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Abstract
Gastric cancer (GC) is highly deadly. Three-dimensional (3D) cancer cell cultures, known as spheroids, better mimic tumor microenvironment (TME) than standard 2D cultures. Cancer-associated fibroblasts (CAF), a major cellular component of TME, promote or restrain cancer cell proliferation, invasion and resistance to drugs. We established spheroids from two human GC cell lines mixed with human primary CAF. Spheroid organization, analyzed by two-photon microscopy, showed CAF in AGS/CAF spheroids clustered in the center, but dispersed throughout in HGT-1/CAF spheroids. Such differences may reflect clonal specificities of GC cell lines and point to the fact that GC should be considered as a highly personalized disease.
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1. Introduction
Gastric cancer (GC) represents the fifth most common malignancy in the world and the third leading cause of cancer-related deaths in both sexes. It is often diagnosed at a late stage, and hence it is highly deadly [1]. Consequently, it is mandatory to develop novel therapeutic strategies aimed at identifying early disease and drug-response markers. One approach to this need is to use three-dimensional (3D) culture models, such as spheroids [2,3]. Indeed, tumor complexity and direct pathophysiology relevance are missed in two-dimensional (2D) cell cultures [4]. By contrast, 3D cell culture models make cell-cell interactions possible in all dimensions. Under such conditions, cells are submitted to more realistic oxygen and nutrient gradients. Spheroids were established as a relevant in vitro model for drug testing in oncology, in view of their ability to reproduce the main features of in vivo solid tumors, i.e. cellular heterogeneity, cell-cell signaling, growth kinetics, gene expression and drug resistance [5]. However, single-cell type tumor spheroids mostly lack extra cellular matrix (ECM) deposition and tumor-stromal cell interactions. Cancer-associated fibroblasts (CAF) are one of the major stromal cell populations in solid tumors. Their role in tumor growth and drug resistance is essential [6–8]. Thus, mixed epithelial/CAF spheroids are a realistic cell combination.
The development of 3D cell culture models has coincided with the progress of 3D optical imaging methods able to characterize the organization of cells in 3D with sub-micrometric spatial resolution [9]. Among these methods, confocal fluorescence, light-sheet and two-photon microscopies, are powerful techniques enabling, in association with optimized sample preparation processes, high throughput visualization of spheroids [10]. Confocal fluorescence microscopy [11–14] is the most widespread modality employed to perform depth-resolved structural analysis of spheroids. However, this technique has some limitations for imaging deep layers, typically located at more than a few tens of µm beyond the surface of large spheroids because of strong light scattering. In addition, photodegradation of the samples, due to short excitation wavelengths, can make 3D imaging challenging. Optical clearing [12,13] is often used before imaging to reduce light scattering and recover relevant visualization of the interior of the spheroids (up to 150-200 µm beyond the surface) but this requires additional sample preparation. Light-sheet microscopy [15] is a good alternative to confocal microscopy as it enables visualization in 3D of large spheroids at high speed with low photodamage. However, observation with a light-sheet microscope requires a more delicate sample preparation process because spheroids need to be mounted in a glass capillary filled with a gel solution for light excitation and detection at orthogonal directions. Two-photon microscopy enables reaching larger imaging depth in uncleared samples in comparison with confocal microscopy and causes less photodegradation, making it a good compromise for the structural analysis of large spheroids. To date, only few studies using two-photon microscopy to analyze 3D cell culture models in cancer research have been reported [16–19], and no structural analysis of GC multicellular spheroids made of tumor cells and CAF has been performed. This may be an important limitation of the model, as it is known that mechanical forces, which are likely contributed by cell-cell interactions, are also important to drive cancer progression towards metastasis. We report here on the structural analysis by two-photon microscopy of living GC multicellular spheroids made from tumor cells and CAF. We have minimized sample preparation steps and the spheroids were directly manipulated. Imaging was performed at different times after spheroids formation, enabling monitoring the organization between CAF and epithelial cancer cells from 2 different human GC cell lines, HGT-1 and AGS. Strikingly, our results revealed different spatial organizations of tumor cells and CAF according to the GC cell line.
2. Materials and methods
2.1 Cell culture, staining and spheroid formation
HGT-1 [20] and AGS [21] human GC cells were grown at 37°C under a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Corning, MA, USA), containing 4.5 g/L glucose and supplemented with 5 or 10% fetal bovine serum (FBS) respectively (Gibco-Invitrogen, Cergy-Pontoise, France) without antibiotics (complete medium).
CAF were obtained from a GC patient-ablated tumor after tissue dissociation with collagenase and growth in culture in complete medium with 10% FBS. After about 10 days in culture, no more epithelial cells adhered to the dish and fibroblasts emerged and kept growing. The population that emerged was apparently homogenous and displayed a characteristic fibroblast morphology. The patient had given informed consent and use of tumor-derived cells was granted by the Ethics Committee of Brest University Hospital.
HGT-1 and AGS sub-populations labeled with eGFP (green) or TdTomato (red) fluorescent tags, respectively, were generated using lentiviral infection. Briefly, self-inactivating HIV-1-based lentiviral vector, pRRL-sin-MND-GFP-IRES-Puro and pRRL-sin-MND-Tomato-IRES-Puro were purchased from VectUB (vectorology platform, University of Bordeaux, France). The vectors express eGFP or TdTomato under control of the MND promoter and co-express the puromycin resistance gene. Forty thousand cells were infected with lentiviruses (at a multiplicity of infection of 2) and selected with puromycin (1 µg/mL). We obtained eGFP fluorescent HGT-1 populations and TdTomato fluorescent AGS populations.
To generate spheroids, 2D cultured HGT-1, AGS and CAF were collected and seeded (in 200 µL complete medium) in ultra-low attachment (ULA) 96-well round bottom microplates (Corning, Amsterdam, Netherlands) allowing formation of uniformly sized spheroids in a scaffold-free model, as recently described [22]. A single spheroid was formed in each well. To obtain the same size range of 6 days-old spheroids, we generated AGS spheroids by seeding 500 cells/well but only 250 cells/well for HGT-1 cells. It is important to consider spheroid sizes when characterizing spheroids, as spheroids with diameters between 400-500 µm sustain oxygen and nutrient gradients associated with specific functional domains [23]. Incubation for 4 days was sufficient to form tightly packed spheroids (Fig.S1).
To obtain bicellular spheroids, cells of each type were added together in a 1:1 ratio (250 HGT-1 + 250 CAF or 500 AGS + 500 CAF) in 200 µL complete medium in ULA 96-well round bottom microplates.
2.2 Two-photon imaging setup
The imaging setup consisted of a two-photon microscope based on a confocal laser scanning unit (FV300, Olympus) mounted on an upright microscope body (BX51WI, Olympus) and a Ti:Sapphire laser oscillator (Chameleon Vision II, Coherent) that can be tuned between 700 nm and 1050 nm, and equipped with a dispersion pre-compensation unit. Light was focused on the samples by a 20X/0.75NA microscope objective (UPlanSApo, Olympus). The microscope objective has a 0.65 mm working distance and is corrected for 0.17 mm-thick glass coverslips. The lateral and axial resolutions (FWHM) were respectively ∼0.5 µm and ∼1.7 µm. Two-photon excitation fluorescence (TPEF) light was epi-collected and detected through BG39 bandpass filters and a 570 nm dichroic mirror (FV300, DM570, Olympus) by two internal photomultiplier tubes (R928, Hamamatsu). Channels 1 and 2 were associated to the detection channels below (“green”) and above (“red”) the cut-off wavelength of the dichroic mirror (570 nm), respectively. TPEF images were acquired with 12-bit intensity resolution and 1024 × 1024 pixel definition. The pixel dwell time was set to the maximum value of 8 µs, resulting in an acquisition time of 9.4 s for a 1024 × 1024 pixel image. The field of view for the images was 710 × 710 µm. Z-stack acquisitions were made on some samples for 3D rendering with 1 µm step and 512 × 512 pixel definition.
2.3 Imaging of spheroids
Two-photon excitation/emission fluorescence spectra of eGFP and TdTomato were taken from [24] and displayed in Fig.S2. EGFP exhibits maximum two-photon absorption at 920 nm and produces maximum fluorescence emission around 510 nm. TdTomato exhibits maximum two-photon absorption at 1050 nm and produces maximum fluorescence emission around 580 nm. Cancer-associated fibroblasts did not need to be stained because they revealed strong autofluorescence at 750 nm excitation wavelength. In addition, the autofluorescence emission spectra of CAF was broad enough to produce approximatively the same intensity on both detection channels. The following process was thus employed to image HGT-1/CAF and AGS/CAF co-culture spheroids. It is illustrated by Fig.S3.
For HGT-1/CAF bicellular spheroids, imaging of HGT-1 cells labelled with eGFP was first performed at 920 nm excitation wavelength and fluorescence light was detected on channel 1. At this excitation wavelength, autofluorescence of CAF is negligible. Imaging of CAF was then realized at 750 nm excitation wavelength and fluorescent light was detected on channel 2 because small residual excitation of eGFP at 750 nm does not produce any significant fluorescent light in channel 2, which is not the case for channel 1 (Fig.S3).
For AGS/CAF bicellular spheroids, imaging of AGS cells stained with TdTomato was first realized at 1000 nm excitation wavelength and fluorescence light was detected on channel 2. At this excitation wavelength, autofluorescence of CAF is negligible. Imaging of CAF was then realized at 830 nm excitation wavelength and fluorescence light was detected on channel 1 for which TdTomato does not produce any significant fluorescence light, whereas autofluorescence from CAF is still conveniently detectable (Fig.S3). The excitation wavelength for CAF imaging was not set to 750 nm as for HGT-1/CAF spheroids because for AGS/CAF co-culture, two-photon excitation of higher electronic transitions of TdTomato starts to produce detectable fluorescence signal on both channels (see Fig.S2).
Laser excitation power was controlled by a Glan-Taylor polarizer and a zero-order half wave plate (WPHSM05-830, Thorlabs) and was measured at the sample plane with a microscope slide thermal sensor (S175C, Thorlabs). It was adjusted to ∼100mW for all experiments and all excitation wavelengths, which gives the best signal-to-noise ratio in the images, while avoiding photodegradation of the samples. The spheroids were stable enough to ensure successive 3D imaging at two different excitation wavelengths.
Images were displayed while encoding the different cell types in selected false colors that produced optimal contrast. HGT-1 cells were encoded in green, AGS cells in red and CAF in yellow.
A custom process for spheroids deposition was implemented (Fig. 1). Approximately 50 µL of medium containing one spheroid was directly collected by aspiration with a micropipette equipped with a glass tip, and gently deposited onto a 0.17 mm-thick glass coverslip stuck on a metallic slide with a large central empty hole. A plastic cuvette was stuck under the metallic slide in order to avoid liquid evaporation. The slide was then quickly reversed and placed on the microscope translation stage. The spheroid was thus stably maintained in a liquid droplet close to the coverslip, enabling 3D imaging with minimal aberrations. The deposition process for one spheroid takes no longer than 2-3 min. Spheroids were kept under standard culture conditions until exploring time.
Fig. 1. Process for two-photon imaging of multicellular spheroids. (a) Schematic representation of the sample holder, composed of 1: 0.17 mm-thick glass coverslip, 2: metallic slide with an central empty hole, 3: liquid, 4: plastic cuvette. The spheroid is represented in gray. (b) 3D image acquisition with the two-photon microscope at two successive excitation wavelengths.
3. Results
3.1 HGT1/CAF spheroid growth monitoring
HGT-1 cancer cells labelled with eGFP were seeded alone or with CAF in ULA plates, and spheroids growth, structure and organization were analyzed over time. Spheroids were initiated at day 0 and were isolated successively at each time point (4, 6, 8 and 11 days thereafter) for imaging. They were manipulated and imaged readily without any additional fixation or staining steps. For each day, three different spheroids were imaged and exhibited very similar behaviors so one representative image was selected. Figures 2 and 3 showed single plane TPEF images taken ∼50 µm below the top of the spheroids. This depth was chosen because it was quite illustrative of the organization between GC cells and CAF inside the spheroids while providing good signal-to-noise ratio in the images. The growth of CAF was much slower, compared to that of HGT-1 cells, hence bicellular spheroids were mainly composed of GC cells after 4 days of culture. We explored the effects of different ratios of GC cells to CAF (1:1, 1:2 and 1:5) on the growth of spheroids. Spheroid organization was similar between all cell ratios (Fig.S4). Therefore, we fixed the ratio to 1:1. Bicellular HGT-1/CAF spheroids were more densely packed in comparison to monocellular HGT-1 spheroids at each time point, from 4 to 11 days after spheroid initiation (Fig. 2, Table S1). The count of eGFP-labelled HGT-1 cells, recovered from monocellular or bicellular spheroids, showed no significant difference between both types of spheroids at the same age (Fig.S5). Two-photon depth imaging of living 4 and 6 days-old HGT-1/CAF spheroids showed a characteristic spatial cell organization with CAF in a crown around the compacted HGT-1 cells and somewhat dispersed in between them (Fig. 2 and Fig. 3). CAF were less present in 8 days-old HGT-1/CAF spheroids, while virtually no more CAF could be seen in 11 days-old spheroids. A second growth monitoring was made (data not shown) and the results were very similar.
Fig. 2. Single plane TPEF images of monocellular HGT-1 spheroids (upper row) and bicellular HGT-1/CI AF spheroids (lower row) at different days (D) after initiation of spheroid formation. HGT-1 cells were labelled with eGFP and appear as green. CAF were detected from their autofluorescence and appear in yellow. Imaging planes were typically located ∼50 µm below the top of the spheroids. Scale bar : 100 µm.
Fig. 3. Single plane TPEF images of an bicellular HGT-1/CAF spheroid 6 days after initiation of spheroid formation. Left : HGT-1 cells only (eGFP display). Middle : CAF only (autofluorescence display). Right : both HGT-1 cells and CAF are displayed. Scale bar : 100 µm.
3.2 AGS/CAF spheroid growth monitoring
We next generated spheroids using the TdTomato-labelled AGS GC cell line. As for HGT-1 cells, AGS cells were seeded alone or with CAF in ULA plates, and spheroids growth, structure and organization were analyzed over time. For each day, three different spheroids were imaged and exhibited very similar behaviors so one representative image was selected. Figures 4 and 5 showed single plane TPEF images taken ∼50 µm below the top of the spheroids. We next explored the effects of different ratio of AGS cells to CAF (1:1, 1:2 and 1:5) on spheroid’s growth and organization. No structural changes were observed between those different ratios (Fig.S4). Hence, we fixed the ratio to 1:1 with 500 AGS + 500 CAF. Bicellular AGS/CAF spheroids appeared very compact compared to monocellular spheroids at each time point, going from 4 to 11 days after spheroid formation (Fig. 4, Table S1). Like for HGT-1/CAF spheroids, cells from AGS/CAF spheroids were firmly attached to each other and were hard to dissociate by mechanical force. We counted TdTomato-labelled AGS cells recovered from monocellular and bicellular spheroids, but we observed no significant difference between both types of spheroids at the same age (Fig.S5). Two-photon depth imaging of AGS/CAF spheroids showed a characteristic spatial cell organization with CAF in the central core surrounded by compacted AGS cells (Fig. 4, Fig. 5). A second growth monitoring was also made (data not shown) and the results were again very similar.
Fig. 4. Single plane TPEF images of monocellular AGS spheroids (upper row) and bicellular AGS/CAF spheroids (lower row) at different days (D) after initiation of spheroid formation. AGS cells were labelled with TdTomato and displayed in red. CAF were detected from their autofluorescence and were displayed in yellow. Imaging planes were typically located ∼50 µm below the top of the spheroids. Scale bar : 100 µm.
Fig. 5. Single plane TPEF images of an bicellular AGS/CAF spheroid 6 days after initiation of spheroid formation. Left : AGS cells only (TdTomato display). Middle : CAF only (autofluorescence display). Right : both AGS cells and CAF are displayed. Scale bar : 100 µm.
3.3 3D rendering of spheroids
In order to explore the volume of the spheroids, we performed 3D TPEF imaging by acquiring several 512 × 512 pixel images at different foci incremented by 1 µm. Three-dimensional images were reconstructed using the ICY software [25] and are displayed for 6 days-old spheroids (Fig. 6). We were able to generate the 3D shape of the imaged part of the spheroids (Fig. 6 and Visualization 1 and Visualization 2). One should note that any fluorescence signal was detected above 100-120µm in depth within the spheroids. However, spheroids diameter measured from 2D images was around 300 µm (Table S1). This may be due to the inability to keep the spherical shape of the spheroid due to the manipulation and deposition process onto the microscope coverslip. In addition, strong light scattering could make it difficult to image beyond 100 µm inside the spheroids, which are thus probably not imaged in full. Nevertheless, 6 different spheroids were imaged at each day (3 per day, 2 growth monitoring) and they always showed the same organization between GC cells and CAF, whatever the orientation of the spheroids. The 3D organization revealed in the images thus reflected the main properties of the entire spheroids. Without clearing manipulation, we could image 3D spheroids and report cells self-organization in such structures. Figure 6(a) and Visualization 1 confirm the organization of CAF in bicellular HGT-1/CAF spheroids, located in crown around a densely packed cancer cell spheroid, as revealed by single plane imaging (Fig. 3). Figure 6(b) and Visualization 2 confirmed the organization of CAF in bicellular AGS/CAF spheroids, located in a central core with cancer cells around it, as revealed by single plane imaging (Fig. 5).
Fig. 6. 3D representation of 6-days old bicellular spheroids. From left to right: GC cells only displayed, CAF only displayed, GC cells and CAF displayed. (a) HGT-1/CAF. HGT-1 cells were displayed in green and CAF in yellow. (b) AGS/CAF. AGS cells were displayed in red and CAF in yellow. The size of the boxes were (a) 710 × 710 × 170 µm and (b) 710 × 710 × 120 µm. See Visualization 1 and Visualization 2 for dynamic visualization of spheroids rotation.
3.4 Infiltration assay
In order to endorse the different spatial cell distributions between GC cell lines, we performed an infiltration assay. Monocellular CAF spheroids were first generated. After 6 days, CAF spheroids were formed and 250 HGT-1 cells or 500 AGS cells were added. Spheroid organization was analyzed by two-photon depth imaging 6 days after. Figure 7 showed the characteristic spatial cell organization of dispersed CAF between HGT-1 cells (Fig. 2), whereas AGS cells surrounded CAF spheroids, as observed during the formation of AGS/CAF spheroids (Fig. 4).
Fig. 7. Single plane TPEF images of (a) monocellular CAF spheroids 6 days after the beginning of spheroid formation; (b) bicellular HGT-1/CAF spheroids 6 days after addition of HGT-1 cells to 6 days-old CAF spheroids; (c) bicellular AGS/CAF spheroids 6 days after addition of AGS cells to 6-days old CAF spheroids. Imaging planes were located between ∼50 µm below the top of the spheroids. Scale bar : 100 µm.
4. Discussion
Cancer cells are strongly influenced by their microenvironment, which modulates local tumor progression and metastasis, and has a significant impact on drug response [26,27]. CAF are one of the major types of stroma cells and play a crucial role in tumor development and survival [28], by their ability to express a variety of growth factors promoting cancer cell proliferation and mediating modifications in ECM composition [29]. The drug resistance phenotype acquired by solid tumors may result, at least in part, from interactions between CAF and tumor cells, which makes the epithelial cancer cells/CAF interaction a promising target for GC therapy [30]. Standard 2D cell cultures have contributed to the development of many cancer therapies. However, this model lacks tumor complexity and direct pathophysiology relevance, which may be one of the main causes of the poor rate of cancer drugs entering early clinical trials, and becoming marketed drugs [31]. Novel preclinical models, such as 3D, better than 2D, cell cultures, can reproduce key factors of tumors, such as cellular organization and cell-cell interactions, and may be more predictive than 2D cell cultures for cancer therapy research [32]. Co-cultured spheroids provide even a closer resemblance to the in vivo tumors [33].
Aiming to explore such a model, we have developed here a 3D co-culture model of epithelial GC cells and CAF well suited to perform 3D optical imaging. Because several optical systems suffer from photodegradation or optical clearing and depend on sample preparation, we set up a simplified method to monitor growth and organization of live spheroids. We used two-photon microscopy, a non-invasive imaging technique well suited to analyze spheroid development and structure in an automated medium throughput format [34]. The low phototoxicity permits longer exposition times that are mandatory for three-dimensional imaging of multicellular spheroids. Applying this method, we were able to image samples containing 2 different cell types without any additional manipulation. In this study, we have generated spheroids in a scaffold-free model, using two different human GC cell lines, carrying a specific fluorescent marker, in combination with human primary CAF that are naturally strongly auto-fluorescent. Scaffold-free models make cultures independent of matrixes (like natural hydrogels or synthetic polymers) that have several disadvantages, like somewhat uncontrollable composition, poor reproducibility, and difficulty to access and harvest single spheroids. These limitations require additional treatments (chemical or physical), which are often time consuming [2]. The organization of single cells within the spheroid itself remains ill-defined, due to the lack of amenability for in-depth microscopy of such opaque structures.
Our results showed that our approach was appropriate to monitor spheroids growth and organizational evolution between day 4 and day 11. It also appeared that cells from bicellular spheroids were more tightened, generating spheroids that are more compact than single cell type spheroids. Interestingly, we observed a different spatial distribution of cells between GC cell lines. In HGT-1/CAF spheroids, a more diffuse distribution of HGT-1 cells was observed with a domination of CAF at the periphery, both when the two cell types were mixed at the beginning or when HGT-1 cells were added when CAF spheroids were already assembled. In contrast, in AGS/CAF spheroids, fibroblasts were concentrated in the central zone leaving cancer cells at the periphery, as previously reported in the context of breast cancer [35]. When added after CAF spheroids were formed, AGS cells also stayed on the surface like when mixed at the same time. Such distinctive behaviors of these cell lines are suggestive of a different migratory / invasive and malignant potential, as reported for other tumor cells [35]. To tackle this possibility, we first analyzed the expression of genes encoding adhesion and/or cytoskeleton molecules such as E-cadherin, epithelial cell adhesion molecule (EpCam), villin and integrins in HGT-1 and AGS cells. Major differences in expression levels of several genes were observed between these two cell lines, the most striking being the total absence of integrin beta-3 mRNA in AGS cells, whereas a fair expression of the gene was observed in HGT-1 cells (Fig.S6). These results suggested that indeed major adhesion molecules showed important differences between HGT-1 and AGS cells. Next, we used a functional assay to look for differences in migration potential of the cells. Using a wound closure assay, it appeared that HGT-1 cells migrated faster than AGS cells (Fig.S7). Taken together, these data strongly suggested that the HGT-1 and AGS cells displayed large differences with respect to their interaction with their environment. These differences could be explained by distinct propensities of these cell lines to undergo epithelial-to-mesenchymal transition progression [36].
Three-dimensional multicellular tumor models, with the co-culture of cancer and stromal cells, have proven well suited to study tumor microenvironments. However, technical complexities restrict the exploration of such models. We used here two-photon fluorescence microscopy to analyze the spatial organization of bicellular spheroids from two different gastric cancer lines, which showed marked differences in structure and behavior when combined with CAF. Two-photon microscopy could also be used in additional applications, such as real-time monitoring of drug response of 3D tumor spheroids. We surmise that such approaches could be applied as well to organoids, in vitro 3D culture models assembled from patients’ primary cancer cells [37].
Funding
INSERM (RAB18153NNA, RAB19101NNA); ERAPerMed (IRSTB111/L3P2131); IBSAM (Institut Brestois Santé Agro Matière).
Acknowledgments
GA received a fellowship from Brest University (“Contrat Doctoral d’Etablissement”). MD, SR and YLG acknowledge the Ministère de l'Économie et des Finances and the Région Bretagne (CPER STIC&ONDES). This work was supported by grants from the INSERM, from Brest University and from the Ligue Contre le Cancer, Comité du Finistère (n° RAB18153NNA and n° RAB19101NNA to CLJC and LC, respectively) and from the ERAPerMed GRAMMY project IRSTB111/L3P2131. All authors acknowledge IBSAM (Institut Brestois Santé Agro Matière) for financial support. All authors thank G. Leroux for technical assistance.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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