Multicellular Tumor Spheroid Formation Research Articles

Long non-coding RNA H19 enhances cell proliferation and anchorage-independent growth of cervical cancer cell lines.

Long non-coding RNA H19 is aberrantly expressed in multiple malignancies and its expression levels correlate with recurrence, metastasis, and patient survival. Despite numerous reports documenting the role of H19 in carcinogenesis, its contribution to cervical cancer development is still largely unknown. In this study, I observed that H19 expression was elevated in cervical cancer cell lines and could be detected in extracellular vesicles in the culture medium. In addition, I demonstrated, by overexpression and knockdown experiments, that H19 promoted cell proliferation and multicellular tumor spheroid formation without significantly affecting apoptosis and cell migration. Finally, treatment with transforming growth factor beta and hypoxia-mimetic CoCl2 could modulate H19 levels in a cell line-specific manner. These findings indicate that H19 promotes both anchorage-specific and -independent growth of cervical cancer cell lines and may serve as a potential target for cancer diagnosis and therapy.

Sialylation transmogrifies human breast and pancreatic cancer cells into 3D multicellular tumor spheroids using cyclic RGD-peptide induced self-assembly.

Multicellular tumor spheroids (MTS) have been at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is highly influenced by aberrant cell surface-specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate MTS formation are not well defined. Matrix-free spheroids from breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD platform of cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK (TPP)). MCF-7 and MCF-7 TMX cells formed tight spheroids both in the classical agarose-and RGD-based platforms while all PANC1 cells formed loose aggregates. Using lectin histochemistry staining, sialidase assay, neuraminidase (Vibrio cholerae) and oseltamivir phosphate (OP) neuraminidase inhibitor treatments, MCF-7 and PANC1 cells and their drug-resistant variants expressed different sialic acid (SA) content on their cell surfaces. α-2,3- and α-2,6-sialic acid surface residues facilitated spheroid formation under cyclo-RGDfK(TPP)-induced self-assembly. Pretreatment with α-2,3- SA specific Maackia amurensis (MAL-II) lectin, α-2,6-SA specific Sambucus nigra (SNA) lectin, and exogenous α-2,6-SA specific neuraminidase (Vibrio cholerae) dose-dependently reduced spheroid volume. OP enhanced cell aggregation and compaction forming spheroids. PANC1 and MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significantly higher levels of α-2,3- SA over α-2,6-SA. MCF-7 spheroids also expressed a high α-2,3-SA to α-2,6-SA ratio. These results suggest that the relative levels of specific sialoglycan structures on the cell surface correlate with the ability of cancer cells to form avascular multicellular tumor spheroids and in vivo xenograft tumors.

Formation of multicellular tumor spheroids induced by cyclic RGD-peptides and use for anticancer drug testing in vitro

Development of novel anticancer formulations is a priority challenge in biomedicine. However, in vitro models based on monolayer cultures (2D) which are currently used for cytotoxicity tests leave much to be desired. More and more attention is focusing on 3D in vitro systems which can better mimic solid tumors. The aim of the study was to develop a novel one-step highly reproducible technique for multicellular tumor spheroid (MTS) formation using synthetic cyclic RGD-peptides, and to demonstrate availability of the spheroids as 3D in vitro model for antitumor drug testing. Cell self-assembly effect induced by addition of both linear and cyclic RGD-peptides directly to monolayer cultures was studied for 12 cell lines of various origins, including tumor cells (e.i. U-87 MG, MCF-7, M-3, HCT-116) and normal cells, in particular L-929, BNL.CL2, HepG2. Cyclo-RGDfK and its modification with triphenylphosphonium cation (TPP), namely cyclo-RGDfK(TPP) in a range of 10–100μM were found to induce spheroid formation. The obtained spheroids were unimodal with mean sizes in a range of 60–120μm depending on cell line and serum content in culture medium. The spheroids were used as 3D in vitro model, in order to evaluate cytotoxicity effects of antitumor drugs (doxorubicin, curcumin, temozolomide). The developed technique could be proposed as a promising tool for in vitro test of novel antitumor drugs.

Role of sialylation in 3D multicellular tumor spheroid formation and in vivo tumors

Role of sialylation in 3D multicellular tumor spheroid formation and in vivo tumors

Abstract 311: High-throughput spheroid formation in a 384-well format using magnetic 3D bioprinting

Abstract Technical limitations in the rapid and reproducible formation of multicellular tumor spheroids prevent their widespread use in high-throughput screening for cancer research and drug development. Existing systems for spheroid formation require lengthy processing times, and make simple tasks like media exchange and sample retention challenging. An ideal 3D cell culture system would print spheroids rapidly, while being easy to handle and process for high-throughput applications. To address this unmet need, we use magnetic 3D bioprinting for high-throughput spheroid formation. Magnetic 3D bioprinting is based on the principle of magnetizing cells using a magnetic nanoparticle assembly. Once magnetized, these cells can be directed and aggregated using magnetic forces; specifically, magnetized cells can be aggregated into spheroids in 384-well plates. These spheroids can be cultured long-term, and, as they are magnetic, the spheroids can be held down or transferred out of the well, avoiding the technical issues of other 3D systems to retain samples. Moreover, the magnetic nanoparticles have no effect on cell behavior and do not interfere with fluorescence. We demonstrated this method by printing spheroids of HepG2, HCT-116, A549, and PANC-1 cancer cells in 384-well plates. We printed spheroids rapidly (15 min) and reproducibly, with cell numbers as small as 100 cells. We assayed the spheroids by using their contraction over time as an endpoint, which we validated as a toxicity endpoint correlating with cell migration, proliferation, and viability. In running these experiments we also demonstrated that these spheroids are amenable to high-content testing, with multiple experiments being performed on the same spheroid to yield more data per test. Thus, this study introduces magnetic 3D bioprinting for high-throughput spheroid formation, where multicellular tumor spheroids that represent native tumor environments can be rapidly and easily printed for cancer research and compound screening. Citation Format: Hubert Tseng, Jacob A. Gage, William L. Haisler, Glauco R. Souza. High-throughput spheroid formation in a 384-well format using magnetic 3D bioprinting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 311. doi:10.1158/1538-7445.AM2015-311

Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids.

This paper describes the impact of different concentrations of single-walled carbon nanotubes (SWCNTs) on cell viability of breast adenocarcinoma, MCF-7 line, and formation of multicellular tumor spheroids (MTS). Chemical composition and purity of nanotubes is controlled by Fourier transform infrared spectroscopy. The strength and direction of the influence of SWCNTs on the tumor cell population was assessed by cell counting and measurement of the volume of multicellular tumor spheroids. Effect of SWCNTs on the formation of multicellular spheroids was compared with the results obtained by culturing tumor cells with ultra dispersed diamonds (UDDs). Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect. The increasing of SWCNTs concentration to 100 to 200 μg/ml stimulated proliferation of tumor cells, especially in suspension fractions. The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples. In result, the median volume of MTS after cultivation with SWCNTs at 100 to 200 μg/ml concentrations is 3 to 5 times greater than that in samples which were incubated with the UDDs and is 2.5 times greater than that in control cultures. So, if SWCNTs reduced cell adhesion to substrate and stimulated formation of tumor cell aggregates volume near 7 · 10−3 mm3, at the same time, UDDs reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids volume no more than 4 · 10−3 mm3. Our results could be useful for the control of cell growth in three-dimensional culture.PACS61. 46 + w; 61.48 + c; 61.48De; 87.15-v; 87.64-t

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids in vitro.

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.

Abstract 3781: A spheroid-based screen identifies mitochondrial targeting as a promising strategy for cancer treatment and drug repositioning

Abstract In the search for novel anticancer agents, the established principle is to harness differences between normal and tumor tissue. Thus, most of current chemotherapeutic agents target fast-proliferating cancerous cells. However, over the years such treatment strategies have proven less effective than initially expected. One of the main reasons for this is that conditions present in solid tumors, such as hypoxia, low glucose availability and high concentrations of metabolites, promote quiescent, highly resistant cell phenotypes. In our research, we aimed to target and exploit these tumor-specific conditions. To mimic the harsh conditions in a tumor, we used multicellular tumor spheroids (MCTS), which are known to simulate tumor microenvironment in vitro. We developed a novel method to easily form MCTS in 384-well format. There is only one spheroid per well formed and all are comparable in terms of size and shape. For the MCTS formation, we used colon carcinoma cell line, HCT116, with constitutive expression of green fluorescent protein (GFP). Then, we performed spheroid-based high-throughput drug screening using 1600 clinically active compounds. As a surrogate marker for cell viability, we measured mean spheroid GFP fluorescence intensity complemented with a standard resazurin-based assay. Active hits were tested in dose-response experiments in both spheroid and monolayer setup. We identified 12 compounds, which showed preferential activity against the MCTS model. We tested them in spheroid-based clonogenic assay and identified five compounds, which after 72 hrs treatment resulted in no clonogenicity at concentrations equal to monolayer-based IC50-values. Interestingly, all of these compounds have been previously reported to impair mitochondrial function. Three of them have been also reported as uncouplers of oxidative phosphorylation. We tested the influence of all five compounds on oxygen consumption rate. All, except one compound, caused irreversible shutdown of mitochondrial function. For further experiments, we decided to choose nitazoxanide - a clinically used anti-parasitic agent with excellent pharmacokinetics, bioavailability and safety profile. We show that treatment with nitazoxanide, at concentrations well below what is reached clinically, results in down regulation of c-myc, mTOR and Wnt signaling. Preliminary in vivo experiments show encouraging results. We conclude that MCTS-based screening identifies mitochondria as a potential target for cancer treatment and the anthelmintic drug nitazoxanide as a promising candidate. Citation Format: Wojciech Senkowski, Xiaonan Zhang, Maria Hägg Olofsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. A spheroid-based screen identifies mitochondrial targeting as a promising strategy for cancer treatment and drug repositioning. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2014-3781

Effects of T-lymphocytes and interferon-γ on different stages of development of multicellular tumor spheroids in vitro

The effect of T lymphocytes and interferon-γ on the vital activity of breast cancer cell population and the formation of multicellular tumor spheroids was studied. The stages of multicellular tumor spheroid formation and the possible ways for realizing the antitumor effects of T lymphocytes and interferon-γ through changes in the expression of estrogen receptors were demonstrated. The results have shown a correlation between the type of humoral effects and the level of expression of estrogen receptor and epidermal growth factor receptor and the intensity of tumor microaggregate formation.

Cell density modulates SHC3 expression and survival of human glioblastoma cells through Fak activation.

Shc3 protein levels are high in human glioblastoma but they decrease in vitro. We found that SHC3 mRNA and protein increased when glioblastoma cells grew as multicellular tumor spheroid (MTS). Shc3 expression was also induced in adherent cultures by increasing cell density. Among the Shc family members, only Shc2 and Shc3 increased with cell density. Shc3 and focal adhesion kinase (Fak) interact as shown by co-immunoprecipitation. Inhibition of Fak activation reduced Shc3 increase and MTS formation and changed Shc3 phosphorylation pattern. Our results suggest that in gliomas cell density modulates Shc3 protein levels and its activity, at least in part, through Fak activation.

Spheroid formation of human thyroid cancer cells under simulated microgravity: a possible role of CTGF and CAV1.

BackgroundMulticellular tumor spheroids (MCTS) formed scaffold-free under microgravity are of high interest for research and medicine. Their formation mechanism can be studied in space in real microgravity or on Earth using ground-based facilities (GBF), which simulate microgravity. On Earth, these experiments are more cost-efficient and easily performable. However, each GBF might exert device-specific and altered superimposingly gravity-dependent effects on the cells.ResultsFTC-133 human thyroid cancer cells were cultivated on a 2D clinostat (CN) and a random positioning machine (RPM) and compared with corresponding 1 g control cells. Harvested cell samples were investigated by microscopy, quantitative realtime-PCR and Multi-Analyte Profiling. Spheroid formation and growth occurred during 72 h of cultivation on both devices. Cytokine secretion and gene activation patterns frequently altered in different ways, when the cells were cultured either on the RPM or the CN. A decreased expression of CAV1 and CTGF in MCTS compared to adherent cells was observed after cultivation on both machines.ConclusionThe development of MCTS proceeds similarly on the RPM and the CN resembling the situation observed under real microgravity conditions, while no MCTS formation was observed at 1 g under identical experimental conditions. Simultaneously, changes in the regulation of CTGF and CAV1 appeared in a comparable manner on both machines. A relationship between these molecules and MCTS formation is discussed.

A novel three-dimensional microfluidic platform for on chip multicellular tumor spheroid formation and culture

The formation of three-dimensional (3D) multicellular cell spheroids such as microspheres and embryoid bodies has recently gained much attention as a useful cell culture technique, but few studies have investigated the suitability of glass for spheroids formation and culture. In this work, we present a novel three-dimensional microfluidic device made of poly(dimethylsiloxane) (PDMS) and glass for the easy and rapid synthesis and culture of tumor spheroid. The cell culture unit is composed of an array of microwells on the bottom of a glass plate, bigger microwells and elastomeric microchannels on the top of a PDMS plate. Cell suspension can be easily introduced into the cell culture unit and exchange with the external liquid environment by the microfluidic channels. A single tumor spheroid can be formed and cultured in each glass cell culture chamber, the surface of which was modified with poly(vinyl alcohol) to render it to be resistant to cell adhesion. As the cell culture medium could be replaced, spheroids of the human breast cancer (MCF-7) cells were cultured on the chip for 3 days, reaching the diameters of about 150 μm. Furthermore, the MCF-7 cells were successfully cultured on the chip in 2D and 3D culture modes. Results have shown that glass is well suitable for multicellular tumor spheroids culture. The established platform provides a convenient and rapid method for tumor spheroid culture, which is also adaptable for anticancer drug screening and fundamental biomedical research in cell biology.

Glycinamide ribonucleotide formyl transferase is frequently overexpressed in glioma and critically regulates the proliferation of glioma cells

AimsCurrent treatments for the most common form of brain tumor, glioma, are disappointing in their effectiveness. Low expression levels of GART, an enzyme in the core nucleotide metabolism, significantly correlate with chemosensitivity, conferring a survival advantage to tumor cells. Our study aimed to explore the expression and function of GART in glioma. MethodsImmunohistochemical and Western blot analysis were performed in 70 cases of human gliomas and normal brain tissues. We mainly used cell growth assay and multicellular tumor spheroid formation assay to evaluate the proliferation and chemosensitivity of glioma cells. ResultsHigh GART expression (most cancer cells cytoplasm stained) was observed in 70 specimens and was related to the grade of malignancy. We also reviewed each grade of tumors separately and investigated whether GART expression predicted patient survival within each subgroup. In brief, GART overexpression was significantly associated with overall survival (P=0.03). Interestingly, transfecting cells with GART-siRNA suppressed proliferation and enhanced temozolomide (TMZ)-induced apoptosis in glioma cells. ConclusionThe current results showed that GART expression was associated with glioma grade and that high GART protein expression might be related to poor outcome.

Multicellular tumor spheroid formation in duplex microcapsules for analysis of chemosensitivity

Multicellular tumor spheroids (MTS) are gaining increased recognition as valuable tools and key elements in anticancer drug discovery and tumor therapy test programs. However, the lack of reproducible and uniform MTS sizes is a major problem for pharmaceutical assays. Here, we show the usefulness of duplex microcapsules with a Ca-alginate gel membrane as a platform for producing MTS with a highly homogeneous size distribution. HeLa cells were enclosed with 86.9% viability within the microcapsules. The enclosed cells grew and formed MTS with the same size as the cavity of the microcapsules by arresting their growth with the microcapsule membrane. The cells in the resultant MTS had a higher proportion in G(0)/G(1) phase (71.2%) than 2-D cultured cells in the stationary phase (64.3%) or those in MTS formed on a non-adherent surface (65.3%) (P < 0.01). Furthermore, the cells in MTS formed within microcapsules showed higher tolerance to mitomycin C (1-1000 nM) and gemcitabine (4.5-4500 nM) than 2-D cultured cells (P < 0.01). In addition, the expression of MDR1, MCT1, HIF-1α, and GRP78 mRNA was 2.9-, 3.2-, 3.8-, and 5.5-fold higher, respectively, than those in 2-D cultured cells (P < 0.04). Cryopreserved encapsulated cells in the microcapsules showed 80.5% viability and formed MTS with a comparable tolerance of 100 and 1000 nM mitomycin C to those that were not cryopreserved (P > 0.09). These findings suggest the duplex microcapsule may be a promising tool for producing MTS for pharmaceutical applications.

Human Lung Cancer cell line SPC-A1 contains cells with characteristics of cancer stem cells

Cancer stem cells (CSCs) play important roles in occurrence, development, recurrence and metastasis of cancer. Isolation and identification of CSCs have been performed from some cancer tissues or cells. In this paper, human lung adenocarcinoma stem cells were induced and isolated from SPC-A1 cells and their characteristics were determined. SPC-A1 cells were cultured in serum-free medium and epidermal growth factor and basic fibroblast growth factor were added into the medium to induce the formation of multicellular tumor spheroids. The results showed that floating multicellular tumor spheroids (named pulmospheres) were formed 5-10 d after the induction of SPC-A1 cells. Real-time PCR analysis showed that in the pulmospheres, the marker of bronchioalveolar stem cells, Clara cell secretary protein and the marker of AT2 cells, alveolar surfactant protein C were highly expressed. Furthermore, such embryonic stem cell markers as octamer-binding transcription factor 4 (OCT-4), Bmi-1, and thyroid transcription factor -1 (TTF-1) were also highly expressed. Some miRNAs as hsa-miR-126, hsa-miR-145, hsa-let-7g, hsa-let-7d, hsa-let-7c, hsa-let-7e and hsa-miR-98, which were lowly expressed in SPC-A1 cells, were not expressed in the pulmospheres. Cell cycle analysis showed that 94.29 % of the pulmosphere cells were in G1 stages. Further study showed that these cells possessed higher proliferation and invasion activity than SPC-A1 cells. Tumorigenicity activity experiments on BALB/c nude mice showed that 1 × 103 of the pulmosphere cells could form tumors with similar pathological features with lung adenocarcinoma. In conclusion, lung adenocarcinoma stem cells were enriched in the pulmosphere cells and were with high tumorigenicity.

Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Moreover, continuous dynamic perfusion allows the establishment of long term cell culture and subsequent multicellular spheroid formation. A droplet-based microfluidic system was used to form alginate beads with entrapped breast tumor cells. After gelation, the alginate beads were trapped in microsieve structures for cell culture in a continuous perfusion system. The alginate environment permitted cell proliferation and the formation of multicellular spheroids was observed. The dose-dependent response of the tumor spheroids to doxorubicin, and anticancer drug, showed multicellular resistance compared to conventional monolayer culture. The microsieve structures maintain constant location of each bead in the same position throughout the device seeding process, cell proliferation and spheroid formation, treatment with drug, and imaging, permitting temporal and spatial tracking.

Biological Implications of Polymeric Microdevices for Live Cell Assays

Lab-on-a-chip technologies have the potential to deliver significant technological advances in modern biomedicine, through the ability to provide appropriate low-cost microenvironments for screening cells. However, to date, few studies have investigated the suitability of poly(dimethylsiloxane) (PDMS) for live cell culture. Here, we describe an inexpensive method for production of reusable, optical-grade PDMS microculture chips which provide a static and self-contained microwell system analogous to conventional polystyrene multiwell plates. We use these structures to probe the effects of PDMS upon live cell culture bioassays, using time-lapse fluorescence imaging to explore the toxicity of the substrate. We use three model systems to explore the efficacy of the microstructured devices: (i) live cell culture, (ii) adenoviral gene delivery to mammalian cells, and (iii) gravity enforced formation of multicellular tumor spheroids (MCTS). Results show that PDMS is nontoxic to cells, as their viability and growth characteristic in PDMS-based platforms is comparable to that of their polystyrene counterparts.

Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies

Three-dimensional (3D) in vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in vivo conditions. In cancer research, the multi-cellular tumor spheroid (MCTS) model is an established 3D cancer model that exhibits microenvironmental heterogeneity close to that of tumors in vivo. However, the established process of MCTS formation is time-consuming and often uncontrolled. Here, we report a method for engineering MCTS using a transient inter-cellular linker which facilitates cell-cell interaction. Using C3A cells (a hepatocellular carcinoma cell line) as a model, we formed linker-engineered spheroids which grew to a diameter of 250 μm in 7 days, as compared to 16 days using conventional non-adherent culture. Seven-day old linker-engineered spheroids exhibited characteristics of mature MCTS, including spheroid morphology, gene expression profile, cell-cell interaction, extracellular matrix secretion, proliferation and oxygen concentration gradients, and cellular functions. Linker-engineered spheroids also displayed a resistance to drug penetration similar to mature MCTS, with dose-dependent extracellular accumulation of the drug. The linker-engineered spheroids thus provide a reliable accelerated 3D in vitro tumor model for drug penetration studies.

Growth kinetics of 2- and 3-D cell models as influenced by the microenvironment

The noncontact cocultivation system was developed for the study of the paracrine interactions between MCF-7 (breast carcinoma cells) and MT-4 (a line of human T-cell leukemia). Viability and proliferation rates were determined in the adhesion and suspension fractions of MCF-7 cells sampled from two model systems: monolayer culture and multicellular tumor spheroids (MTS). Cocultivation with MT-4 reduced the number of MCF-7 cells in the adhesion fraction and had no effect upon the suspension fraction, despite an increase in the total population of MCF-7 cells. The two model systems displayed a substantial difference in cell viability, alone and in the presence of MT-4 cells — the fraction of viable cells in the monolayers was greater than in the spheroids. It is suggested that cocultivation with MT-4 stimulates proliferation of MCF-7 cells via a paracrine mechanism, reduces adhesion to the substrate, and leads to MTS formation.

Role of E-cadherin in the induction of apoptosis of HPV16-positive CaSki cervical cancer cells during multicellular tumor spheroid formation

Multicellular tumor spheroids (MCTS) are three dimensional cell culture systems induced by suspension culture. MCTS are widely used in cancer research because of their similarity to solid tumors. CaSki cells are derived from a metastatic cervical cancer containing human papillomavirus 16 (HPV16). Cell death of CaSki cells in MCTS has been previously reported, and our model is used to better characterize the mechanisms of cell death of HPV16-positive keratinocytes. In this study, we found that apoptosis of CaSki cells was induced by suspension culture along with the formation of MCTS after 24 h of incubation. In suspended CaSki cells, monoclonal antibodies blocking E-cadherin function inhibited MCTS formation and suppressed suspension-induced apoptosis in a dose-dependent manner. Western blot for E-cadherin detected upregulation of the authentic 120 kDa band from MCTS of CaSki cells as well as a shorter 100 kDa band. Addition of EGF, whose receptor is known to form a complex with E-cadherin, abrogated apoptosis of suspended CaSki cells in a dose-dependent manner. These findings suggest that E-cadherin-dependent cell-cell contact, directly or indirectly, mediates the signal to undergo apoptosis of CaSki cells during MCTS formation, and thus provides new information on the role of E-cadherin in cervical cancer cell apoptosis.