Pro-invasive role of activated hepatic stellate cells in experimental uveal
melanoma metastasis
Research article
Auteurs : Ioana Fugaru1-3, Julie Bérubé1-3, Stéphanie Proulx1,2,4, Solange Landreville1-3
1 CUO-Recherche et Axe médecine régénératrice, Centre de recherche du CHU de Québec-Université Laval,
Hôpital du Saint-Sacrement, CHU de Québec; 2 Centre de recherche en organogénèse expérimentale de
l’Université Laval/LOEX; 3 Centre de recherche sur le cancer de l’Université Laval; 4 Département
d’ophtalmologie et d’ORL-CCF, Faculté de médecine, Université Laval Les auteurs n’ont aucun conflit d’intérêt à déclarer.
2.1 Résumé:
Le mélanome uvéal est la tumeur primaire intraoculaire la plus fréquente chez l’adulte. C’est une tumeur sporadique qui découle de la transformation maligne des mélanocytes de l’uvée. Plusieurs approches thérapeutiques permettent un contrôle satisfaisant de la tumeur au niveau local. Cependant, la survie à long terme des patients atteints du mélanome uvéal est altérée par le haut risque de développer des métastases: 34% des patients développent des métastases hépatiques dans les 10 ans suivant le diagnostic initial. Au stade métastatique, le taux de mortalité atteint environ 80% après 12 mois. Les mécanismes à l’origine du tropisme particulier du mélanome uvéal pour le foie n’ont pas encore été élucidés. Les cellules stellaires hépatiques activées sont impliquées dans la fibrose hépatique lors de la progression métastatique des cancers colorectal et pancréatique via la sécrétion de cytokines et de protéines de la matrice extracellulaire, favorisant ainsi l’invasion maligne. Les interactions entre les cellules stellaires hépatiques et les cellules cancéreuses de mélanome uvéal seraient déterminantes dans le développement des métastases hépatiques du mélanome uvéal de ce type de cancer. Dans ce projet, nous avons étudié les interactions bilatérales entre les cellules stellaires hépatiques et les cellules cancéreuses de mélanome uvéal, ainsi que leur remodelage de la matrice extracellulaire hépatique.
Nous avons utilisé des cellules cancéreuses de mélanome uvéal primaires dérivées de patients ayant développé un mélanome uvéal métastatique ainsi que des cellules stellaires hépatiques primaires. Nous avons effectué des co-cultures en inserts avec des cellules stellaires hépatiques et des cellules cancéreuses de mélanome uvéal, puis analysé les profils d’expression génique ainsi que l’expression directe de cytokines par analyses en protéomique. Nous avons également étudié la capacité de migration des cellules stellaires hépatiques et des cellules cancéreuses de mélanome uvéal en co-culture par rapport à leur monoculture. Nous avons créé le premier modèle tridimensionnel de stroma de cellules stellaires hépatiques comme substitut hépatique dans lequel nous avons ensemencé des cellules cancéreuses de mélanome uvéal pour étudier le remodelage de la matrice extracellulaire.
Nos travaux ont permis d’identifier plusieurs variations dans l’expression de cytokines et d’autres protéines associées à l’angiogenèse, à la défense cellulaire et à la matrice extracellulaire. La capacité de migration des cellules stellaires hépatiques en co-culture avec des cellules cancéreuses de mélanome uvéal était plus importante que celle des cellules stellaires hépatiques cultivées seules. Notre modèle tridimensionnel de stroma hépatique produit avec des cellules stellaires hépatiques a démontré une désorganisation matricielle importante lors de l’ensemencement de cellules cancéreuses de mélanome uvéal.
Les interactions entre les cellules stellaires hépatiques et les cellules cancéreuses de mélanome uvéal sont des médiateurs majeurs du développement des métastases hépatiques du mélanome uvéal.
2.2 Abstract:
Uveal melanoma is the most common primary intraocular tumor in adults. Uveal melanoma is a sporadic tumor that arises from the malignant transformation of melanocytes found in the uveal tract. Different treatments allow for a satisfactory local tumor control. However, long-term survival of uveal melanoma patients is threatened by the high risk of developing metastases: 34% of patients develop hepatic metastases within 10 years of the initial diagnosis. At the metastatic stage the death rate reaches 80% at 12 months. However, the mechanisms underlying the specific tropism of uveal melanoma for the liver have yet to be understood. Activated hepatic stellate cells are involved in hepatic fibrosis during the metastatic progression of colorectal and pancreatic cancers through the secretion of cytokines and extracellular matrix proteins that facilitate the malignant invasion. In this study, we will investigate the bilateral interactions between hepatic stellate cells and uveal melanoma cells and their remodeling of the extracellular matrix.
We used primary uveal melanoma cell lines derived from patients who developed metastatic uveal melanoma, as well as primary hepatic stellate cells. We co-cultured in inserts hepatic stellate cells and uveal melanoma cells, and then analyzed the gene expression profiles and direct expression of cytokines using cytokine arrays. We also studied the migration capacity of hepatic stellate cells and uveal melanoma cells in co-cultures compared to their monoculture. We engineered the first tridimensional stroma of hepatic stellate cells as hepatic substitute into which we embedded uveal melanoma cells in order to study the remodeling of the extracellular matrix.
Our work allowed to identify many variations in the expression of cytokines and other proteins associated to angiogenesis, host defense, and extracellular matrix. The migration capacity of hepatic stellate cells in co-culture with uveal melanoma cells was greater than that of hepatic stellate cells grown alone. Our tridimensional model of hepatic stroma produced with hepatic stellate cells demonstrated an important matrix disorganization after the seeding of uveal melanoma cells.
Interactions between hepatic stellate cells and uveal melanoma cells are major mediators of the development of hepatic metastases of uveal melanoma.
2.3 Introduction
Uveal melanoma (UM) is the most common primary intraocular tumor in adults, although a rare disease with 5 new cases per million each year in the USA (Krantz et al., 2017; Singh & Topham, 2003). UM arises from the malignant transformation of melanocytes found in the uveal tract (choroid, iris and ciliary body), with the choroid being the most frequent site (90%) (Kaliki & Shields, 2017). UM is a sporadic tumor and although its etiology remains to be clarified, risk factors such as light iris color and fair skin have been identified (Kaliki & Shields, 2017; Nayman et al., 2017). Different treatments, using radiation therapy or surgical eye removal, have allowed for satisfactory local tumor control (Kaliki & Shields, 2017; Wilson & Hungerford, 1999). Great advances in early diagnosis and in prognostic determination of UM have been made over the last few years, such as the identification of key chromosomal abnormalities and driver mutations, as well as the development of the DecisionDx®-UM gene expression profile test that provides risk stratification (Decatur et al., 2016; Harbour, 2012; Harbour et al., 2010; Landreville et al., 2008; Onken et al., 2008; Prescher et al., 1996; Sisley et al., 1997; Van Raamsdonk et al., 2010). However, long-term survival of UM patients is threatened by the high risk of developing metastases. In fact, 34% of patients develop metastases within 10 years of the initial diagnosis, and at this stage the death rate reached 80% at 12 months (Collaborative Ocular Melanoma Study, 2001; Diener-West et al., 2005). About 90% of advanced UM patients present hepatic metastases (Collaborative Ocular Melanoma Study, 2001, Diener-West et al., 2005). UM survival rates have yet to improve, as no intervention is available to prevent or restrain on a long period the growth of hepatic metastases (Augsburger et al., 2009; Singh & Topham, 2003; Singh et al., 2011).
A new understanding of tumorigenesis has been gained over the past decades with the growing characterization of the interactions between malignant cells and their surrounding microenvironment (Dvorak, 1986; Fidler, 2002, 2003; Hanahan & Coussens, 2012; Hart & Fidler, 1980; Joyce & Pollard, 2009; Langley & Fidler, 2007; Shuman Moss et al., 2012; Spano & Zollo, 2012). Tumor microenvironment remodeling has been associated with cancer progression (Hanahan & Weinberg, 2000; Johnsen et al., 1998; Werb et al., 1996; Wernert, 1997; Wong & Rustgi, 2013). In UM, various observations suggest the presence of circulating tumor cells or micrometastases at the time of the initial diagnosis and post-enucleation. For instance, tumor doubling time estimations have pointed towards the existence of subclinical, dormant, single or micrometastatic nests in the liver at the time of diagnosis (Singh, 2001). Moreover, post-mortem analysis of liver biopsies from UM patients has objectified the presence of micrometastases (Borthwick et al., 2011; Grossniklaus, 2013). This phenomenon, known as tumor dormancy, seems to involve a quiescent state of the malignant cells (Blanco et al., 2012; Borthwick et al., 2011). Several explanations have been suggested for dormancy such as cell cycle arrest, immune surveillance and down-regulation of pro-angiogenic cytokines by the tissue microenvironment
(Farrar et al., 1999; Naumov et al., 2002; Suzuki et al., 2006; Takahashi et al., 1996; Townson & Chambers, 2006). However, key triggers and role of the surrounding stroma in awakening UM dormant cells have yet to be characterized.
Hepatocytes (parenchymal cells), hepatic stellate cells (HSCs; drivers of liver fibrosis and regeneration), Kupffer cells (innate defense cells) and sinusoidal endothelial cells make up the main cellular populations in the liver (Friedman, 2008b). The hepatic microenvironment is composed of secreted cytokines, growth factors, extracellular matrix (ECM) components (proteoglycans, collagens, laminins, fibronectin, tenascin, elastin, fibrin, thrombospondins), as well as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs; tissue inhibitors of metalloproteases) (Friedman, 1993, 2008b). Quiescent HSCs (qHSCs) are vitamin A- and fat-storing cells, while their activation by stimuli such as alcohol or toxins, results in contractile, matrix- producing myofibroblasts (positive for alpha-smooth muscle actin, α-SMA) that are responsible for the excessive fibril-forming collagen deposition (types I, III and IV), and secretion of inflammatory cytokines (Friedman, 1993, 2008a; Friedman et al., 1985; Greenwel et al., 1991; Yin et al., 2013). Indeed, ECM stiffness has been associated with increased metastatic rates through enhanced growth, survival and migration of tumor cells (Barkan et al., 2010; Butcher et al., 2009; Lo et al., 2000). In hepatocellular carcinoma (HCC), the activation of HSCs drives fibrosis, which increases the ECM stiffness that provides a reservoir for bound growth factors, promotes angiogenesis and enhances the survival of preneoplastic hepatocytes and HSCs. These fibrotic changes can modulate the activation of other HSCs and the activity of inflammatory liver cells, and thus decrease the immune surveillance (Gortzen et al., 2015; Zhang & Friedman, 2012). In addition, an excess of pro-angiogenic and pro-inflammatory cytokines produced by HSCs have been shown to play an important role in tumor initiation and progression (Zhu et al., 2011).
In this study, we investigated the crosstalk between HSCs and UM cells (UMCs). First, we characterized the gene and cytokine expression profiles in HSC-UMC co-cultures. Next, we studied the migration potential of HSCs when exposed to UMCs cellular fronts. Finally, we developed a 3D model of hepatic stroma to investigate the remodeling of the ECM by UMCs. In summary, our study provides additional insights into UMCs and host cell interactions, and how this crosstalk promotes the development of metastases.
2.4 Materials and methods 2.4.1 Tissue culture
This study was approved by our institutional human experimentation committee (Centre de recherche du CHU de Québec-Université Laval, Quebec City, QC) and was conducted in accordance with the tenets of the Declaration of Helsinki. Primary HSCs were acquired from ScienceCell, and were identified as HSC1 (3- year-old male) and HSC2 (15-year-old female). Their activated state was confirmed by microscopic inspection, and positivity for α-SMA and synaptophysin (Dako, Mississauga, ON) using 5 µm-thick cell-block sections (Figure 1). UM cell lines T128 and T131 were isolated from primary tumors of enucleated patients who died of metastases (Clinique des tumeurs oculaires du CHU de Québec, Quebec City, QC) (Landreville et al., 2011; Mouriaux et al., 2016). The UM cell line H79 was derived from a hepatic metastasis of a patient diagnosed with UM (Berube et al., 2005). Clinicopathological characteristics and survival data are shown in Table 2.1. Written informed consent was obtained from the subjects. HSC and UMC cultures were grown at 37 °C in a 5% CO2
atmosphere in Dulbecco's Modified Eagle's Medium (DMEM; Wisent, St-Bruno, QC) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and 50 µg/mL gentamicin. All cell lines were tested routinely for mycoplasma infection by PCR (ATCC, Manassas, VA).
Cell
lines Sex/Age
Date of
biopsy Cell type Localization
Follow-up* (months)
Last status**
T128 M/45 10/2008 Epithelioid Choroid 5 DOM
T131 M/54 12/2008 Epithelioid Choroid 32 DOM
H79 F/76 08/1998 Epitheloid Liver 28 DOM
*Follow-up: period from surgery until patient death or last visit **Last status: DOM, dead of metastasis
Table 2.1. Summary of clinicopathological features and survival data of UM patients. 2.4.2 Co-culture experiments
H79 cells and HSCs were grown alone or co-cultured together for 48h in serum-free DMEM using compatible 6-well plates (Falcon, Corning, NY) and 1 µm pore-size culture inserts (Falcon), Corning, NY), which allow for the diffusion of medium components but prevent cellular migration. The layout of the co-culture conditions can be found in the Supplementary Figure 2.1. The controls consisted of seeding one cell type both on the insert and in the bottom well. The seeding density for H79 was 1.12 x 105 cells on the insert (indicated
as H79i) or 2.25 x 105 cells in the bottom well. The seeding density for HSCs was 1.5 x 105 cells on the insert
(indicated as HSCi) or 3 x 105 cells in the bottom well, in order to account for their slower growth.
Supplementary Figure 2.1. Layout of HSC/H79 co-culture conditions. Hepatic stellate cells seeded onto the
insert (HSCi; upper chamber) secrete their factors into the lower chamber where the UM cell line H79 is growing (A). This co-culture was compared to the H79 monoculture. H79 cells seeded onto the insert (H79i; upper chamber) secrete their factors into the lower chamber where the HSCs are growing (B). This co-culture was compared to the HSC monoculture.
2.4.3 Gene profiling
Total RNA was extracted from H79 or HSC cells in the lower well of the co-culture systems using the RNeasy Mini Kit (Qiagen, Toronto, ON). The quantity and quality of total RNA were assessed using the 2100 Bioanalyzer and RNA 6000 Nano LabChip Kit (Agilent Technologies, Mississauga, ON). Gene expression profiling was performed by the Plateforme de génétique moléculaire du CUO-Recherche (Quebec City, QC) using a SurePrint G3 Human GE 8×60K array slide (60,000 probes; Agilent Technologies). Cyanine 3-CTP labeled cRNA targets were prepared from 25 ng of total RNA using the Agilent One-Color Microarray-Based Gene Expression Analysis kit (Agilent Technologies). Then, 600 ng cRNA were incubated on the array slide. The slide was scanned on an Agilent SureScan Scanner according to the manufacturer’s instructions (Agilent Technologies). A single technicate replicate was performed. Data were analyzed using the ArrayStar version 12 software (DNASTAR, Madison, WI) and were preprocessed using robust multiarray analysis and quantile normalization to obtain an expression measure for each probe set to generate scatter plots and heatmaps of mRNAs of interest. The color scale used in heatmaps to display the log2 expression level values was
determined by the Hierarchical clustering algorithm of the Euclidian metric distance between genes. The housekeeping mRNAs golgin A1 (GOLGA1) and beta-2-microglobulin (B2M) were used as internal controls to
normalize the linear signals of the selected mRNAs (Lee et al., 2007).
2.4.4 Cytokine arrays
The conditioned medium from the bottom well of each co-culture/monoculture was collected after 48 hours, and the expression of 105 cytokines/chemokines (spotted in duplicate) was determined by chemiluminescence using a commercially available Cytokine Array Kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The pixel density in each spot of the array was collected from developed X-ray films and analyzed using a transmission-mode scanner and the image analysis software Quick Spots (Western Vision Software, Salt Lake City, UT). The average pixel density of the pair of duplicate spots representing the negative controls (average background signal) was subtracted from the average pixel density of the pair of duplicate spots representing each cytokine. The relative expression in percentage was determined for each cytokine with respect to its control.
2.4.5 Cellular migration assay
The cell migration in co-cultures/monocultures was evaluated using Culture-Insert 2 Well with a defined 500 µm cell-free gap (IBIDI, Fitchburg, WI). Pictures of the gap were taken (10x objective) at various time points: 0 (T0), 12 (T12) and 26 (T26) hours using a phase contrast microscope. The influence of co- culture combinations on the migration speed of HSCs and UMCs was determined by measuring the cell- covered area in the pictures using the ImageJ software (Straatman, 2008). The percentage area represented by the gap out of the total surface was determined for each time point.
2.4.6 3D model of hepatic stroma
3D hepatic stromas were reconstructed using HSCs following an adaptation of the self-assembly approach (Supplementary Figure 2.2) described previously (Auger et al., 2002; Carrier et al., 2008; L'Heureux et al., 1998). This cell sheet technology based on mesenchymal cells stimulation with ascorbic acid leads to the superimposition of ECM-rich cell sheets to create 3D constructs maturing over time in culture. HSCs were seeded at a density of 3 x 105 cells in T25 flask and cultured for 18 to 25 days in DMEM supplemented with
10% FBS (Wisent, St-Bruno, QC), 50 µg/mL gentamicin, and 50 µg/mL ascorbic acid (Sigma-Aldrich, Oakville, ON) to promote the secretion and accumulation of ECM proteins (Carrier et al., 2008; Geesin et al., 1988; Hata & Senoo, 1989). The HSC cell sheets were mounted on paper anchors, peeled from the flasks and superimposed together to form a stroma. This stroma was kept in culture for an additional week, after which, 3 x 105 UMCs were seeded on top of the stroma and cultured in submerged conditions. Tissue-engineered
stromas with or without UMCs were fixed and embedded in paraffin. Hematoxylin and eosin (H&E) and Masson’s trichrome stainings were performed.
Supplementary Figure 2.2. Production of 3D tissue-engineered bilayer stromas with HSCs using the self-
assembly approach. HSCs were plated and grown in the presence of ascorbic acid (50 µg/mL). When a cellular sheet has formed, two layers were mounted into a stroma, which was then left in culture for an additional 7 days. Afterwise, UMCs were added onto the stroma. The stroma seeded with UMCs modeled metastatic uveal melanoma.
2.4.7 Indirect immunofluorescence
After a week of culture in the presence of UMCs, the 3D stromas were embedded in Optimal Cutting Temperature compound (OCT; Somagen, Edmonton, AB), frozen in liquid nitrogen and then stored at −80 °C until use. Cryosections of 5 µm were cut and fixed with acetone, then incubated at room temperature for 45 minutes with antibodies diluted in PBS (137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 0.9 mM CaCl2,
0.48 mM MgCl2 containing 1% BSA) as described previously (Proulx et al., 2010). Primary antibodies used
were: monoclonal mouse anti-collagen I (COL1, 1:600; Sigma-Aldrich), anti-tenascin C (TNC, 1:500, Thermo- Fisher Scientific, Rockford, IL) and anti-α-SMA (1:100; Dako, Santa Clara, CA). A goat anti-mouse IgG antibody conjugated with Alexa 488 (Invitrogen, 1:500) was used as secondary antibody. Cell nuclei were
counterstained with Hoechst (1:100; Sigma-Aldrich). The fluorescent signal was observed using a LSM-800 confocal microscope (Zeiss, Toronto, ON).
2.4.8 Statistical analysis
Experimental data are presented as column bar graphs (mean ± standard error of the mean [SEM]) or heatmaps using Prism 7 software (GraphPad Software, La Jolla, CA). The Student’s t-test or ANOVA were performed to determine the statistical significance, and differences were considered statistically significant at a
P value of less than 0.05. The sample size included data from two independent experiments performed in
triplicate with biological replicates. Cytokine arrays were performed with duplicate biological replicates.
2.5 Results
To study the interactions between UMCs and HSCs in our 2D and 3D models, we performed our experiments with human primary cultures of HSCs instead of the widely used immortalized LX-2 cell line. These HSC cultures underwent a change in morphology into a myofibroblast phenotype according to phase contrast microscopy (Figure 2.1A, D). They also expressed moderately to strongly α-SMA (Figure 2.1B, E), the single most reliable marker of HSC activation (Friedman, 2008a; Ramadori et al., 1990; Yoshino et al., 2006). Both HSC cultures showed a weak to moderate staining for synaptophysin (Figure 2.1C, F), a marker of quiescent and activated HSCs (Cassiman et al., 1999). HSC1 and HSC2 cell lines thus represented reliable models of activated HSCs to assess their synergistic interactions with UMCs.
Figure 2.1. Characteristics of HSC cell lines. Phase contrast micrographs of monolayer cultures of HSC1 and
HSC2 (A, D). Both HSC cell lines were positive (dark brown) for α-SMA (B, E) and synaptophysin (C, F) by immunohistochemistry on cell blocks. Scale bars: 50 µm.
We first analyzed the gene expression profile of HSC1 and HSC2 in monoculture or co-cultured with the UMC line H79 (Figure 2.2). H79 cells were seeded into the insert (H79i, “i” stands for “insert position”), and their secretome was released for 48h towards the HSC1 or HSC2 cells (Supplementary Figure 2.1B), before RNA was isolated from the bottom well to study the impact of the H79 secretome on the HSC transcriptome.
Figure 2.2. Gene expression profiling of HSCs co-cultured with UMCs. Scatter plot of pooled log2 signal
intensity from targets covering the entire human transcriptome of H79 cells co-cultured with HSCs (y-axis) plotted against the monoculture of HSCs (x-axis) (A). The twofold change in intensity lines is shown in green. The displacement of the linear regression curve (dashed purple) away from the central axis reflects the degree of divergence in the pattern of gene expression between the two conditions (R2 value, coefficient of
determination). Heatmap representation of the twofold or more variations for the H79/HSC co-culture