Developing tumor microenvironment in rotating human melanoma cell cultures: study of novel preclinical model

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Stępień This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4884972/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract More than 97% of anticancer drugs under investigation fail in vitro evaluation, while only 0.4% of drug candidates that pass this stage proceed to the clinical trials. The differences between cell morphology and physiology observed in vitro and in vivo make the selection of a drug candidate problematic: traditional in vitro cultures do not reflect tissue-like conditions. Here we aimed for developing and characterizing human melanoma tumorspheres cultured in rotating bioreactors as an alternative for in vitro modeling. Tumorspheres were characterized by in-depth confocal imaging and image cytometry, followed by quantitative analysis that was used for whole tumorspheres characterization. Cell viability and changes in proteins expression were investigated in single-cell analysis through the spectral flow cytometry followed by STRING interaction networks assessment. The tumorspheres showed the ability to grow for at least one month to reach millimeter sizes. In this way, it was possible to improve the morphology of tumorspheres and to observe changes in tumor microenvironment (TME) and the expression of key proteins. The advantage of the described models is the creation of perspectives for further development of maintaining cellular models that are hybrid systems combining the features of spheroids and organoids for preclinical and translational research. Cell & Tissue Engineering Cancer Biology Oncology Biochemical Research Methods 3D-cell cultures bioreactor confocal imaging imaging cytometry organoids tumorspheres Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Significance One of the most important and unmet challenges in drug development is an in vitro - in vivo gap caused by inadequate cell models. In this study, we identified and described the differences in tumor development and progression under different culture conditions. We focused on morphological and physiological features at various cellular levels, which are currently a less explored area of research oriented towards new approaches to biological modeling. This study is one of the first to examine melanoma in-depth development and assessment in vitro and indicates that the resulting models shows favorable characteristics compared to those currently used in preclinical studies. Research Highlights Rotating human melanoma cell cultures shows a great prospects to make a step forward in preclinical tumor modeling. Longer culturing leads to improved morphology and TME, thus enabling to achieve more clinically-relevant melanoma models. 1. Introduction Significant differences at various cellular levels between in vitro, in vivo and clinical tumor models are one of the major challenges hindering the progress in cancer diagnosis and therapy. In vitro assessment, as preliminary evaluation step, has a critical impact reflecting further stages success rates as they are used not only for in vitro evaluation, but are subsequently applied for tumor inoculation in animal model (Dowden & Munro, 2019 ; Law et al., 2021 ; Stępień et al., 2020 ). Applicability range of traditional cell cultures enables only basic research, such as drug uptake or cell survival. Two-dimensional growth in unnatural flattened shape impairs the cell morphology, while uniform distribution of nutrients and medium results in high homogeneity. Synchronized cell cycles contribute to changes in cellular response and growth, leading to abnormal outcomes. Furthermore, cell-to-cell interactions, limited to the boundary of the cell outline, lead to changes in genes and protein expression, also affecting intercellular signaling, and more importantly the development of tumor microenvironment (TME). Consequently, these models due to numerous morphological and physiological shortcomings do not effectively assembles the clinical tissues (Kapałczyńska et al., 2018 ). Introducing a spatial, three-dimensional (3D) cell culture models, classified as spheroids (cell culture-derived) or organoids (tissue-derived), offer an advantages over the traditional ones (Han et al., 2021a ). This environment permits cells to preserve or restore their inherent morphological characteristics, thereby facilitating the establishment of a sophisticated cancer microenvironment ( milieu ) exhibiting a higher degree of complexity relative to its two-dimensional (2D) counterpart. More pronounced cell differentiation coupled with improved intercellular interactions via ions, small molecules and electrical signals, makes them more suitable for simulating tumor tissue (Durak-Kozica et al., 2023 ). The expression of genes and proteins can be similar to in vivo conditions (Jensen & Teng, 2020 ) and may allow the introduction tumor growth factors, such as gravity and mechanical stimulation, which are unavailable in 2D cultures (Guillaume et al., 2019 ; Huang et al., 2021 ). The main disadvantage of typical 3D cell cultures is the limited culture time (~ several days) and poor model reproducibility, having a primary importance for the extensive preclinical evaluation various types of novel therapeutic approaches (Wawrowicz et al., 2023 ; Żelechowska-Matysiak et al., 2023 ). Additionally, reduced nutrients and oxygen penetration, insufficient elimination of waste and metabolic products are another limitations of typically used methods. These features as well as economic aspects do not offer encouraging benefits that could support their wide implementation into preclinical protocols (Katt et al., 2016 ). One of major requirements that could lead to improve the effectiveness of drug discovery is a holistic approach. Redefining model types and their use must be the first step towards improving the quality of preclinical evaluation. Furthermore, recent FDA regulations also force the replacement of in vivo models with advanced 3D cell cultures (Meredith Wadman, 2023 ), emphasizing the importance of rapid progress in such systems. Consequently, there is an urgent need to develop and characterize highly advanced 3D models for preclinical research, both upgrading in vitro models and/or replacing animals. Addressing those needs, with this study we aimed for developing and characterizing of human melanoma tumorspheres cultured with rotating bioreactors as promising alternative for in vitro tumor modeling (Fig. 1 ). 2. Materials and methods 2.1. Cell lines Two human melanoma cell lines were used – primary (FM55p) and malignant (WM266-4) purchased from ESTDAB Melanoma Cell Bank (Tübingen, Germany) and cultured according to the manufacturer recommendations as we previously described (Karimi et al., 2020 ). 2.2. Spheroids pre-incubation Seven days before the experiment, FM55p and WM266-4 cells (10 3 cells in 250 µL/per well) were seeded into 96-well U-bottom SPL3D™ Cell Floater plates (SPL Life Sciences Co., Ltd., Pocheon-si, South Korea). The goal of this step was to ensure better models reproducibility of the models by pre-cultivating the cells until they initially form spheroids before transferring to the bioreactor. During this initial period of incubation (37 o C, 5% CO 2 ), medium renewal was performed every two days. Structures cultured in bioreactor are further referred to as tumorspheres , while plate-based models are further referred to as spheroids . 2.3. Large-sized spheroids culturing Culture of large-sized spheroids was performed using the ClinoStar® (CelVivo, Inc, Chevy Chase, MD, USA) system designed for generating in vitro models under conditions resembling the tumor environment in a living organism. For this purpose, one day before the transferring of spheroids, the bioreactor chamber was equilibrated with 25 mL of sterile water (4 o C, overnight) to hydrate the moisture beads. Then, cell culture chamber was washed twice with complete medium. Finally, the chamber was filled with ~ 6 mL of culture medium and placed in the ClinoStar® incubator (37 o C, 5% CO 2 ) for at least 2 hours with gentle rotation (15 rpm). All procedures involving injection, replacement and rising of the medium were performed with using a 20 mL syringe with a needle to minimize the risk of contamination. After equilibration, the medium was removed and 5 mL of fresh complete medium was added. Afterwards, 75 to 96 spheroids (per one bioreactor) were inoculated. The cell chamber was filled with culture medium to avoid the formation of air bubbles. During the experiment, the rotation speed ranged from 15 to 35 rpm, and the adjustment depended on the size of the tumorspheres separately for each bioreactor. The day after the spheroid transfer, all aggregates were removed from the chamber to maintain homogeneity and optimal growing conditions. The medium was replaced every 2 days, and the bioreactor was replaced after 14 days, according to the manufacturer’s recommendations. Control spheroids (75–96 per group), growing in the 96-well U-bottom plates were also transferred to new plates after day 14 and subjected to medium replacement every 2 days. 2.4. Spheroids imaging - morphology Each week, starting on day 0, growth, shape, and physiological activity were evaluated. For this purpose, a Celigo bright-field (BF) and fluorescence imaging cytometer (Nexcelom Biosciences, Lawrence, MA, USA) was used. Prior to each imaging, spheroids were transferred to the SPL3D™ Cell Floater plates and washed twice with culture medium, to remove any cell fragments and debris . The acquisition setup was adjusted to the cell line type, shape and size of the spheroid, and included pre-filtering to determine analysis parameters such as colony diameter, tumorsphere area and tumorsphere intensity range to exclude artefacts. 2.5. Necrotic core staining Propidium iodide staining of the necrotic core ( PI , Merck & Co., Inc., Kenilworth, NJ, USA) is one of the most widely used techniques to visualize necrotic cells located deep in the spheroid structure (Dini et al., 2016 ). For this purpose, the spheroids were stained with a 0.5 mg/mL PI solution in Mg 2+ /Ca 2+ -free PBS. After transfer to a 96-well U-bottom plate and complete removal of the medium, the spheroids were resuspended in 50 µL of PBS, and then 50 µL of PI solution was added. After 30 min, the spheroids were washed three times with PBS and finally resuspended in 100 µL of PBS. Fluorescence imaging was performed using a Celigo cytometer, with a pre-filtering step as described above. The resulting images were analyzed via Celigo 5.3.0.0 and FIJI software. 2.6. In-depth tumorsphere analysis with confocal imaging For post-experimental sectioning, spheroids were fixed in 2.5% of glutaraldehyde (ThermoFisher Scientific) and snap frozen with liquid nitrogen in Tissue Freezing Medium (Leica Microsystems, Wetzlar, Germany). The spheroids were then cut into 5 µm thick sections in the CM 1900 cryostat (CM 1900; Leica Microsystems) at − 20°C. Sections were mounted on poly-lysine coated coverslips and examined using a CKX 41 Olympus inverted phase-contrast microscope (Olympus, Tokyo, Japan). Entire spheroids imaging was achieved by immunolabeling and clearing using CytoVista 3D culture Clearing kit (ThermoFisher Scientific, Waltham, MA, USA). Fixed spheroids were permeabilized in increasing concentrations of methanol at 4°C: 50% methanol in PBS, 80% methanol in deionized water, 100% methanol. Subsequently, they were washed in 20% DMSO/methanol, then in: 80% MeOH/H 2 O; in 50% MeOH/PBS; 100% PBS, and finally in PBS with 2% Triton X-100. Samples were incubated CytoVista Antibody Penetration Buffer for 30 minutes and blocked in CytoVista Blocking Buffer for 24 hours at 4°C. Spheroids were incubated with primary antibody diluted in CytoVista Antibody Dilution Buffer (VEGF-β Monoclonal Antibody, ThermoFisher Scientific) for 24 hours at 4°C. The spheroids were then washed and incubated with Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (ThermoFisher Scientific) for 24 hours at 4°C. Cell nuclei were counterstained with Hoechst 33342 (ThermoFisher Scientific), washed and dehydrated with increasing concentrations of methanol (50% MeOH/PBS; 80% MeOH/H 2 O; 100% MeOH). After methanol removal, the spheroids were placed in an 8-well chambered µ-Slide 8 coverslip (Ibidi, Gräfelfing, Germany) and cleared with 3D Cell Culture Clearing Reagent. z-stacks of spheroids were obtained with FV-1000 confocal microscope (Olympus Corporation, Tokyo, Japan) and reconstructed with FIJI software. 2.7. Single-cell tumorspheres analysis with spectral flow cytometry Between 25–50 tumorspheres per group have been collected and precipitated. Subsequently, growing medium was removed and 200 µL of Accutase (Merck & Co., Inc., Kenilworth, NJ, USA) was added to ensure structures disintegration (5 min, 37 o C). After complete cell detachment 800 µL of complete growing medium was added and samples were centrifuged (250 g , 5 min). Cells were reconstituted in 1 mL of PBS/5% FBS and 30 µL sample was collected for cell counting and trypan blue exclusion staining and counted with Luna-II Automated Cell Counter (Logos Biosystems, Dongan-gu Anyang-si, Gyeonggi-do 14055 South Korea). For spectral flow cytometry following antibodies from BioLegend (San Diego, CA, USA) were used: VE-cadherin (PE anti-human CD144, clone BV9), E-cadherin (PE/Dazzle TM 594 anti-mouse/human CD324, clone DECMA-1, isotype: Rat IgG1, κ), CD44 (BV711 anti-mouse/human, clone IM7, isotype: Rat IgG2b, κ), Vimentin (AF647 anti-Vimetin, clone W16220A, isotype Rat IgG2a, κ), MCAM (FITC anti-human CD146, clone P1H12, isotype: Mouse IgG1, κ). For viability, Annexin-V-pacific blue conjugate (Life-technologies, Eugene, OR, USA) and PI were used. As isotype control following antibodies were used: PE clone MOPC-173 mouse IgG2a, κ; PE/Dazzle TM 594, clone RTK2071, isotype: Rat IgG1, κ; BV711 clone IM7, Rat IgG2b, κ; AF647 clone RTK2758, isotype Rat IgG2a, κ; FITC clone MOPC-21, mouse IgG1, κ. All single and mixed (A-V + PI and CD44 + MCAM + Vimentin) were performed according to manufacturer protocol without any modifications. Samples were analyzed with ID7000 Sony Spectral Flow cytometer (Sony Biotechnology, San Jose, CA, USA). The ID7000 used in this study was equipped with four lasers 405/488/561/637 nm, PMT gains/voltages were independently adjusted for each laser. For each sample, a total of 10 4 cells were analyzed. Before analysis, the Sony ID7000 was calibrated using alignment checks (Sony Biotechnology Inc AlignCheck Flow Cytometer Alignment Beads 10 7 /mL 10 µm, 2 mL, cat. no AE700510) and the 8-peak performance beads (Sony Biotechnology Inc 8 Peak Bead cat. no AE700522, 10 7 /mL 3.1 µm, 5 mL cat. no AE700510), following the instrument supplier’s guidelines. 2.8. Statistical and protein interactions analysis GraphPad Prism v.8 Software (GraphPad Software, San Diego, CA, USA) was used for statistical interpretation of obtained data. Results are presented as means with standard deviation, with p values corresponding to: (*) p ≤ 0.05, (**) p ≤ 0.01, (***) p ≤ 0.001, and (****) p ≤ 0.0001. For prediction of common protein interactions two proteins were analyzed by means of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (Szklarczyk et al., 2019 ). 3. Results 3.1. Evaluation of growth and morphology changes in primary and metastatic melanoma Significant differences in FM55p tumorspheres structure and growth were observed since 14th day of experiment (Fig. 2 A-C and table S2, supplementary information ). Exceptionally big-sized FM55p tumorspheres after 28 days reached the unique diameter ~ 2 mm (2000 ± 65 µm) and a perimeter exceeding 7 mm. Interestingly, complete decomposition of control spheroids was observed as a consequence of widespread cell death, as revealed in living cells counting (Fig. 2 D). Doubling-time (DT) calculations based on living cells population and tumorspheres volume (Karimi et al., 2023 ) showed that those cultured in bioreactor had significantly ( p = 0.004 and p = 0.0007 respectively, Fig. 2 E) reduced DT when compared to the control. As shown in Fig. 3 , any disintegration or spheroid death was not observed for metastatic melanoma models. However, meaningful differences were noticed in growth and morphology starting from first week after inoculation in bioreactor (Fig. 3 A-C and table S3 supplementary information ). Tumorspheres reached an average diameter of 1432 ± 54 µm being approximately 33% larger than control. Enhanced growth was associated with significantly ( p ≤ 0.0001) increased number of living cells found in bioreactor-based tumorspheres and 25–50% shortened doubling time depending on cell number or tumor volume calculations (Fig. 3 D /E ). 3.2. In-depth tumorspheres analysis Remarkably expressed morphological differences between tested methods inspired us to verify how does the external changes links to the tumor inside. To date, in-depth in vitro 3D tumor assessment is rarely executed, mostly due to numerous challenges of such examination (Han et al., 2021b ). Confocal bright-field (BF) visualization of tumors cross-sections as well as fluorescent imaging of whole tumors (Fig. 4 ) was performed at day 14 when FM55p control spheroids disintegration was noticed and the aim of this imaging was to identify possible reasons of this phenomenon. Internal structure of tumorspheres from bioreactor was compact with high cell density and only single stripped of cells areas (Fig. 4 A /E , marked with white arrows). We did not identify any changes in compactness with increasing section depth. Notably, high tumor structure homogeneity is an important future of clinically observed solid tumors and conversely to traditional models was found to be achievable in bioreactor-based models. Despite the satisfactory external morphology of WM266-4 control group as well, we found there much more structural diversities e.g. , empty spaces and looser cells-cell connections (Fig. 4 D). These observations were in line with necrotic core staining that revealed increased dead cells content located internally. In particular, PI signal from necrotic cells in both (FM55p and WM266-4) control spheroids was intensive and widespread among tumors (Fig. 4 B /G ). An increase in fluorescence was found in larger clusters of organized cells, but PI-positive cells were also detected in the area of single cell layers. Conversely, tumorspheres from bioreactor revealed only trace PI + cells content ( p = 0.002 and p = 0.0004 for FM55p and WM266-4 respectively) localized mostly in form of small foci in midsection. Subsequently, we visualized the expression of vascular endothelial growth factor type β (VEGF-β) (Fig. 4 C /F ). VEGF-β is one of major hallmarks describing development of the tumor tissue, providing an information about the capability to vascularization, crucial in predicting tumor growth (Claffey et al., 1996 ; Wierzbicki et al., 2018 ). The 3D z-stack reconstructed images revealed, that externally located cells show high expression of VEGF-β both in clinoreactor and the plate-based control. Quantitative fluorescence analysis showed that signal intensity from VEGF-β, after prior normalization per structure volume, in tumorspheres was significantly ( p ≤ 0.05) higher when compared to the control. More interestingly, no statistical differences were observed between primary and metastatic tumorspheres group, showing only slight variances ( p = 0.89). 3.3. Protein expression and epithelial-to-mesenchymal (EMT) transition Distribution analysis of FM55p living cells evaluated with Annexin-V and PI single-cell staining showed notably higher A-V (+) and PI (+) cells content (Fig. 5 A /C ) when compared to the tumorspheres from bioreactors (Fig. 5 B /C ), especially at day 14. In general, living cells content assessed with flow cytometry was in line with trypan-blue exclusion staining, indicating elevated number of dead cells in control spheroids. Increase of PI + cells number in tumorspheres at day 21 is probably an aftermath of larger tumor size when compared to day 14, however as we showed previously, it did not impaired or inhibited tumorspheres growth (Fig. 2 A /B ). Protein expression analyses performed with reference to the level of respective proteins in 2D cell cultures (Fig. 5 D) showed low levels of E-cadherin and VE-cadherin (< 2.5%), hence did not justify quantitative analysis of epithelial-to-mesenchymal transition (EMT), which is one of major hallmarks of cancer progression and metastatic sites formation. Interestingly, meaningful alterations in proteins expression were found in all of other markers. Progressing over time changes in expression of MCAM and CD44 in tumorspheres were identified mostly at day 21 ( p = 0.0005 for MCAM and p = 0.0010 for CD44) while vimentin level was downregulated to 3.6 ± 1.6% at day 21, probably due to suppressing the EMT (Dou et al., 2014 ) (Fig. 5 E). The number of living cells in WM266-4 tumorspheres was significantly higher when compared not only to the control, but also in reference to FM55p (Fig. 6 A /C ). Conversely to the primary melanoma, WM266-4 showed reduced PI signal, especially at day 21 when FM55p and WM266-4 control revealed an progressing over time increase of necrotic cells. Based on protein expression analyses, levels of MCAM, vimentin and CD44 expression were significantly elevated when compared to control. Similarly to previously observed changes, WM266-4 tumorspheres also downregulated vimentin expression during last week of growth, but interestingly also slight reduction of CD44 level was noted (Fig. 6 D /E ). 3.4. Protein co-expression and STRING analysis Proteome changes of desired makers may additionally allow to make a step forward in understating interactions and their implications in tumor in vitro modeling especially when their up- or downregulation is linked with other molecular markers. For this purpose we firstly examined the CD44, MCAM, and vimentin simultaneous expression in both of tested melanomas. As shown in Fig. 7 A, both FM55p and WM266-4 tumorspheres cultured in bioreactor showed overall significantly higher proteins co-expression than control groups, regardless of the markers being considered. Moreover, plate-based spheroids did not differ significantly between each other ( p = 0.8629), ultimately showing limitations and disadvantages of traditional model. Interestingly, statistical analyses revealed that in primary melanoma simultaneous expression occurs more frequently when compared to the metastatic models (88% vs. 60%) but any specific correlation was not identified among tested subgroups including CD44 + vimentin, CD44 + MCAM and vimentin + MCAM respectively. STRING analysis (Fig. 7 B) of co-expressed proteins revealed that dual upregulation of CD44 and MCAM is frequently associated with various interactions mediated by laminins (LAMA1-5; LAMB1-3; LAMC1-3) and collagens (COL4A3 and COL15A1). Conversely, any correlation with vimentin as a third of co-expressed protein was not found. Therefore, based on protein profile of analyzed tumorspheres it can be concluded, that higher development of single cells translates to higher maturity of entire system. 4. Discussion Implementing a bioreactor offers several technological advantages, stating form suspending the cells in a static orbit during rotation that provides protective function against shear stress. Increased access to nutrients and oxygen ensures a better supply for the vital elements, while effective waste elimination prevents undesirable cell poisoning as a result of cumulated metabolic products. Consequently, strengthening of cell-to-cell interactions is observed and advanced model development can be achieved. Discussed morphological and physiological changes plays an important role in the intact expression of proteins and genes, thus evolving TME leads to achievement more clinically-like tumor models (El Harane et al., 2023 ; Pittman, 2013 ). The obtained tumorspheres showed unprecedented size and more importantly, preserved proliferation capacity and biochemical functions during month-long experiment on population of 1100 tumorspheres originated from primary and metastatic melanoma. While the diameter of most commonly used spheroids is in the range of 250–700 µm (Singh et al., 2020 ) and usually does not reach 1000 µm (Mahmoud et al., 2023 ), in our study we obtained large 3D tumorspheres with over 2 mm (2.3 mm maximum) diameter corresponding to 5.0 ± 0.9 mm 3 of the estimated volume, which was previously impossible in vitro (WU et al., 2024 ; Zanoni et al., 2016 ). Apart from the morphological maturity that was enabled due to higher cell viability, tumor microenvironment was found to be in higher development level as well. Increased VEGF-β expression proved that vascularization capacity is enhanced, thus in vivo angiogenesis shows a great prospects for undergo efficient modulation with discussed models (Silvestre et al., 2003 ). Co-expression MCAM and CD44 found in over 95% (FM55p) and 65% (WM266-4) of cells is indirect but solid evidence that tumorspheres from bioreactor can also restore or acquire the tissue-like functions. Our predictions for CD44 and MCAM proteins revealed the involvement of two extracellular matrix (ECM) protein pathways: the laminin 332 gene family and type IV collagen alpha chain (COL4A). LM 332 (formerly called LM 5), as a specific variant of laminin glycoprotein, is a heterotrimer of α3, β3 and γ2 chains, encoded by LAMA3, LAMB3 and LAMC2. LM332 forms the epithelial-basement membrane (BM) and supports the main function of epithelial tissue such as formation, healing and regeneration. LM332 is also considered a hallmark of cancer development, and its expression is impaired in various cancer cells (invasive mammary, colon, melanoma, and sarcoma) (Rousselle & Scoazec, 2020 ). COL4A is also a major component of BM, and its role in tumor angiogenesis and progression has been widely investigated (Zeng et al., 2020 ). Overexpression of these two ECM components was observed in our study as strong intercellular interactions in the tested spheroids, which made cell separation difficult (it was necessary to use accutase to digest the spheroids) and constituted a mechanical barrier for dyes and antibodies used in the visualization of spheroids. Similar problems regarding the Ab-based detection in 3D models were previously reported by Mitrakas (Mitrakas et al., 2023 ), thus it was not possible to detect VEGF-β deeper in whole tumorsphere structure, enabling only boundary Ab penetration. In general, discussed approach can be easily and repeatedly used for other cellular models. Low SD’s with highly representative tumorspheres population were observed not only within the experimental groups, but more importantly between separate experimental runs. Due to high morphological and physiological development, presented models can serve not only as convenient in further in vitro research, but principally as standalone biological model that may provide complementary data at various stages of drug development process. Possible implications of presented in vitro models may primarily fill the gap in biological examination in preclinical stage, but also shows great potency to be applicable for analyzes with clinically used diagnostic systems. This includes particular molecular imaging specifically for research into the development of new radiopharmaceuticals and multiphoton imaging in novel positron emission tomography (PET) and single photon emission computed tomography (SPECT) devices and may provide completely new view of the field, regarding intratumoral markers expression, heterogeneity and metabolism based on currently developed approaches (Shimazoe & Uenomachi, 2022 ; Uenomachi et al., 2022 ). Available to date in vitro models due to numerous features do not support such interdisciplinary examination. Our findings presented in this article have proven that it is possible to design and develop remarkable biological models which can bridge the gap between in vitro and in vivo systems and become the basis of future cancer research. The results showed important characteristics of spheroid properties obtained for the first time in millimeter-range size, with retained growth ability over several weeks of culture and most importantly highly established TME. Expanding of 3D spheroids or organoids themselves is a major strategy used in the study of cellular components. Presented results shows a great prospects to develop hybrid 3D cellular systems combining the features of spheroids and organoids for preclinical research. Declarations Founding This work was supported by the National Science Centre of Poland through grants no. 2022/47/I/NZ7/03112 to E.Ł.S., the Jagiellonian University via projects CRP/0641.221.2020, SciMat and qLife Priority Research Areas budget under the program Excellence Initiative - Research University. Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authors contributions Conceptualization: K.W., E.Ł.S; Formal analysis K.W., M.D.-K., M.W.; Funding acquisition: E.Ł.S.; Investigation: K.W., M.D.-K., M.W., E.Ł.S.; Methodology: K.W., M.D.-K., M.W., E.Ł.S.; Project administration: E.Ł.S.; Resources: E.Ł.S; Supervision: E.Ł.S.; Visualization: K.W., M.W.; Writing - original draft: K.W., E.Ł.S; and Writing - review & editing: K.W., M.D.-K., E.Ł.S. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4884972","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":338005381,"identity":"c880bd76-0072-4458-8072-4b1bede872e0","order_by":0,"name":"Kamil Wawrowicz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYBACCSA+8ADGS2BgkAPyGRh4gBw+fFoSkLQYw7Ww4dHCkIAkkNgAIvFpkWw/nQi05bC8we3DxyQe1NSlbzh4gPHB2zaGPFxapHlyN4C0GG44l5YmkXCMDcg9wGw4t42hGJcWOQawltuM287wmN1IbACZcIBNmreNIbENlxb+t2At9lAtEukGBw6w/8anRVoCYksiVItBAlALGzM+LZIzQLYY/E/ef4Yt/UfCsQTDmQcONkvOOSeB0y8S53M3f/hQkWY7s4f5sOGPmjp5vhuHD354U2aTx49DCwQYoJhysAFEJuDVgQr4G8AUKVpGwSgYBaNgeAMAW1tiHK4sn9wAAAAASUVORK5CYII=","orcid":"","institution":"Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland","correspondingAuthor":true,"prefix":"","firstName":"Kamil","middleName":"","lastName":"Wawrowicz","suffix":""},{"id":338005382,"identity":"60a4ebf3-113b-4ee1-a8c3-f4c238c277f7","order_by":1,"name":"Martyna Durak-Kozica","email":"","orcid":"https://orcid.org/0000-0002-5275-6462","institution":"Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland","correspondingAuthor":false,"prefix":"","firstName":"Martyna","middleName":"","lastName":"Durak-Kozica","suffix":""},{"id":338005383,"identity":"07334df6-20f1-4da7-98ac-c240fc6a3b5e","order_by":2,"name":"Mateusz Wierzbicki","email":"","orcid":"https://orcid.org/0000-0003-3623-8929","institution":"Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland","correspondingAuthor":false,"prefix":"","firstName":"Mateusz","middleName":"","lastName":"Wierzbicki","suffix":""},{"id":338005384,"identity":"67931dd0-cc2d-4755-8271-f5724ac820f2","order_by":3,"name":"Ewa Ł. Stępień","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYPACCQYG9gYGAwYGZiCHuYGQcsYGsBaeAzAtjERpAVmUACKJ0MLPwP78wccdFnLmkm8MChgqrBMbpBvxa5Fs4DFsnHlGwthydo6BAcOZ9MQGmYP4tRgc4GFs5m2TSNxwG6iFse1wYoNEIn4t9gfYHzb/bZOo33DzDFDLPyK0AAPJsJmxTSLB4AYPUEsDEVokDvMYzuw9I2G44UxagUHCsXTjNkJ+4W9vf/Dh5446eYPjh7cZfKixlu2Xbj6AVwtyRLAZJIBICfwaIACqhfkBxK3EaBkFo2AUjIKRBAD/xkZ1jkruYQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3589-1715","institution":"Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Kraków, Poland","correspondingAuthor":true,"prefix":"","firstName":"Ewa","middleName":"Ł.","lastName":"Stępień","suffix":""}],"badges":[],"createdAt":"2024-08-09 06:59:24","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4884972/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4884972/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62322336,"identity":"8d3bbf48-9591-4daa-8cfd-9604705e8b87","added_by":"auto","created_at":"2024-08-13 02:03:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161811,"visible":true,"origin":"","legend":"\u003cp\u003eStudy design and outline.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/49dde3e822ace75507b0c984.png"},{"id":62321884,"identity":"7ef02717-e55d-4395-a13a-75f7d0bd0fce","added_by":"auto","created_at":"2024-08-13 01:55:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":473428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrimary melanoma (FM55p) tumorspheres growth\u003c/strong\u003e. Morphological changes (\u003cstrong\u003eA\u003c/strong\u003e) and tumor growth rates differences between control and tested group (\u003cstrong\u003eB\u003c/strong\u003e) – due to the decomposition of control spheroids data presented in part A does not contain relevant measurements for reference spheroids; example of bright-field images used for analysis with imaging cytometry (\u003cstrong\u003eC\u003c/strong\u003e); distribution of living cells in tumorspheres (\u003cstrong\u003eD\u003c/strong\u003e); spheroid doubling time calculations based on single-cell analysis and tumor volume (\u003cstrong\u003eE\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/aba7b7676d4c097e54d1ffaf.png"},{"id":62321878,"identity":"61b9615d-5e89-42fa-b589-56be82467051","added_by":"auto","created_at":"2024-08-13 01:55:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":499017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetastatic melanoma (WM266-4) tumorspheres growth\u003c/strong\u003e. Morphological changes (\u003cstrong\u003eA\u003c/strong\u003e) and tumor growth rates differences between control and tested group (\u003cstrong\u003eB\u003c/strong\u003e); example of bright-field images used for analysis with imaging cytometry (\u003cstrong\u003eC\u003c/strong\u003e); distribution of living cells in tumorspheres (\u003cstrong\u003eD\u003c/strong\u003e); spheroid doubling time calculations based on single-cell analysis and tumor volume (\u003cstrong\u003eE\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/043319b5489f7be3d3aaee0e.png"},{"id":62321877,"identity":"b3d1bc2d-4c14-441a-9994-e178e46f2ebf","added_by":"auto","created_at":"2024-08-13 01:55:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":223714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn-depth tumorspheres visualization.\u003c/strong\u003e Parts \u003cstrong\u003eA-C\u003c/strong\u003ecorresponds to FM55p, while \u003cstrong\u003eD-G\u003c/strong\u003e to WM266-4. Bright-field tumorspheres imaging followed by confocal visualization of top and middle part cross-sections (\u003cstrong\u003eA, D, E\u003c/strong\u003e); propidium iodide fluorescent images and integrated fluorescence (\u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eG\u003c/strong\u003e); z-stack reconstructed confocal images of VEGF-β expression (\u003cstrong\u003eC,F\u003c/strong\u003e). Scale bar in images \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003ecorresponds to 500 µm. Due to the decomposition of control spheroids data presented in part C does not contain relevant measurements for reference spheroids\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/8f326afcc8b2186ebbb0499b.png"},{"id":62321880,"identity":"34c7f7f6-6d83-4a9a-b420-cb05e5274477","added_by":"auto","created_at":"2024-08-13 01:55:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":737088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpectral flow cytometry single-cell analyses – FM55p.\u003c/strong\u003e Annexin-V- and propidium iodide-positive cells distribution in control (\u003cstrong\u003eA\u003c/strong\u003e) and bioreactor-based tumorspheres (\u003cstrong\u003eB\u003c/strong\u003e); examples of spectra used for viability analyses (\u003cstrong\u003eC\u003c/strong\u003e); desired protein expression in 2D cell culture and 3D plate/bioreactor-based models (\u003cstrong\u003eD\u003c/strong\u003e) with representative examples spectra acquired at day 14 for each marker (\u003cstrong\u003eE\u003c/strong\u003e). Black frames in figure \u003cstrong\u003eE \u003c/strong\u003eindicates part of spectrum used for gating and further quantitative analyses presented in right side histograms. Due to the decomposition of control spheroids data presented in part D does not contain relevant measurements for reference spheroids performed at day 21.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/82fb415d8b793921ed219a75.png"},{"id":62321881,"identity":"7e67ff5d-98d0-4d10-8504-6dfdb0c6d230","added_by":"auto","created_at":"2024-08-13 01:55:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":749896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpectral flow cytometry single-cell analyses – WM266-4.\u003c/strong\u003e Annexin-V- and propidium iodide-positive cells distribution in control (\u003cstrong\u003eA\u003c/strong\u003e) and bioreactor-based tumorspheres (\u003cstrong\u003eB\u003c/strong\u003e); examples of spectra used for viability analyses (\u003cstrong\u003eC\u003c/strong\u003e); desired protein expression in 2D cell culture and 3D plate/bioreactor-based models (\u003cstrong\u003eD\u003c/strong\u003e) with representative examples of spectra acquired at day 14 for each marker (\u003cstrong\u003eE\u003c/strong\u003e). Black frames in figure \u003cstrong\u003eE \u003c/strong\u003eindicates part of spectrum used for gating and further quantitative analyses presented in right side histograms.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/7a7a0502804139c837b8404c.png"},{"id":62322337,"identity":"1320f2bb-c1f6-4e50-9525-03aaa5c85819","added_by":"auto","created_at":"2024-08-13 02:03:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":314236,"visible":true,"origin":"","legend":"\u003cp\u003eCD44, vimentin and MCAM simultaneous expression in FM55p and WM266-4 obtained structures – simultaneous expression of CD44/vimentin/MCAM was assessed with mixed (triple) staining and analyzed with spectral flow cytometry (\u003cstrong\u003eA\u003c/strong\u003e); STRING analysis of protein-protein interaction network based on identified simultaneous expression – confidence level equal or higher than 0.700 (Szklarczyk et al., 2019) (\u003cstrong\u003eB\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/2fa43c1d1cdd64d78e6e1314.png"},{"id":62323301,"identity":"368ba908-083d-4abc-95e2-8282937dd246","added_by":"auto","created_at":"2024-08-13 02:11:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3856959,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4884972/v1/717424a1-ac3e-4fb3-b17b-4d365cbb66ca.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDeveloping tumor microenvironment in rotating human melanoma cell cultures: study of novel preclinical model\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Significance","content":"\u003cp\u003eOne of the most important and unmet challenges in drug development is an \u003cem\u003ein\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e-\u003cem\u003ein vivo\u003c/em\u003e gap caused by inadequate cell models. In this study,\u0026nbsp;we identified and described the differences in tumor development and progression under different culture conditions. We focused on morphological and physiological features at various cellular levels, which are currently a less explored area of research oriented towards new approaches to biological modeling. This study is one of the first to examine melanoma in-depth development and assessment \u003cem\u003ein vitro\u003c/em\u003e and indicates that the resulting models shows favorable characteristics compared to those currently used in preclinical studies.\u003c/p\u003e"},{"header":"Research Highlights","content":"\u003cul\u003e\n \u003cli\u003eRotating human melanoma cell cultures shows a great prospects to make a step forward in preclinical tumor modeling.\u003c/li\u003e\n \u003cli\u003eLonger culturing leads to improved morphology and TME, thus enabling to achieve more clinically-relevant melanoma models.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eSignificant differences at various cellular levels between \u003cem\u003ein vitro, in vivo\u003c/em\u003e and clinical tumor models are one of the major challenges hindering the progress in cancer diagnosis and therapy. \u003cem\u003eIn vitro\u003c/em\u003e assessment, as preliminary evaluation step, has a critical impact reflecting further stages success rates as they are used not only for \u003cem\u003ein vitro\u003c/em\u003e evaluation, but are subsequently applied for tumor inoculation in animal model (Dowden \u0026amp; Munro, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Law et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Stępień et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApplicability range of traditional cell cultures enables only basic research, such as drug uptake or cell survival. Two-dimensional growth in unnatural flattened shape impairs the cell morphology, while uniform distribution of nutrients and medium results in high homogeneity. Synchronized cell cycles contribute to changes in cellular response and growth, leading to abnormal outcomes. Furthermore, cell-to-cell interactions, limited to the boundary of the cell outline, lead to changes in genes and protein expression, also affecting intercellular signaling, and more importantly the development of tumor microenvironment (TME). Consequently, these models due to numerous morphological and physiological shortcomings do not effectively assembles the clinical tissues (Kapałczyńska et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIntroducing a spatial, three-dimensional (3D) cell culture models, classified as spheroids (cell culture-derived) or organoids (tissue-derived), offer an advantages over the traditional ones (Han et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). This environment permits cells to preserve or restore their inherent morphological characteristics, thereby facilitating the establishment of a sophisticated cancer microenvironment (\u003cem\u003emilieu\u003c/em\u003e) exhibiting a higher degree of complexity relative to its two-dimensional (2D) counterpart. More pronounced cell differentiation coupled with improved intercellular interactions \u003cem\u003evia\u003c/em\u003e ions, small molecules and electrical signals, makes them more suitable for simulating tumor tissue (Durak-Kozica et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The expression of genes and proteins can be similar to \u003cem\u003ein vivo\u003c/em\u003e conditions (Jensen \u0026amp; Teng, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and may allow the introduction tumor growth factors, such as gravity and mechanical stimulation, which are unavailable in 2D cultures (Guillaume et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The main disadvantage of typical 3D cell cultures is the limited culture time (~\u0026thinsp;several days) and poor model reproducibility, having a primary importance for the extensive preclinical evaluation various types of novel therapeutic approaches (Wawrowicz et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Żelechowska-Matysiak et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, reduced nutrients and oxygen penetration, insufficient elimination of waste and metabolic products are another limitations of typically used methods. These features as well as economic aspects do not offer encouraging benefits that could support their wide implementation into preclinical protocols (Katt et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of major requirements that could lead to improve the effectiveness of drug discovery is a holistic approach. Redefining model types and their use must be the first step towards improving the quality of preclinical evaluation. Furthermore, recent FDA regulations also force the replacement of \u003cem\u003ein vivo\u003c/em\u003e models with advanced 3D cell cultures (Meredith Wadman, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), emphasizing the importance of rapid progress in such systems. Consequently, there is an urgent need to develop and characterize highly advanced 3D models for preclinical research, both upgrading \u003cem\u003ein vitro\u003c/em\u003e models and/or replacing animals. Addressing those needs, with this study we aimed for developing and characterizing of human melanoma tumorspheres cultured with rotating bioreactors as promising alternative for \u003cem\u003ein vitro\u003c/em\u003e tumor modeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell lines\u003c/h2\u003e \u003cp\u003eTwo human melanoma cell lines were used \u0026ndash; primary (FM55p) and malignant (WM266-4) purchased from ESTDAB Melanoma Cell Bank (T\u0026uuml;bingen, Germany) and cultured according to the manufacturer recommendations as we previously described (Karimi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Spheroids pre-incubation\u003c/h2\u003e \u003cp\u003eSeven days before the experiment, FM55p and WM266-4 cells (10\u003csup\u003e3\u003c/sup\u003e cells in 250 \u0026micro;L/per well) were seeded into 96-well U-bottom SPL3D\u0026trade; Cell Floater plates (SPL Life Sciences Co., Ltd., Pocheon-si, South Korea). The goal of this step was to ensure better models reproducibility of the models by pre-cultivating the cells until they initially form spheroids before transferring to the bioreactor. During this initial period of incubation (37\u003csup\u003eo\u003c/sup\u003eC, 5% CO\u003csub\u003e2\u003c/sub\u003e), medium renewal was performed every two days. Structures cultured in bioreactor are further referred to as \u003cem\u003etumorspheres\u003c/em\u003e, while plate-based models are further referred to as \u003cem\u003espheroids\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Large-sized spheroids culturing\u003c/h2\u003e \u003cp\u003eCulture of large-sized spheroids was performed using the ClinoStar\u0026reg; (CelVivo, Inc, Chevy Chase, MD, USA) system designed for generating \u003cem\u003ein vitro\u003c/em\u003e models under conditions resembling the tumor environment in a living organism. For this purpose, one day before the transferring of spheroids, the bioreactor chamber was equilibrated with 25 mL of sterile water (4\u003csup\u003eo\u003c/sup\u003eC, overnight) to hydrate the moisture beads. Then, cell culture chamber was washed twice with complete medium. Finally, the chamber was filled with ~\u0026thinsp;6 mL of culture medium and placed in the ClinoStar\u0026reg; incubator (37\u003csup\u003eo\u003c/sup\u003eC, 5% CO\u003csub\u003e2\u003c/sub\u003e) for at least 2 hours with gentle rotation (15 rpm). All procedures involving injection, replacement and rising of the medium were performed with using a 20 mL syringe with a needle to minimize the risk of contamination. After equilibration, the medium was removed and 5 mL of fresh complete medium was added. Afterwards, 75 to 96 spheroids (per one bioreactor) were inoculated. The cell chamber was filled with culture medium to avoid the formation of air bubbles. During the experiment, the rotation speed ranged from 15 to 35 rpm, and the adjustment depended on the size of the tumorspheres separately for each bioreactor. The day after the spheroid transfer, all aggregates were removed from the chamber to maintain homogeneity and optimal growing conditions. The medium was replaced every 2 days, and the bioreactor was replaced after 14 days, according to the manufacturer\u0026rsquo;s recommendations. Control spheroids (75\u0026ndash;96 per group), growing in the 96-well U-bottom plates were also transferred to new plates after day 14 and subjected to medium replacement every 2 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Spheroids imaging - morphology\u003c/h2\u003e \u003cp\u003eEach week, starting on day 0, growth, shape, and physiological activity were evaluated. For this purpose, a Celigo bright-field (BF) and fluorescence imaging cytometer (Nexcelom Biosciences, Lawrence, MA, USA) was used. Prior to each imaging, spheroids were transferred to the SPL3D\u0026trade; Cell Floater plates and washed twice with culture medium, to remove any cell fragments and \u003cem\u003edebris\u003c/em\u003e. The acquisition setup was adjusted to the cell line type, shape and size of the spheroid, and included pre-filtering to determine analysis parameters such as colony diameter, tumorsphere area and tumorsphere intensity range to exclude artefacts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Necrotic core staining\u003c/h2\u003e \u003cp\u003ePropidium iodide staining of the necrotic core (\u003cem\u003ePI\u003c/em\u003e, Merck \u0026amp; Co., Inc., Kenilworth, NJ, USA) is one of the most widely used techniques to visualize necrotic cells located deep in the spheroid structure (Dini et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For this purpose, the spheroids were stained with a 0.5 mg/mL PI solution in Mg\u003csup\u003e2+\u003c/sup\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e-free PBS. After transfer to a 96-well U-bottom plate and complete removal of the medium, the spheroids were resuspended in 50 \u0026micro;L of PBS, and then 50 \u0026micro;L of PI solution was added. After 30 min, the spheroids were washed three times with PBS and finally resuspended in 100 \u0026micro;L of PBS. Fluorescence imaging was performed using a Celigo cytometer, with a pre-filtering step as described above. The resulting images were analyzed \u003cem\u003evia\u003c/em\u003e Celigo 5.3.0.0 and FIJI software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. In-depth tumorsphere analysis with confocal imaging\u003c/h2\u003e \u003cp\u003eFor post-experimental sectioning, spheroids were fixed in 2.5% of glutaraldehyde (ThermoFisher Scientific) and snap frozen with liquid nitrogen in Tissue Freezing Medium (Leica Microsystems, Wetzlar, Germany). The spheroids were then cut into 5 \u0026micro;m thick sections in the CM 1900 cryostat (CM 1900; Leica Microsystems) at \u0026minus;\u0026thinsp;20\u0026deg;C. Sections were mounted on poly-lysine coated coverslips and examined using a CKX 41 Olympus inverted phase-contrast microscope (Olympus, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eEntire spheroids imaging was achieved by immunolabeling and clearing using CytoVista 3D culture Clearing kit (ThermoFisher Scientific, Waltham, MA, USA). Fixed spheroids were permeabilized in increasing concentrations of methanol at 4\u0026deg;C: 50% methanol in PBS, 80% methanol in deionized water, 100% methanol. Subsequently, they were washed in 20% DMSO/methanol, then in: 80% MeOH/H\u003csub\u003e2\u003c/sub\u003eO; in 50% MeOH/PBS; 100% PBS, and finally in PBS with 2% Triton X-100. Samples were incubated CytoVista Antibody Penetration Buffer for 30 minutes and blocked in CytoVista Blocking Buffer for 24 hours at 4\u0026deg;C. Spheroids were incubated with primary antibody diluted in CytoVista Antibody Dilution Buffer (VEGF-β Monoclonal Antibody, ThermoFisher Scientific) for 24 hours at 4\u0026deg;C. The spheroids were then washed and incubated with Goat anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody, Alexa Fluor\u0026trade; 594 (ThermoFisher Scientific) for 24 hours at 4\u0026deg;C. Cell nuclei were counterstained with Hoechst 33342 (ThermoFisher Scientific), washed and dehydrated with increasing concentrations of methanol (50% MeOH/PBS; 80% MeOH/H\u003csub\u003e2\u003c/sub\u003eO; 100% MeOH). After methanol removal, the spheroids were placed in an 8-well chambered \u0026micro;-Slide 8 coverslip (Ibidi, Gr\u0026auml;felfing, Germany) and cleared with 3D Cell Culture Clearing Reagent. z-stacks of spheroids were obtained with FV-1000 confocal microscope (Olympus Corporation, Tokyo, Japan) and reconstructed with FIJI software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Single-cell tumorspheres analysis with spectral flow cytometry\u003c/h2\u003e \u003cp\u003eBetween 25\u0026ndash;50 tumorspheres per group have been collected and precipitated. Subsequently, growing medium was removed and 200 \u0026micro;L of Accutase (Merck \u0026amp; Co., Inc., Kenilworth, NJ, USA) was added to ensure structures disintegration (5 min, 37\u003csup\u003eo\u003c/sup\u003eC). After complete cell detachment 800 \u0026micro;L of complete growing medium was added and samples were centrifuged (250 \u003cem\u003eg\u003c/em\u003e, 5 min). Cells were reconstituted in 1 mL of PBS/5% FBS and 30 \u0026micro;L sample was collected for cell counting and trypan blue exclusion staining and counted with Luna-II Automated Cell Counter (Logos Biosystems, Dongan-gu Anyang-si, Gyeonggi-do 14055 South Korea).\u003c/p\u003e \u003cp\u003eFor spectral flow cytometry following antibodies from BioLegend (San Diego, CA, USA) were used: VE-cadherin (PE anti-human CD144, clone BV9), E-cadherin (PE/Dazzle\u003csup\u003eTM\u003c/sup\u003e594 anti-mouse/human CD324, clone DECMA-1, isotype: Rat IgG1, κ), CD44 (BV711 anti-mouse/human, clone IM7, isotype: Rat IgG2b, κ), Vimentin (AF647 anti-Vimetin, clone W16220A, isotype Rat IgG2a, κ), MCAM (FITC anti-human CD146, clone P1H12, isotype: Mouse IgG1, κ). For viability, Annexin-V-pacific blue conjugate (Life-technologies, Eugene, OR, USA) and PI were used. As isotype control following antibodies were used: PE clone MOPC-173 mouse IgG2a, κ; PE/Dazzle\u003csup\u003eTM\u003c/sup\u003e594, clone RTK2071, isotype: Rat IgG1, κ; BV711 clone IM7, Rat IgG2b, κ; AF647 clone RTK2758, isotype Rat IgG2a, κ; FITC clone MOPC-21, mouse IgG1, κ. All single and mixed (A-V\u0026thinsp;+\u0026thinsp;PI and CD44\u0026thinsp;+\u0026thinsp;MCAM\u0026thinsp;+\u0026thinsp;Vimentin) were performed according to manufacturer protocol without any modifications. Samples were analyzed with ID7000 Sony Spectral Flow cytometer (Sony Biotechnology, San Jose, CA, USA). The ID7000 used in this study was equipped with four lasers 405/488/561/637 nm, PMT gains/voltages were independently adjusted for each laser. For each sample, a total of 10\u003csup\u003e4\u003c/sup\u003e cells were analyzed. Before analysis, the Sony ID7000 was calibrated using alignment checks (Sony Biotechnology Inc AlignCheck Flow Cytometer Alignment Beads 10\u003csup\u003e7\u003c/sup\u003e/mL 10 \u0026micro;m, 2 mL, cat. no AE700510) and the 8-peak performance beads (Sony Biotechnology Inc 8 Peak Bead cat. no AE700522, 10\u003csup\u003e7\u003c/sup\u003e/mL 3.1 \u0026micro;m, 5 mL cat. no AE700510), following the instrument supplier\u0026rsquo;s guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical and protein interactions analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism v.8 Software (GraphPad Software, San Diego, CA, USA) was used for statistical interpretation of obtained data. Results are presented as means with standard deviation, with \u003cem\u003ep\u003c/em\u003e values corresponding to: (*) \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, (**) \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, (***) \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001, and (****) \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001.\u003c/p\u003e \u003cp\u003eFor prediction of common protein interactions two proteins were analyzed by means of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (Szklarczyk et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Evaluation of growth and morphology changes in primary and metastatic melanoma\u003c/h2\u003e \u003cp\u003eSignificant differences in FM55p tumorspheres structure and growth were observed since 14th day of experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C \u003cb\u003eand table S2, supplementary information\u003c/b\u003e). Exceptionally big-sized FM55p tumorspheres after 28 days reached the unique diameter\u0026thinsp;~\u0026thinsp;2 mm (2000\u0026thinsp;\u0026plusmn;\u0026thinsp;65 \u0026micro;m) and a perimeter exceeding 7 mm. Interestingly, complete decomposition of control spheroids was observed as a consequence of widespread cell death, as revealed in living cells counting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Doubling-time (DT) calculations based on living cells population and tumorspheres volume (Karimi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) showed that those cultured in bioreactor had significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007 respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) reduced DT when compared to the control.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, any disintegration or spheroid death was not observed for metastatic melanoma models. However, meaningful differences were noticed in growth and morphology starting from first week after inoculation in bioreactor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C \u003cb\u003eand table S3 supplementary information\u003c/b\u003e). Tumorspheres reached an average diameter of 1432\u0026thinsp;\u0026plusmn;\u0026thinsp;54 \u0026micro;m being approximately 33% larger than control. Enhanced growth was associated with significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001) increased number of living cells found in bioreactor-based tumorspheres and 25\u0026ndash;50% shortened doubling time depending on cell number or tumor volume calculations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e/E\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. In-depth tumorspheres analysis\u003c/h2\u003e \u003cp\u003eRemarkably expressed morphological differences between tested methods inspired us to verify how does the external changes links to the tumor inside. To date, in-depth \u003cem\u003ein vitro\u003c/em\u003e 3D tumor assessment is rarely executed, mostly due to numerous challenges of such examination (Han et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Confocal bright-field (BF) visualization of tumors cross-sections as well as fluorescent imaging of whole tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) was performed at day 14 when FM55p control spheroids disintegration was noticed and the aim of this imaging was to identify possible reasons of this phenomenon.\u003c/p\u003e \u003cp\u003eInternal structure of tumorspheres from bioreactor was compact with high cell density and only single stripped of cells areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e/E\u003c/b\u003e, marked with white arrows). We did not identify any changes in compactness with increasing section depth. Notably, high tumor structure homogeneity is an important future of clinically observed solid tumors and conversely to traditional models was found to be achievable in bioreactor-based models.\u003c/p\u003e \u003cp\u003eDespite the satisfactory external morphology of WM266-4 control group as well, we found there much more structural diversities \u003cem\u003ee.g.\u003c/em\u003e, empty spaces and looser cells-cell connections (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These observations were in line with necrotic core staining that revealed increased dead cells content located internally. In particular, PI signal from necrotic cells in both (FM55p and WM266-4) control spheroids was intensive and widespread among tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e/G\u003c/b\u003e). An increase in fluorescence was found in larger clusters of organized cells, but PI-positive cells were also detected in the area of single cell layers. Conversely, tumorspheres from bioreactor revealed only trace PI\u0026thinsp;+\u0026thinsp;cells content (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004 for FM55p and WM266-4 respectively) localized mostly in form of small foci in midsection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we visualized the expression of vascular endothelial growth factor type β (VEGF-β) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e/F\u003c/b\u003e). VEGF-β is one of major hallmarks describing development of the tumor tissue, providing an information about the capability to vascularization, crucial in predicting tumor growth (Claffey et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Wierzbicki et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The 3D z-stack reconstructed images revealed, that externally located cells show high expression of VEGF-β both in clinoreactor and the plate-based control. Quantitative fluorescence analysis showed that signal intensity from VEGF-β, after prior normalization per structure volume, in tumorspheres was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) higher when compared to the control. More interestingly, no statistical differences were observed between primary and metastatic tumorspheres group, showing only slight variances (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.89).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Protein expression and epithelial-to-mesenchymal (EMT) transition\u003c/h2\u003e \u003cp\u003eDistribution analysis of FM55p living cells evaluated with Annexin-V and PI single-cell staining showed notably higher A-V (+) and PI (+) cells content (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e/C\u003c/b\u003e) when compared to the tumorspheres from bioreactors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e/C\u003c/b\u003e), especially at day 14. In general, living cells content assessed with flow cytometry was in line with trypan-blue exclusion staining, indicating elevated number of dead cells in control spheroids. Increase of PI\u0026thinsp;+\u0026thinsp;cells number in tumorspheres at day 21 is probably an aftermath of larger tumor size when compared to day 14, however as we showed previously, it did not impaired or inhibited tumorspheres growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e/B\u003c/b\u003e). Protein expression analyses performed with reference to the level of respective proteins in 2D cell cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) showed low levels of E-cadherin and VE-cadherin (\u0026lt;\u0026thinsp;2.5%), hence did not justify quantitative analysis of epithelial-to-mesenchymal transition (EMT), which is one of major hallmarks of cancer progression and metastatic sites formation. Interestingly, meaningful alterations in proteins expression were found in all of other markers. Progressing over time changes in expression of MCAM and CD44 in tumorspheres were identified mostly at day 21 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005 for MCAM and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0010 for CD44) while vimentin level was downregulated to 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% at day 21, probably due to suppressing the EMT (Dou et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThe number of living cells in WM266-4 tumorspheres was significantly higher when compared not only to the control, but also in reference to FM55p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e/C\u003c/b\u003e). Conversely to the primary melanoma, WM266-4 showed reduced PI signal, especially at day 21 when FM55p and WM266-4 control revealed an progressing over time increase of necrotic cells. Based on protein expression analyses, levels of MCAM, vimentin and CD44 expression were significantly elevated when compared to control. Similarly to previously observed changes, WM266-4 tumorspheres also downregulated vimentin expression during last week of growth, but interestingly also slight reduction of CD44 level was noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e/E\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Protein co-expression and STRING analysis\u003c/h2\u003e \u003cp\u003eProteome changes of desired makers may additionally allow to make a step forward in understating interactions and their implications in tumor \u003cem\u003ein vitro\u003c/em\u003e modeling especially when their up- or downregulation is linked with other molecular markers. For this purpose we firstly examined the CD44, MCAM, and vimentin simultaneous expression in both of tested melanomas. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, both FM55p and WM266-4 tumorspheres cultured in bioreactor showed overall significantly higher proteins co-expression than control groups, regardless of the markers being considered. Moreover, plate-based spheroids did not differ significantly between each other (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8629), ultimately showing limitations and disadvantages of traditional model. Interestingly, statistical analyses revealed that in primary melanoma simultaneous expression occurs more frequently when compared to the metastatic models (88% vs. 60%) but any specific correlation was not identified among tested subgroups including CD44\u0026thinsp;+\u0026thinsp;vimentin, CD44\u0026thinsp;+\u0026thinsp;MCAM and vimentin\u0026thinsp;+\u0026thinsp;MCAM respectively.\u003c/p\u003e \u003cp\u003eSTRING analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) of co-expressed proteins revealed that dual upregulation of CD44 and MCAM is frequently associated with various interactions mediated by laminins (LAMA1-5; LAMB1-3; LAMC1-3) and collagens (COL4A3 and COL15A1). Conversely, any correlation with vimentin as a third of co-expressed protein was not found. Therefore, based on protein profile of analyzed tumorspheres it can be concluded, that higher development of single cells translates to higher maturity of entire system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eImplementing a bioreactor offers several technological advantages, stating form suspending the cells in a static orbit during rotation that provides protective function against shear stress. Increased access to nutrients and oxygen ensures a better supply for the vital elements, while effective waste elimination prevents undesirable cell poisoning as a result of cumulated metabolic products. Consequently, strengthening of cell-to-cell interactions is observed and advanced model development can be achieved. Discussed morphological and physiological changes plays an important role in the intact expression of proteins and genes, thus evolving TME leads to achievement more clinically-like tumor models (El Harane et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pittman, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe obtained tumorspheres showed unprecedented size and more importantly, preserved proliferation capacity and biochemical functions during month-long experiment on population of 1100 tumorspheres originated from primary and metastatic melanoma. While the diameter of most commonly used spheroids is in the range of 250\u0026ndash;700 \u0026micro;m (Singh et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and usually does not reach 1000 \u0026micro;m (Mahmoud et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), in our study we obtained large 3D tumorspheres with over 2 mm (2.3 mm maximum) diameter corresponding to 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mm\u003csup\u003e3\u003c/sup\u003e of the estimated volume, which was previously impossible \u003cem\u003ein vitro\u003c/em\u003e (WU et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zanoni et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApart from the morphological maturity that was enabled due to higher cell viability, tumor microenvironment was found to be in higher development level as well. Increased VEGF-β expression proved that vascularization capacity is enhanced, thus \u003cem\u003ein vivo\u003c/em\u003e angiogenesis shows a great prospects for undergo efficient modulation with discussed models (Silvestre et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Co-expression MCAM and CD44 found in over 95% (FM55p) and 65% (WM266-4) of cells is indirect but solid evidence that tumorspheres from bioreactor can also restore or acquire the tissue-like functions. Our predictions for CD44 and MCAM proteins revealed the involvement of two extracellular matrix (ECM) protein pathways: the laminin 332 gene family and type IV collagen alpha chain (COL4A). LM 332 (formerly called LM 5), as a specific variant of laminin glycoprotein, is a heterotrimer of α3, β3 and γ2 chains, encoded by LAMA3, LAMB3 and LAMC2. LM332 forms the epithelial-basement membrane (BM) and supports the main function of epithelial tissue such as formation, healing and regeneration. LM332 is also considered a hallmark of cancer development, and its expression is impaired in various cancer cells (invasive mammary, colon, melanoma, and sarcoma) (Rousselle \u0026amp; Scoazec, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). COL4A is also a major component of BM, and its role in tumor angiogenesis and progression has been widely investigated (Zeng et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Overexpression of these two ECM components was observed in our study as strong intercellular interactions in the tested spheroids, which made cell separation difficult (it was necessary to use accutase to digest the spheroids) and constituted a mechanical barrier for dyes and antibodies used in the visualization of spheroids. Similar problems regarding the Ab-based detection in 3D models were previously reported by Mitrakas (Mitrakas et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), thus it was not possible to detect VEGF-β deeper in whole tumorsphere structure, enabling only boundary Ab penetration.\u003c/p\u003e \u003cp\u003eIn general, discussed approach can be easily and repeatedly used for other cellular models. Low SD\u0026rsquo;s with highly representative tumorspheres population were observed not only within the experimental groups, but more importantly between separate experimental runs. Due to high morphological and physiological development, presented models can serve not only as convenient in further \u003cem\u003ein vitro\u003c/em\u003e research, but principally as standalone biological model that may provide complementary data at various stages of drug development process. Possible implications of presented \u003cem\u003ein vitro\u003c/em\u003e models may primarily fill the gap in biological examination in preclinical stage, but also shows great potency to be applicable for analyzes with clinically used diagnostic systems. This includes particular molecular imaging specifically for research into the development of new radiopharmaceuticals and multiphoton imaging in novel positron emission tomography (PET) and single photon emission computed tomography (SPECT) devices and may provide completely new view of the field, regarding intratumoral markers expression, heterogeneity and metabolism based on currently developed approaches (Shimazoe \u0026amp; Uenomachi, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Uenomachi et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Available to date \u003cem\u003ein vitro\u003c/em\u003e models due to numerous features do not support such interdisciplinary examination.\u003c/p\u003e \u003cp\u003eOur findings presented in this article have proven that it is possible to design and develop remarkable biological models which can bridge the gap between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e systems and become the basis of future cancer research. The results showed important characteristics of spheroid properties obtained for the first time in millimeter-range size, with retained growth ability over several weeks of culture and most importantly highly established TME. Expanding of 3D spheroids or organoids themselves is a major strategy used in the study of cellular components. Presented results shows a great prospects to develop hybrid 3D cellular systems combining the features of spheroids and organoids for preclinical research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFounding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Science Centre of Poland through grants no. 2022/47/I/NZ7/03112 to E.Ł.S., the Jagiellonian University via projects CRP/0641.221.2020, SciMat and qLife Priority Research Areas budget under the program Excellence Initiative - Research University.\u003c/p\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthors contributions\u003c/h2\u003e \u003cp\u003eConceptualization: K.W., E.Ł.S; Formal analysis K.W., M.D.-K., M.W.; Funding acquisition: E.Ł.S.; Investigation: K.W., M.D.-K., M.W., E.Ł.S.; Methodology: K.W., M.D.-K., M.W., E.Ł.S.; Project administration: E.Ł.S.; Resources: E.Ł.S; Supervision: E.Ł.S.; Visualization: K.W., M.W.; Writing - original draft: K.W., E.Ł.S; and Writing - review \u0026amp; editing: K.W., M.D.-K., E.Ł.S.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe Authors would like to express sincere gratitude for Olena Bohomolova for her help in laboratory procedures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eClaffey KP, Brown LF, de Agulia LF, Tognazzi K, Yeo K-T, Manseau EJ, Dvorak HF (1996) \u003cem\u003eGrowth, Angiogenesis, and Experimental Metastasis Endothelial Growth Factor by Melanoma Cells Increases Tumor Expression of Vascular Permeability Factor/Vascular Citing Articles E-mail alerts\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cancerres.aacrjournals.org/content/56/1/172http://cancerres.aacrjournals.org/content/56/1/172#related-urls\u003c/span\u003e\u003cspan address=\"http://cancerres.aacrjournals.org/content/56/1/172http://cancerres.aacrjournals.org/content/56/1/172#related-urls\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDini S, Binder BJ, Fischer SC, Mattheyer C, Schmitz A, Stelzer EHK, Bean NG, Green JEF (2016) Identifying the necrotic zone boundary in tumour spheroids with pair-correlation functions. 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Translational Cancer Res 9(9):5218\u0026ndash;5232. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21037/tcr-20-517\u003c/span\u003e\u003cspan address=\"10.21037/tcr-20-517\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Informations","content":"\u003cp\u003eSupplementary Information is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"c897d035-ee04-473c-9f74-009415a3b566","identifier":"10.13039/501100004281","name":"Narodowe Centrum Nauki","awardNumber":"2022/47/I/NZ7/03112 ","order_by":0},{"identity":"07ffc009-476b-41ab-88d4-d9c29a92d2f1","identifier":"10.13039/501100007088","name":"Uniwersytet Jagielloński w Krakowie","awardNumber":"CRP/0641.221.2020, SciMat and qLife Priority Research Areas budget under the program Excellence Initiative - Research University","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Jagiellonian University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"3D-cell cultures, bioreactor, confocal imaging, imaging cytometry, organoids, tumorspheres","lastPublishedDoi":"10.21203/rs.3.rs-4884972/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4884972/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMore than 97% of anticancer drugs under investigation fail \u003cem\u003ein vitro\u003c/em\u003e evaluation, while only 0.4% of drug candidates that pass this stage proceed to the clinical trials. The differences between cell morphology and physiology observed \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e make the selection of a drug candidate problematic: traditional in vitro cultures do not reflect tissue-like conditions. Here we aimed for developing and characterizing human melanoma tumorspheres cultured in rotating bioreactors as an alternative for \u003cem\u003ein vitro\u003c/em\u003e modeling.\u003c/p\u003e \u003cp\u003eTumorspheres were characterized by in-depth confocal imaging and image cytometry, followed by quantitative analysis that was used for whole tumorspheres characterization. Cell viability and changes in proteins expression were investigated in single-cell analysis through the spectral flow cytometry followed by STRING interaction networks assessment.\u003c/p\u003e \u003cp\u003eThe tumorspheres showed the ability to grow for at least one month to reach millimeter sizes. In this way, it was possible to improve the morphology of tumorspheres and to observe changes in tumor microenvironment (TME) and the expression of key proteins. The advantage of the described models is the creation of perspectives for further development of maintaining cellular models that are hybrid systems combining the features of spheroids and organoids for preclinical and translational research.\u003c/p\u003e","manuscriptTitle":"Developing tumor microenvironment in rotating human melanoma cell cultures: study of novel preclinical model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-13 01:55:28","doi":"10.21203/rs.3.rs-4884972/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62e92bd2-a75e-42ca-b00f-326723e27431","owner":[],"postedDate":"August 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35807289,"name":"Cell \u0026 Tissue Engineering"},{"id":35807290,"name":"Cancer Biology"},{"id":35807291,"name":"Oncology"},{"id":35807292,"name":"Biochemical Research Methods"}],"tags":[],"updatedAt":"2024-08-13T01:55:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-13 01:55:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4884972","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4884972","identity":"rs-4884972","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}