Spheroid Culture Models Imitating the Tumor Microenvironment of Renal and Melanoma Cancer

doi:10.21203/rs.3.rs-693762/v1

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Spheroid Culture Models Imitating the Tumor Microenvironment of Renal and Melanoma Cancer

https://doi.org/10.21203/rs.3.rs-693762/v1

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Background:

Tumor development studies need higher degree of adaptation to the cancer cells specific mechanisms of plasticity in connection with their microenvironment. It appears that standard two-dimensional (2D) cultures and gas composition are not relevant to the real cancer environment. Existing three-dimensional (3D) models are often requiring difficult sophisticated conditions.

Methods:

Two distinct cancer models were chosen: melanoma (B16F10) and kidney cancer (RenCa) for their different biological reactions in terms of cancer progression. We proposed 3D method which brings simplicity, reproducibility and remarkable mimicry of the in vivo tumor reactions. We characterize and compare the 3D models with standard 2D culture in normoxic and hypoxic condition, depending on presence of hypoxia related genes/proteins and aggressiveness mechanism (EMT- Epithelial Mesenchymal Transition and CSC- Cancer Stem Cells). We validate proposed 3D method by comparing it with in vivo obtained tumors.

Results:

The spheroid formation by both lines indicated a strong potency of melanoma cells to form and grow in 3D structures. These spheroid-growing cells reached a cell cycle arrest at the G0/G1 phase and showed the formation of a hypoxic core while a similar development time produced renal cancer (RCC) 3D cells in growing phase and delayed hypoxia, as shown by hypoxia-related genes/proteins expression. A comparable response to hypoxia was observed in 3D spheroids and in vivo tumors for both lines as opposed to monolayer cultures. RCC model showed induction of CSC characteristic whereas, melanoma spheres contribute to EMT-related mechanism. Such distinction in the use of separate aggressiveness-leading pathways and response to hypoxia was observed in in vivo melanoma vs kidney tumors.

Conclusion:

This work shows that effects of the structural parameters and reaction to hypoxia uncover that these two cell models use distinct mechanisms to reach aggressiveness: epithelial to mesenchymal transition (EMT), cancer stem cells (CSC) by B16F10 and RenCa cells, respectively. These data on aggressiveness pathways and reaction to hypoxia confirm and validate the proposed 3D models for further design of alternative methods.

Cancer Biology

Oncology

Spheroids

EMT

CSC

hypoxia

melanoma

RCC

cancer

Despite advantages in cancer treatment research, this disease is still the leading cause of death worldwide (1). Treating and studies on cancers are challenging due to complexity of tumor site (2). This complex system is composed of cancer and many other non-cancer cells, which all have specific role in the functioning of the tumor (3). Along with the development of cancer cells the mass gets hypoxic and this conditions the development of a microenvironment of the tumor cells (4). Such condition is defined as lower oxygen tension than it is observed in non-pathologic conditions (pO₂ 1 %) (5). Hypoxia induces the expression of Hypoxia-inducible factor 1α (HIF-1α). Transcription of HIF-1 in turn, induces angiogenic factors such as Vascular endothelial growth factor A (VEGF-A) (6–8). This mechanism is controlled by the tumor suppressor von Hippel-Lindau (VHL) (9–12). In physiological oxygen tension, VHL binds to HIF-1α and promotes degradation of HIF-1α in proteasome (13). In RCC cancers mutation of VHL gene is often occurring, which promotes a constant pathological hypoxic condition (10, 11). Hypoxia represents poor prognosis factor in many cancers, it is linked with cancer aggressiveness, invasion, and metastasis (4, 6, 14). Epithelial Mesenchymal Transition (EMT) process is one of the initiating steps of cancer invasion, during which tumor cells dedifferentiate by losing epithelial markers (e.g. E-cadherin) in favor of mesenchymal ones (e.g. N-cadherin, ß-catenin, Vimentin) (15). To study the biology of cancer cells in vitro the development of 3D cultures has been It has been shown that spheroid cultures induce expression of EMT markers (16, 17).

Metastasis process is also linked with the selection of highly aggressive cells - Cancer Stem Cells (CSC) (16). These cells are characterized with stem-like features (18). It has been shown that CSCs are responsible for tumor initiation, progression, and disease relapse (16, 19). They are also thought to be responsible for the development of resistance to cancer treatment (19). CSCs could be found in melanoma and renal cancer, they were characterized with the expression of cell surface markers specific to the type of cancer, such as CD105 (20, 21), CD133 (22, 23), Aldehyde dehydrogenase 1 (ALDH1) (24, 25).

Mimicking real cancer microenvironment is crucial in cancer research (2). Syngeneic in vivo murine models are highly valuable tools in terms of recreating the full spectrum of tumor (26). However, animal models cannot always be used in each phase of pre-clinical studies. Therefore, most initial research is performed with the use of monolayer cell culture. Standard two-dimensional (2D) cell culture methods are highly limited, they are lacking the three-dimensional shape of the tumor which largely modulate the cell to cell interactions and limits oxygen availability thus lowering oxygen tension (27). Hypoxic conditions can be reproduced in 2D cultures by usage of hypoxic chambers (28), however the oxygen distribution will not reconstitute natural oxygen gradient as set in the tumor. Along with the spheroid development its 3D structure allows such natural establishment of hypoxia (29). There are already developed different type 3D culture methods with a usage of scaffold and non-scaffold methods (30). However, by various factors, e.g., cost, special equipment and reproducibility of the highly complicated methods, widespread implementation of 3D's into cell-based studies has been limited (3, 30–32). Commercially available products for the 3D cell culture (e.g. hanging drop or scaffold plates) are also not always well optimized and due to the lack of standardized protocols interpretation of the data can be challenging (31)

The aim of the present studies was to optimize and validate simple in vitro models which will correspond to the characteristic observed in the in vivo tumor mass, such as: three-dimensional shape of the tumor, proper oxygen tension and cellular responses (3, 15). We used two different cell lines to establish an universal protocol allowing sphere formation. We characterize the obtained 3D structures compared to standard monolayer culture with a special emphasis on EMT and CSC related markers. The goal of the study was to assess whether cells undergo similar molecular changes in spheroids as in the tumor mass in vivo. Such well-characterized models may contribute to obtaining more reliable data in easy way, which will bring research tools to provide data closer to the possible translation into clinical practice.

1. Cell culture

The tests were carried out with murine renal carcinoma RenCa (CRL-2947 ™, ATCC, USA) and melanoma B16-F10 (given by Prof. J. Dulak, Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland; authenticated in 2021, ATCC) cell lines. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ in appropriate media - RPMI-1640 GlutaMaxTM medium (ThermoFisher Scientific, Waltham, Massachusetts, USA), with 10% FBS (vol/vol) (ThermoFisher Scientific). After confluence reached 70–80% all cell lines were passaged by detaching with Accutase solution (Biolegend, San Diego, California, USA). ). Cells used in the experiments were Mycoplasma free as assayed with PCR Mycoplasma Test (Biomedica, Poland) and did not exceed 15th passage.

1.1. Two-dimensional cell culture

Two-dimensional cultures were carried out under two oxygen partial pressures “normoxia” (18,75% O2)- 2DN and hypoxia (1% O2)- 2DH. Cells were seeded at distinct densities: RenCa − 6600 cells/cm², B16F10 3300 cells/cm². 24 hours after plating, hypoxia intended cell cultures were transferred to hypoxia chambers (BioSpherix, Xvivo System Model X3, USA), while changing the cell medium to fresh hypoxia-balanced medium. Normoxic cell cultures were kept under standard aerobic conditions, after switching to fresh, normoxic medium. The cultures were pursued for 72 hours.

1.2. Three-dimensional cell cultures by spheroid formation

500 cells resuspended in 20 µL medium supplemented with 0.25% methylcellulose (vol/vol in medium), (R&D Systems, USA) were seeded as a single hanging drop, on the cover of culture plate in standard culture conditions (5% CO₂, 21% pO₂). After 72h drops were individually transferred to a 96-well plate previously covered with 1% agarose dissolved in double distilled water (w/vol, VWR, Belgium), and 50 µL of fresh medium (supplemented with 0,25% methylcellulose, vol/vol) were added concomitantly. Cells were allowed to expand for another four days period.

1.3. In vivo tumor implantation and development

The experiments were carried out with female mice of the C57Bl6 strain - syngeneic model of melanoma and BALB /c - syngeneic model of kidney tumor. Cancer cell suspensions: RenCa − 100,000 cells; B16F10–200,000 cells in 100 µL of PBS mixed with Matrigel™ (50%, vol/vol) (Corning, USA) and maintained at 4֠֠ C, were subcutaneously administered to the animal's thigh. The renal cancer model experiment was terminated after 22 days, while the melanoma cancer model experiment was completed 18 days after the injection of the tumor cells. Experimental groups consisted of 3–4 mice kept in one cage, and experiments was performed using two separate sets of animals. All experiments were conducted after obtaining approval for procedures from Second Warsaw Local Ethics Committee for Animal Experimentation (no. WAW2/76/2017) and following Directive 2010/63/EU regulations.

3. Western blot

The two-dimensional cell cultures were washed twice with ice-cold PBS and collected by scraping. Harvested and PBS-washed spheroids were dispersed, and cell suspensions were centrifuged before lysing by RIPA lysis buffer complemented with proteases and phosphatases inhibitors (both from ThermoFisher Scientific, USA). Fragments of neoplastic tissues were homogenized using TissueLyser II (Qiagen) in RIPA lysis buffer with proteases/phosphatases inhibitors. Insoluble material was removed by lysates centrifugation (14,000 × g for 10 min at 4°C). Equal amount of total protein were separated on 12% SDS polyacrylamide gels for 40 min at a constant 200 V using a Bio-Rad Minigel System, followed by transfer to nitrocellulose membranes (GE Healthcare life science, Germany). Membranes, after blocking, were probed with primary antibodies to detect: ALDH1A1 (1:250 Polyclonal; LSBio), VHL (1:200 anti-mo; Clone: FL-181; Santa Cruz Biotechnology), HIF1α (1:100; Clone: 28b; Santa Cruz Biotechnology), Vimentin (1:1000 anti-rb, clone: D21H3; Cell Signaling Technology) and Vinculin (1:1000 anti-mo, clone: V284; Santa Cruz Biotechnology), which served as loading control. Membranes were further treated with secondary antibodies conjugated with horseradish peroxidase-HRP (goat anti - mouse/anti-rabbit, 1: 10,000; Vector Laboratories, USA). Bands were detected using Luminol as HRP substrate (Santa-Cruz, USA) with X-ray films. Bands were analyzed using ImageJFiji.

4. Flow cytometry detection of intracellular enzymes activity and cell surface antigens

Cell suspensions from 2D cultured cells were obtained using Accutase solution (Biolegend, USA). Individually cultured spheroids were treated with 0,1 mg/ml collagenase type II (Gibco by Life Technologies) and incubated at 37° C with simultaneous stirring until the spheres disintegrated into single cells.

4.1. Aldehyde dehydrogenase- 1 activity assessment

The ALDH1 enzyme activity was measured intracellularly with the help of Aldefluor fluorescent reagent. It is a BODIPY-aminoacetaldehyde fluorescent ALDH substrate able to diffuse into intact and viable cells. Converted into BODIPY-aminoacetate by ALDH in cells, the negatively charged product is retained inside the cells increasing fluorescence for flow cytometry analysis (λex max = 493 nm, λem max = 588 nm). Protocol was followed according to the manufacturer’s recommendations (STEMCELL Technologies). Briefly, 0.5 x 10⁶ cells were suspended in 500 µL of Aldefluor assay buffer. Assay samples received 2,5 µl of the Aldefluor reagent, mixed and immediately 250 µl of the suspension was taken out and put in the control tube with the DEAB (N, N’-dimethylaminobenzaldehyde) inhibitor 2,5 µL. Cells were further incubated for 45 minutes at 37°C in the dark. After washing cells, viability was determined by staining with 7-Amino-Actinomycin D (BD Biosciences) a membrane permeant dye which is excluded from viable cells, it binds to double stranded DNA of dead cells (λex max = 546 nm, λem max = 647 nm). All samples were analyzed using a Calibur flow cytometer (from Becton Dickinson, Sunnyvale Ca, USA). Cells treated with inhibitor were used to set the gate for ALDHneg population and positive cells were analyzed.

4.2. Surface antigens detection by fluorescent antibodies

The evaluation of the expression of CD105 and CD133 was determined using commercially available antibodies: PerCP-eFluor710 conjugate rat-anti-mo (IgG1) CD133 (Clone: 13A4; Thermo Fisher Scientific, USA) and PE conjugate rat-anti-mo (IgG2a) CD105 (Clone: MJ7/18; Thermo Fisher Scientific, USA). 500,000 cells were resuspended in PBS (200 µl) and first stained for viability determination by LIVE/DEAD™ Fixable Dead Cell Stain Sampler Kit, 488 nm excitation (Thermo Fisher Scientific, USA) for fluorescence spectra compatibility (for 15 min). The dye reacts with free amines both inside and on the surface of dead cells, yielding to an intense fluorescent staining. The cells were washed and resuspended in Stain Buffer – BSA (BD Pharmingen), then the labelling for antigens was performed by incubation of the cells in the presence of fluorescent antibodies for 2h at 4°C in the dark. Cell analysis was performed using Calibur flow cytometer (Becton and Dickinson, USA).

5. Reverse Transcriptase-Polymerase Chain Reaction

RNA samples from in vitro and in vivo cultures were isolated using the commercially available RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacture protocol. The obtained cell lysates were either stored at -80 ° C or RNA isolation was started. The isolated RNA was then purified using the TURBOTM DNA-free Kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. The concentration and purity of RNA were determined by measuring the absorbance in the wavelength range: 230, 260 and 280 nm. RNA integrity was assessed using a fluorometer based on the RIN (RNA integrity number) coefficient (acceptable values 7–9). After obtaining high purity mRNA, a reverse transcription process, using a commercially available High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) was performed according to the manufacturer's instructions. The resulting single-stranded cDNA was used as real-time polymerase chain reaction templates.

RT-PCR reactions were performed using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, USA) together with commercially available probes all from Thermo Fisher Scientific: Vhl: Mm00494137, Vegf-a: Mm00437306, Hif-1α Mm00468869, Actinβ Mm02619580. The samples run on Bio-Rad CFX384 qPCR System or CFX Connect qPCR System (Bio-Rad). Amplification conditions were as follows: 50°C (2 min), 95°C (10 min) and 40 cycles of 95°C (15 s) and 60°C (1 min). The quantity of mRNA was calculated using the 2(-Delta C(T)) method and normalized to β-actin. All reactions were performed as triplicates.

6. Next Generation Sequencing (NGS)

NGS was performed outsourced service by Lexogen GmbH (Vienna, Austria) with NextSeq 500 system (Illumina, USA). Briefly, RNA was extracted and purified using methods described in Reverse Transcriptase-Polymerase Chain Reaction section. RNA BR Assay Kit (Thermo Fisher Scientific, USA) was used to measure total RNA concentration and Qubit RNA IQ Assay Kit (Thermo Fisher Scientific, USA) to measure RNA integrity and quality. To obtain cDNA libraries 1µg of total RNA with IQ > 8,5 was used. Preparation of libraries was performed according to manufactures instructions as followed: The NEBNext® Poly(A) mRNA Magnetic Isolation Module - mRNA isolation and fragmentation (New England Biolabs) Ultra RNA Library Prep Kit for Illumina - cDNA Library synthesis, New England Biolabs); NEBNext Multiplex Oligos for Ilumina - adding Adaptors (New England Biolabs). All steps were performed along with the purification of the reaction products using NEBNext Sample purification Beads (New England Biolabs). Libraries were qualified on Bioanalyzer (Agilent, USA), with High Sensitivity DNA Kit (Agilent, USA). Differentially expressed genes (DEGs) were classified according to -1,5 < logFC > 1,5 and p-value < 0.5. Gene symbols were translated into UniProt accession numbers using the UniProt Knowledgebase (UniProtKB). Protein networks were retrieved from the STRING (Search Tool for the Retrieval of Interacting Genes) database using the list of protein accession numbers as a query and then analyzed using the Cytoscape software.

Microscopy Imaging

3D cultures were performed according to the above-mentioned procedure. Imaging to assess live and dead cells in the spheres was performed after labelling by calcein acetoxymethyl ester (Calcein AM, Biolegend) which is hydrolyzed by cellular esterases to give fluorescent calcein in the cytoplasm of live cells and propidium iodide (Exbio, Czech Republic) DNA intercalating agent which is highly fluorescent after intercalation. Briefly, after adding the dyes spheroids and cells grown in two-dimensions were incubated for 15 minutes in the dark at room temperature. Images were acquired using a Zeiss AxioObserver.7, fluorescence, and inverted microscope (5X magnification) and analysis performed with the Zen 2.6 blue edition software (Zeiss, Germany).

Statistical Analysis

The results are shown as a mean +/- SEM. All experiment was performed at least in 3 biological replicates. All statistical analyses were performed using GraphPad Prism 9.0 software (RRID:SCR_002798). Depending on the gaussian distribution we performed One-Way Analysis of variance (ANOVA) with Tukey post-hoc test or Kruskal-Wallis with post-hoc Dunn’s test.

Optimization, characterization, and comparison of 3D model with 2D culture models.

Establishment of 3D cultures

Irregular cell aggregates were observed especially for RCC model after using standard culture methods, e.g.: hanging drop in regular culture medium (Supplementary Fig. 1A). Consistent spheroids from renal and melanoma murine cancer cells in their long-term culture in normoxic conditions were obtained by combining the hanging drop method with culture in semi-solid matrix (Fig. 1B). 500 cells in a 20 µL drop were seeded for both cell lines to compare the proliferative capacity of the cells during the sphere’s formation. Spheroids were cultured for seven days in total: three days in hanging drops and then four days in agarose coated bottom plate in standard culture medium supplemented with methylcellulose (Fig. 1A). After three days of culture in hanging drops irregular cell aggregates were observed for both cell lines: RenCa and B16F10 (Supplementary Fig. 1B). Round and regular spheroids were obtained after seven days of culture were completed (Fig. 1. B.). As measured by diameter, the RCC cells at the end of culture formed smaller spheroids (~ 400µm) as compared to spheres (~ 600 µm) (Fig. 1C) from B16F10 despite the initial number of seeded cells was the same. This suggests a slower growth of RenCa cells in 3D. Using flow cytometry, we determined the cell viability in 2D, and 3D cultures conducted in normoxic and hypoxic conditions. Higher proportion of viable cells for both cell lines was recovered from two-dimensional cultures, than from spheroids in both oxygen tension conditions (Fig. 1D). Microscopic observation of spheres stained by calcein and propidium iodide showed a necrotic core of 3D structures in normoxia (Fig. 1.E). The cells from spheroids cultured in hypoxia showed a very low viability (Fig. 1.D; Supplementary Fig. 1C) and were smaller than spheres cultured in normoxia. It suggests that hypoxia which is known to develop in the center of the sphere may be influencing the viability of cells recovered from 3D structures where cells have an impaired access to oxygen (29). Consequently, spheroid culture in additional/external hypoxic conditions may cause an intense cellular stress drastically reducing the cell survival. As very few cells were recovered from hypoxia cultured spheres, we decided to conduct further research on cells cultured under normoxic condition which seems more appropriately reconstitute natural development of a tumor.

3D culture condition induces melanoma cancer cell cycle arrest at the G0/G1 phase

Spheroid culture affected the cell cycle distribution in both cell lines with no direct effect of hypoxia only (Fig. 2A-D). In the case of B16F10 cells, 3D culture caused accumulation of cells in G0/1 phase with concurrent reduction of S phase mostly (Fig. 2A, C). On the contrary, spheroid recovered RenCa cells were enriched in proliferating, G2/M cells with non-significant drop of cells in both G0/1 and S phase (Fig. 2B, D). As activation of p53 tumor suppressor can lead to the cell cycle arrest (33) we checked the expression of p53 and its inhibitor mdm2. For both cancer models we observed a similar downregulation of p53 expression by both hypoxia and 3D formation, as compared with 2D normoxic culture (Fig. 2E). Expression of mdm2 in melanoma cancer for 2D hypoxia and 3D was down regulated as compared with 2D normoxia, with a significantly stronger effect due to the spheroid type of culture (Fig. 2E). For RCC cultures no statistically significant changes of mdm2 expression were observed (Fig. 2E).

3D melanoma cancer model induces hypoxia.

Presence of necrotic core in 3D structures of RCC and melanoma models after staining with calcein and propidium iodide suggested the induction of hypoxia in the middle of the spheres (29). We checked several hypoxia related genes/proteins such as: HIF-1α, vegf-a, and VHL (Fig. 3. A, B). In 3D melanoma model we observed upregulation of hif-1α expression and a tendency for higher protein level as compared to both 2D normoxia and hypoxia (Fig. 3. A, B). Vhl expression was not affected by any of tested culture conditions. Vegf expression increased both in 3D and 2D culture in hypoxia, although in the later condition, no HIF-1α upregulation occurred (Fig. 3. A, B). In RCC model we observed an opposite tendency; 3D culture downregulated hif1α, vhl and vegf-a genes, however hypoxia alone tended to increase HIF-1α protein and vhl and vegf gene expression (Fig. 3. A, B). Global gene expression analysis with NGS was performed for RCC model to identify hypoxia gene signatures in 3D as compared with 2D cultures (Fig. 3. C). String protein networks indicated upregulation of Endothelial PAS Domain Protein 1 (Epas1) for 3D culture as compared with both 2D normoxia and hypoxia culture conditions. Also, upregulation of matrix metalloproteinase-10 (Mmp10), Matrix metallopeptidase 13 (Mmp13) and Lysyl oxidase (Lox) was observed as compared to 2DN culture (Fig. 3. C).

Renal cancer spheroids show upregulation of a cancer stem like cell – CSC- population

Upregulation of cells arrested in G0/G1 phase may suggest the presence of CSC (34), and hypoxia was shown to induce selection of CSC in cancer foci (29). Therefore, the levels of several potential CSC markers such as: ALDH1, CD133 and CD105 were evaluated (Fig. 4). In the case of B16F10 cells with G0/G1 arrest, we could not observe CSCs induction; CD105 positive cells dropped in 3D culture while CD133 positive cells remained unchanged (Fig. 4D, E). Similarly, ALDH1 was not significantly altered in those cells (Fig. 4A, B). Surprisingly, in spheroid growing RCC cells, that were characterized by increased G2/M accumulation, a strong increase of ALDH1 protein level and activity were observed (Fig. 4A, B). Analysis of String protein networks also indicated the upregulation of Aldehyde dehydrogenase 2 (ALDH2) expression in 3D (Fig. 4C). Additionally, in RenCa cell line an increased number of CD133 positive cells in 2D cell cultures in hypoxia and in 3D cultures was shown, whereas no statistically significant changes for CD105 levels were observed (Fig. 4. D, E). Remarkedly, an opposite tendency was displayed by B16F10 cells.

3D induces EMT in melanoma model

Another mechanism of cancer aggressiveness is EMT (35). We tested whether this process is induced by spheroid culture by assessing the main EMT markers: Vimentin, N-cadherin, and B-catenin (Fig. 5). In the B16 F10 melanoma model, the upregulation of N-cadherin, Vimentin and B-catenin was observed in spheroids. In the RenCa model B-catenin in spheroids was downregulated and N-Cadherin could not be detected in any tested culture conditions (Fig. 5). Changes for vimentin expression in RCC models were not significant, however tendencies were opposite than those observed in melanoma model (Fig. 5), confirming the distinct reactions uncovered by the 3D mode of culturing the two cancer cell types.

3D spheroid formation as an alternative model to in vivo murine tumors

To assess for the significance of spheroid formation to mimic some tumor characteristics, we compared the levels of above tested markers in 3D models to in vivo tumors, comparatively to standard monolayers type of culture in normoxia (2DN).

3D cell culture models induce similar pattern of expression of cancer suppressor genes as in vivo growing tumor

Melanoma and RCC spheroids and their corresponding tumors, showed similar level of downregulation of p53 expression as compared to monolayer cultured cells (Fig. 6). While, in the melanoma model a similar downregulation pattern was observed for mdm2 expression, this was not the case in the RCC model as mdm2 expression was not significantly altered (although tended to increase) (Fig. 6).

3D cultures and in vivo tumors show a similar expression pattern of proteins / genes associated with hypoxia

Melanoma spheroids and tumors displayed a similar upregulation of hif1α and vegf-a genes expression but the HIF-1 protein increase in spheroids was not as strong as in tumors (Fig. 7A-C). A statistically significant downregulation of vhl was observed only for its transcripts in tumor (Fig. 7A). On the other hand, there was no effect on the corresponding protein level both in spheroid and tumor (Fig. 7B, C). For RCC we observed that 3D and tumors show similar changes in expression when compared to monolayer, but tendencies were opposite to melanoma model: both in tumor and 3D a downregulation of hif1α, vhl and vegf-a was observed (Fig. 7A-C). On the level of proteins, VHL increased in tumors only; in spheroid culture this upregulation did not reach a statistical significance (Fig. 7B, C).

RCC spheroids and tumors show a similar expression pattern of markers associated with stemness

Previously observed ALDH1 induction in renal cancer spheroids was also seen tumors; ALDH1 protein level was increased similarly in 3D culture as compared to monolayer (Fig. 8). Also, melanoma tumors were characterized by a very high expression of this protein, although in this model, spheroid culture could not upregulate ALDH1 significantly (Fig. 8).

Melanoma and RCC spheroids and tumors show a similar expression pattern of markers associated with EMT

Previously observed ß-catenin induction in melanoma and downregulation in renal cancer spheroids was also observed tumors (Fig. 8). The same tendencies for vimentin expression in 3D and the tumor was also observed for both models, were we observed upregulation of this protein in melanoma and downregulation in RCC model (Fig. 8).

To increase validity and success rate of cancer research, it is necessary to handle parameters that mimic the most important features of the disease. Monolayer cell cultures provide useful and homogenous model, however many studies showed that 3D culture models display features that are closer to in vivo tumor characteristics (29, 30). 3D models using B16F10 cells do exist for murine melanoma, but there are no well-optimized models for murine kidney cancer using RenCa cells for studies (29, 36–38). The present work performed long long-term 3D cultures allowing comparative models for melanoma and renal cancer and characterized them in terms of response to hypoxia, CSC selection and EMT induction. Additionally, we demonstrated the validity of the spheroid type of culture to reflect the mechanisms observed in tumors in vivo. The designed method does not require any special equipment nor expensive laboratory consumables and pipetting was reduced to minimum. We exploited 96-well format that can be easily used for cost- and time-effective drug screening but provide sufficient cellular material for molecular experiments (western blot, qPCR or flow cytometry can be performed using 96 spheroids for each technique) (Fig. 1A).

It was shown that size of the sphere influences the cellular processes induced within. The dimensions of spheroids are usually correlated with seeding density (32), however in our study we point out also intrinsic features of used cells for the outcome of 3D culture. The study, although restricted to two murine cancer cell type to reduce the variability of spheroid formation, confirms the universality of the method. Indeed, the present modification of hanging drop method was efficiently inducing 3D formation by both cell lines, despite the poor RenCa ability for sphere formation (Supplementary Fig. 1A, B) (Fig. 1C). RCC spheroids were significantly smaller than melanoma’s, although initial cell number was same for both cell lines. Such differences may result from different proliferation capacity of cells, resulting is a less apparent necrotic core (Fig. 1E). Ability to form spheroids corelates with cancer stem-like cells characteristics of cell lines (39, 40). We showed that melanoma cell line is initially characterized by higher levels of ALDH1 (protein / ALDH1 activity) and CD133 positive cells than RenCa cells (Fig. 4) what relates with the higher capacity of B16F10 cells to form spheroids.

Cells recovered from spheroids were less viable probably due to a necrotic core formed in 3D structures as observed by microscopy (Fig. 1E). This suggests that hypoxia develops in the center of the sphere (41). Indeed, in the melanoma spheres, we observed upregulation of Hif1α (42), inducer of the main proangiogenic factor VEGF-A, (Fig. 3A). In RCC 3D model we observed downregulation hif1α, vhl and vegf-a expression (not statistically significant), which suggest that hypoxic processes were not effectively induced probably because spheres were smaller (Fig. 3A, B). However, the same pattern of gene expression was observed in the subcutaneous tumor obtained in vivo (Fig. 7). This shows that 3D culture induces cellular mechanisms that are cell-specific, in addition to the physical parameters that universal and due to spheroid architecture. This strongly support the conclusion that, in many aspects, spheroids are sufficient to model tumor physiology.

Further characterization of RenCa spheroid model revealed upregulation of Epas1 (Hif2α) transcript (Fig. 3C). Studies using in vivo and in vitro models indicated that HIF-1α acts as a tumor suppressor, whereas HIF-2α has oncogenic potential (12). Together with increased expression of Hif2α, increase of Mmp10, Mmp13 and Lox expression was observed (Fig. 3C). These proteins are associated with cancer progression and metastasis in various types of tumors (43–45). Taken together that RCC spheroids have population of aggressive, highly proliferative cells that are selected upon 3D formation. Indeed, RenCa spheroids were enriched in proliferating, G2/M cells (Fig. 2), cancer stem-like cells (upregulation of ALDH1, ALDH2 expression and CD133 + increased population) (Fig. 4) and metastasis linked proteins. While the induction of Hif2α down regulator for p53 (46, 47) was observed in both models (Fig. 2E), induction of pro-metastatic EMT-linked processes was not observed (35) in RCC spheres (Fig. 5).

In contrast melanoma spheroids were characterized by a reduced percentage of proliferating cells (G2/M) with a simultaneous G0/G1 phase shift in the cell cycle, despite p53 downregulation together with its regulator mdm2 (Fig. 2). G0/G1 phase arrest also suggests the presence of quiescent cancer stem cells (48), although other markers of CSC phenotype, as ALDH1 and CD133, were not induced in B16F10 spheroids (Fig. 4A, B, D) or down regulated as CD105 positive cells (49) (Fig. 4E). CD105 downregulation in these cells causes reduction of proliferative capacity, and promotes spheroids compaction (21). This may explain cell cycle arrest in G0/G1 phase of cells in the melanoma spheres (Fig. 2A-D), and shape differences of both cell models (Fig. 1B, E). Although we do not observe any changes in CSC markers expression in melanoma spheres, EMT markers were upregulated (Fig. 5). The use of the 3D type of culture uncovered the distinct molecular mechanisms set by the melanoma and the RCC cells to reach a higher aggressiveness. As EMT is induced by hypoxia in several types of cancer (14), this might have not been reached by RenCa spheres in contrast to B16F10. Although RenCa spheroids are smaller and less hypoxic, their structure allowed a positive CSC selection and proliferation. B16F10 form bigger and hypoxic spheres which possess EMT features. Our result correspond with what was shown previously, that melanoma intra-tumor heterogeneity does not rely on the CSC hypothesis, but rather on EMT features (50).

To validate the developed models for mimicking the most important features of cancer, we compared the levels of the above tested markers in 3D models with in vivo tumors. Melanoma and RCC spheroids showed a similar pattern of regulation of p53 oncogene and its regulator – mdm2 (Fig. 6), which may indicate similar proliferative capacities of tumors and spheres (51). Hypoxia markers found in the melanoma spheroids were expressed at similar levels but the HIF1 protein, for which increase in spheroid lower than in the tumor (Fig. 7). This indicates that melanoma 3D culture is indeed able to mimic the features of tumor hypoxia. RCC spheres also show similar changes as observed in the corresponding tumor, although these two cancer types were often oppositely modulating molecular pathways (Fig. 7). The 3D type of cultures clearly bring the conditions much closer to in vivo ones. Indeed, the induction of CSC in RCC model was as efficient as in the tumor confirming the similarities between 3D and the tumor, in contrast to the melanoma where the CSC induction was lower than in the tumor but in both cases the EMT processes were largely mimicked.

In our studies, we observed that 3D models can mimic some features of the tumor such as hypoxia and CSC induction, adequately to the characteristics of corresponding in vivo tumor mass. Spheroid models of renal and melanoma cancers induce different mechanisms, related to the progression of cancer. RCC model displayed induction of CSC characteristic whereas, melanoma spheres contribute to EMT-related mechanism. Changes in cellular processes in these two cell lines in spheroids compared to standard monolayer were not universal, however were reproduced in the in vivo subcutaneous model, proving the validity of spheroid cultures to model cancer physiology.

two-dimensional culture

2DH

two-dimensional culture in hypoxia

2DN

two-dimensional culture in normoxia

three-dimensional culture

ALDH1

Aldehyde dehydrogenase 1

ALDH1

Aldehyde dehydrogenase 2

ANOVA

Analysis of variance

CSC

Cancer Stem Cells

EMT

Epithelial Mesenchymal Transition

EPAS1

Endothelial PAS Domain Protein 1

HIF1α

Hypoxia Inducible Factor 1α

Lox

Lysyl oxidase

Mmp10

matrix metalloproteinase-10

Mmp13

Matrix metallopeptidase 13

NGS

Next Generation Sequencing

pO2

partial pressure of oxygen

RCC

renal cancer

VEGF-A

Vascular endothelial growth factor A

VHL

the tumor suppressor von Hippel-Lindau

Ethics approval and consent to participate:

All experiments were conducted after obtaining approval for procedures from Second Warsaw Local Ethics Committee for Animal Experimentation (no. WAW2/76/2017) and following Directive 2010/63/EU regulations.

Consent for publication: Not applicable

Availability of data and materials:

The datasets generated and/or analyzed during the current study are available in the [Mendeley Data] repository, [doi:10.17632/9259s6c46f.1].

Competing interests:

The authors declare that they have no competing interests.

Funding: The research was funded by National Scientific Center grant no 2016/23/B/NZ1/03211. 3D culture optimalization was performed within Military Institute of Medicine intramural grant no 5/8958 (467). AF was supported by European Social Fund: POWER “Next generation sequencing technologies in biomedicine and personalized medicine”

Autor contributions: AF, KB, AM performed experiments and analyzed the data. CK developed conception of the research and work hypothesis. All authors contributed to the writing of this manuscript

Acknowledgements: Not applicable.

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Spheroid Culture Models Imitating the Tumor Microenvironment of Renal and Melanoma Cancer

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Version 1

Abstract

Figures

Background

Material And Methods

1. Cell culture

1.1. Two-dimensional cell culture

1.2. Three-dimensional cell cultures by spheroid formation

1.3. In vivo tumor implantation and development

3. Western blot

4. Flow cytometry detection of intracellular enzymes activity and cell surface antigens

4.1. Aldehyde dehydrogenase- 1 activity assessment

4.2. Surface antigens detection by fluorescent antibodies

5. Reverse Transcriptase-Polymerase Chain Reaction

6. Next Generation Sequencing (NGS)

Microscopy Imaging

Statistical Analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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Version 1