thus preventing anoikis [15,43]. This metabolic shift is also indicated in the MCTS in the present study. Malignant cells have been shown to provide and utilize fatty acids . The lipogenic profile of the MCTS-derived cells reveals an increase in the synthesis of cholesterol, a trait associated with cancer aggressiveness [38,44,45]. Depletion of cholesterol has been shown to result in anoikis-like cell death . Whether the lipogenic switch seen in the MCTS in the present study is valid for in vivo disseminated UM cells remains to be revealed. Lipogenic targeting could be advantageous for solid tumors. The present study showed abundant LDs in the MCTS and in the primary tumor. The presence of LDs in UM has been described in the literature previously, as a response to radiation and in the untreated tumor tissue [47,48]. UM is characterized by its poor response to chemotherapeutics, and FAO has been shown to fuel chemoresistant cancer cells . Several FAO inhibitors have shown promising results in mice models, although chemosensitization by FAO and cholesterol synthesis inhibitors might be even more favorable [50-52]. The present results imply that ANGPTL4 might be a key player in orchestrating lipid metabolism in MCTS. ANGPTL4 has previously been shown to play a role in anoikis resistance and in angiogenesis and oncogenesis of several cancers, including UM [34,53-55]. The link between ANGPTL4 and EMT has recently been highlighted as a decrease in EMT markers and aggressiveness after silencing of ANGPTL4 in non-small cell lung cancer . Although most publications indicate an oncogenic function of ANGPTL4, the opposite has been shown in gastric cancer, where it is a proposed tumor suppressor . These conflicting findings suggest that further characterization of ANGPTL4 in UM is needed. The present results suggest that ANGPTL4 could be an attractive target in UM and possibly a way to target disseminated cancer cells.
Another compelling finding in the present study is the marked upregulation of SSX4. SSXs show tissue-restricted expression and are therefore regarded as attractive targets for cancer therapy [40,58]. The proteins are implied to be involved in proliferation and survival in cancer cells and formed a transient complex with beta-catenin thus altering the expression of genes involved in EMT . SSXs are localized to the nucleus and contains two different repressor domains: a Krüppel associated box (KRAB) domain and a potent repressor domain (RD) [60,61]. SSXs have a close connection with the Polycomb repressive group of proteins [62,63]. SSX2 (a homologous SSX group member) has been shown to antagonize BMI1 and EZH2 through an indirect mechanism, thus activating repressed genes. Additionally, SSX2 has been shown to have DNA-binding properties and negatively regulate the distribution of histone mark H3K27me3, implying that SSX2 plays a role in the regulation of chromatin structure and function . The exact function of SSXs in UM is not known, although the link between EMT and SSXs highlights a potential role in anoikis resistance. Disseminated cancer cells are likely to have an altered metabolic state as a survival strategy, and SSXs with their gene-regulating properties might be essential for these alterations. The synovial sarcoma fusion protein SS18-SSX2 has been associated with induction of cholesterol synthesis . Whether there is a direct link between lipid metabolism and SSXs in UM is yet to be unveiled. SSX4 has been shown to be expressed in 21% of skin melanomas; however, SSX4 expression in UM has not yet been assessed . If SSXs are highly expressed in disseminated cancer cells, it would make them valuable targets for immunotherapy. The restricted tissue expression of SSXs might lead to less severe side effects than targeting molecules and pathways involved in normal cellular homeostasis.
Cell culturing of UM is often hampered by tumor size and growth properties. A limitation of the present study is the low number and histological homogeneity of the donors included. In our experience, spindle cell tumors are more challenging to cultivate, thus making it difficult to run extensive genomic analyses on this cell type. Tumor size is an important aspect in UM research as the relative size of the tumors is small compared to other cancers, such as colon and breast. The diagnostic assessment should always be prioritized, meaning that miniscule amounts of tissue are available for research if the primary tumor is small. Unfortunately, small tissue samples (as often seen in spindle cell UM) also show greater clonal homogeneity upon expansion provided that the same number of cells is needed for downstream analyses. By using samples from larger tumors and early cell culture passages, we hope to better reflect the innate properties of the primary tumor. Epitheloid and mixed tumors are more prone to metastasis. The donors D1, D2, and D3 all had confirmed liver metastases. The selection of tumors analyzed in this study therefore is highly representative of aggressive UMs. Whether these results are valid for all UMs or solely the aggressive UMs is yet to be revealed, although there are indications that tumors with a low metastatic risk profile are more difficult to cultivate using the present protocol. The optimization of culture conditions would enable us to conduct further experiments for extensive verification of results and unravelling of epigenetic pathways.
In conclusion, we found that UM MCTS cultures undergo a metabolic shift. The MCTS display traits associated with anoikis resistance, including a shift toward a lipogenic profile. Targeting of lipid metabolism as a method to kill disseminated cancer cells could be a compelling new therapy in UM and needs further investigation. Additionally, the present study showed marked upregulation of SSXs, transcriptional repressors related to the PcG proteins that modulate epigenetic silencing of genes. SSXs have been implied in the process of EMT, and their expression could be increased in cells that have conferred anoikis resistance, thus serving as a potential target for disseminated cancer cells. UM MCTS could be a suitable model to reveal novel candidate targets for treatment of UM dissemination.
Appendix 1. J-Express and rank product (RP) analysis of differentially expressed genes (≥2 fold up/down, q≤ 0.05) in multicellular tumor spheroids (MCTS) versus uncultured tumor biopsies vs adherent primary cultures.
We would like to acknowledge all personnel at the Center for Eye Research and at the Dep. of Ophthalmology OUS that contributed in this project. We thank the personnel at the Genomics Core Facility, OUS and Helse Sør-Øst for performing microarray analysis and helping out with J-express analysis. We would also like to thank Sverre-Henning Brorson at the Dep. of Pathology, OUS for helping with TEM imaging. The work was funded by the South-Eastern Norway Regional Health Authority (Helse Sør-Øst) project 2012104, Norwegian Cancer Society project 5808589 and supported by grants from Arthur and Odd Clausons ophthalmological fund, Aase and Knut Tønjums ophthalmological fund, Futura fund, Unifor Frimed, Norwegian Association of the Blind and Partially Sighted, Inger Holms memorial fund, Stiftelsen for fremme av kreftforskning at University of Oslo and Legat til fremme av kreftforskning. All authors contributing to the study have read and approved the manuscript. There are no conflicts of interest for any of the authors.