Activity Of Spheroids Research Articles

Analysis of Matrix Metalloproteinase Activity and Inhibition in Cancer Spheroids.

The utilization of tumor spheroids and organoids has greatly facilitated mechanistic understanding of tumor growth and invasion and lead to more effective high-throughput analysis of potential chemotherapeutic agents. In spheroid and organoid systems, tumor invasion occurs in three dimensions and monitoring this behavior can be data intensive. Quantitative correlation of tumor invasion with protease activity can further exacerbate data storage issues. The present method utilizes the "Hit Pick" approach to provide quantitative analysis and correlation of tumor invasion and membrane type 1 matrix metalloproteinase (MT1-MMP) activity in a rapid fashion with greatly reduced data storage requirements compared with standard image analysis approaches. Inhibition of MT1-MMP activity in spheroids can also be monitored by the present approach.

The Ultrastructural Analysis of Human Colorectal Cancer Stem Cell-Derived Spheroids and Their Mouse Xenograft Shows That the Same Cells Types Have Different Ratios.

Simple SummaryA comparative ultrastructural and flow cytometric analysis of colorectal cancer-derived spheroids and their mouse xenografts showed that they both contain the same cell types but with different ratios, reflecting the interaction of cancer cells, respectively, with the in vitro and in vivo microenvironment.Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.

Abstract B16: STAT3 promotes ovarian cancer spheroid growth and metastasis

Abstract High-grade serous ovarian cancer remains deadly for the majority of woman who are diagnosed every year. The high mortality rate necessitates new treatment options for physicians and patients to consider. To produce new therapies, different signaling pathways that are activated in ovarian cancer need to be identified. In order to mimic the growth of ovarian cancer in the body, 3D culturing systems were used to grow clusters of ovarian cells (spheroids). Comparing gene expression from cells grown in 2D and 3D, we identified the STAT3 signaling pathway as being highly enriched in ovarian spheroids. STAT3 is a transcription factor that is activated by tyrosine phosphorylation. Once phosphorylated, STAT3 dimerizes, translocates to the nucleus, and regulates the expression of many genes involved in cell survival and migration. To determine if STAT3 was activated in 3D growing cells, we analyzed protein from cells grown in 2D and 3D and determined that STAT3 was tyrosine phosphorylated in cells grown in 3D We have found that the increase in STAT3 phosphorylation in 3D grown cells was associated with increased expression of STAT3 target genes. Consistent with this, we found enhanced STAT3 DNA binding to regulatory regions of STAT3 target genes. Recognizing that STAT3 activation is upregulated in 3D, we wanted to know if STAT3 is necessary for growth in 3D. By using siRNA to reduce the expression of STAT3, we were able to hinder the ability for ovarian cancer to grow in 3D. Moreover, reducing the expression of various components in the STAT3 signaling pathway also reduced the growth of ovarian spheroids. One of the major steps in ovarian cancer metastasis is the clearance of mesothelial cells by the cancer cells. To assess the effects of enhanced STAT3 activity in spheroids, we performed a mesothelial clearance assay with spheroids containing and lacking STAT3 expression. We found that ovarian spheroids with reduced STAT3 expression had a decreased ability to metastasize. This suggests that the STAT3 signaling pathway is a critical pathway involved in the growth and metastasis of ovarian cancer spheroids. This raises the possibility of using STAT3 inhibitors by themselves or in conjunction with standard-of-care therapeutics in the treatment of advanced ovarian cancer. Thus, targeting signaling pathways important in 3D growth may be beneficial for the treatment of ovarian cancer. Citation Format: David F. Walker, Sarah J. Lacroix, Zachary T. Giaccone, David A. Frank, Sarah R. Walker. STAT3 promotes ovarian cancer spheroid growth and metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B16.

Characterization of Placental Mesenchymal Stem Cells Spheroids after Generation, Hypothermic and Subnormothermic Storage

Background. Placental mesenchymal stem cells application is a promising area of cell therapy for number diseases. Their using in the form of spheroids results in enhancement of the therapeutic potential, improve the cells survival after transplantation. Medical purposes require the characterization and development of spheroids storage technology. Objective. The aim of the paper is to characterize placental mesenchymal stem cells spheroids after generation and low-temperature storage. Methods . Spheroids were produced by a hanging drop method from placental mesenchymal stem cells, chara­cterized after production and after subnormothermic (20 °C) and hypothermic (4 °C) storage for up to 96 h. The structure, metabolic activity of spheroids according to the data of the MTT test and the resazurin restoration test were studied. Results . The hanging drop method makes it possible to produce from 1×10 6 placental mesenchymal stem cells about 825.3 ± 56.2 spheroids in the size of 50–150 microns and 62.3 ± 5.1 spheroids of 150–300 microns in size, having a spherical shape, not stained with trypan blue, stained with neutral red and fluorescein diacetate. The spheroids adhere to the plastic and destroying through short-time centrifugation. During subnormothermic storage spheroids, structural characteristics and metabolic activity change after 48 h, during hypothermic – after 24 h of storage. Conclusions. The hanging drop method makes it possible to produce about 900 spheroids from 1 million placental MSCs with stable metabolic activity. For medical purposes, subornorthomeric (20 °C) storage not more than 24 h is recommended for placental MSC spheroid. Longer storage leads to a sharp decreasing of their metabolic activity and destroying to the separated cells.

The FAK inhibitor BI 853520 inhibits spheroid formation and orthotopic tumor growth in malignant pleural mesothelioma

No tyrosine kinase inhibitors are approved for malignant pleural mesothelioma (MPM). Preclinical studies identified focal adhesion kinase (FAK) as a target in MPM. Accordingly, we assessed the novel, highly selective FAK inhibitor (BI 853520) in 2D and 3D cultures and in vivo. IC50 values were measured by adherent cell viability assay. Cell migration and 3D growth were quantified by video microscopy and spheroid formation, respectively. Phosphorylation of FAK, Akt, S6, and Erk was measured by immunoblot. The mRNA expression of the putative tumor stem cell markers SOX2, Nanog, CD44, ALDH1, c-myc, and Oct4 was analyzed by qPCR. Cell proliferation, apoptosis, and tumor tissue microvessel density (MVD) were investigated in orthotopic MPM xenografts. In all 12 MPM cell lines, IC50 exceeded 5 μM and loss of NF2 did not correlate with sensitivity. No synergism was found with cisplatin in adherent cells. BI 853520 decreased migration in 3 out of 4 cell lines. FAK phosphorylation was reduced upon treatment but activation of Erk, Akt, or S6 remained unaffected. Nevertheless, BI 853520 inhibited spheroid growth and significantly reduced tumor weight, cell proliferation, and MVD in vivo. BI 853520 has limited effect in adherent cultures but demonstrates potent activity in spheroids and in orthotopic tumors in vivo. Based on our findings, further studies are warranted to explore the clinical utility of BI 853520 in human MPM.Key messagesResponse to FAK inhibition in MPM is independent of NF2 expression or histotype.FAK inhibition strongly interfered with MPM spheroid formation.BI 853520 has been shown to exert anti-tumor effect in MPM.

Bystander Effects of Hypoxia-Activated Prodrugs: Agent-Based Modeling Using Three Dimensional Cell Cultures.

Intra-tumor heterogeneity represents a major barrier to anti-cancer therapies. One strategy to minimize this limitation relies on bystander effects via diffusion of cytotoxins from targeted cells. Hypoxia-activated prodrugs (HAPs) have the potential to exploit hypoxia in this way, but robust methods for measuring bystander effects are lacking. The objective of this study is to develop experimental models (monolayer, multilayer, and multicellular spheroid co-cultures) comprising ‘activator’ cells with high expression of prodrug-activating reductases and reductase-deficient ‘target’ cells, and to couple these with agent-based models (ABMs) that describe diffusion and reaction of prodrugs and their active metabolites, and killing probability for each cell. HCT116 cells were engineered as activators by overexpressing P450 oxidoreductase (POR) and as targets by knockout of POR, with fluorescent protein and antibiotic resistance markers to enable their quantitation in co-cultures. We investigated two HAPs with very different pharmacology: SN30000 is metabolized to DNA-breaking free radicals under hypoxia, while the dinitrobenzamide PR104A generates DNA-crosslinking nitrogen mustard metabolites. In anoxic spheroid co-cultures, increasing the proportion of activator cells decreased killing of both activators and targets by SN30000. An ABM parameterized by measuring SN30000 cytotoxicity in monolayers and diffusion-reaction in multilayers accurately predicted SN30000 activity in spheroids, demonstrating the lack of bystander effects and that rapid metabolic consumption of SN30000 inhibited prodrug penetration. In contrast, killing of targets by PR104A in anoxic spheroids was markedly increased by activators, demonstrating that a bystander effect more than compensates any penetration limitation. However, the ABM based on the well-studied hydroxylamine and amine metabolites of PR104A did not fit the cell survival data, indicating a need to reassess its cellular pharmacology. Characterization of extracellular metabolites of PR104A in anoxic cultures identified more stable, lipophilic, activated dichloro mustards with greater tissue diffusion distances. Including these metabolites explicitly in the ABM provided a good description of activator and target cell killing by PR104A in spheroids. This study represents the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the power of combining mathematical modeling of pharmacokinetics/pharmacodynamics with multicellular culture models to dissect bystander effects of targeted drug carriers.

Formation of spheroids by dental pulp cells in thepresence of hypoxia and hypoxia mimetic agents.

To evaluate the impact of hypoxia and hypoxia mimetic agents (HMA) on the formation and activity of spheroids by dental pulp cells (DPC). DPC on agarose-coated plates were treated with hypoxia and the HMA dimethyloxallyl glycine (DMOG), desferrioxamine (DFO) and L-mimosine (L-MIM). Images of spheroids were taken directly after seeding and at 6h and 24h. Spheroid sizes were quantified by area measurement with ImageJ software. Viability was assessed with Live-Dead staining, MTT and resazurin-based toxicity assay. Production of VEGF, IL-8 and SDF-1 was evaluated using immunoassays. Data were analysed using Kruskal-Wallis test and post hoc Mann-Whitney U-test. DPC formed spheroids in the presence of hypoxia, HMA and combined treatment with hypoxia and HMA. No pronounced difference in spheroid size was found in the groups treated with hypoxia, DMOG, DFO, L-MIM and the combination of hypoxia and the HMA relative to their normoxic controls (P>0.05). Spheroids appeared vital in Live-Dead and MTT staining and the resazurin-based toxicity assay. Evaluation of protein production with immunoassays revealed significantly enhanced levels of VEGF and IL-8 (P<0.05), but there was no significant effect on SDF-1 production (P>0.05). Treatment with a combination of hypoxia and HMA did not further boost VEGF and IL-8 production (P>0.05). Pre-conditioning with hypoxia and HMA increased the pro-angiogenic capacity of spheroids whilst not interfering with their formation. Pre-clinical studies will reveal whether pre-conditioning of spheroids with hypoxia and HMA can effectively improve the efficiency of cell transplantation approaches for regenerative endodontics.

Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids.

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5'-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.

Abstract A47: The LKB1-AMPK pathway mediates the metabolic stress response of dormant ovarian cancer spheroids

Abstract Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse throughout the peritoneum via ascites to seed secondary lesions. We hypothesize that EOC spheroids are key mediators of metastasis, which use specific signaling pathways to alter cell metabolism for increased survival. Our lab discovered that AKT signaling is reduced during spheroid formation leading to cellular quiescence and autophagy. Given the induction of quiescence and autophagy in EOC spheroids, we are studying the 5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response and dormant phenotype of EOC spheroids. We demonstrate AMPK activity and its upstream kinase LKB1 are increased in quiescent EOC spheroids compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Targeted knockdown of STK11, encoding LKB1, reduces cell viability in ovarian cancer cell line spheroids; PRKAA1 (AMPKα1) knockdown has little to no effect on EOC cell or spheroid viability. Combination of STK11 knockdown with carboplatin treatment leads to a synergistic enhancement in EOC spheroid cell death. In contrast, AICAR treatment of proliferating adherent ovarian cancer cell lines and primary EOC cells induces AMPK activity and causes either cytostasis or cell death. In addition, AICAR treatment of spheroids during reattachment decreases the dispersion capacity of migrating EOC cells. These results offer a glimpse of the potential important contributions of LKB1-AMPK pathway in stress signaling related to EOC cell survival during metastasis. In addition, downstream effectors of upregulated LKB1-AMPK signalling may provide additional mechanisms by which EOC evades chemotherapy. It is foreseeable that these findings will allow us to identify new therapeutic targets within this pathway critical to EOC progression for ultimate translation to the clinic. Citation Format: Teresa Peart, Elena Fazio, Yudith Ramos-Valdes, Monique Bertrand, Jacob McGee, Michel Prefontaine, Akira Sugimoto, Gabriel E. DiMattia, Trevor G. Shepherd. The LKB1-AMPK pathway mediates the metabolic stress response of dormant ovarian cancer spheroids. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A47.

Noninvasively measuring respiratory activity of rat primary hepatocyte spheroids by scanning electrochemical microscopy

Construction of an in vitro drug screening method for evaluating drug metabolism and toxicity by using cells is required instead of the conventional in vivo one that uses animals. In order to realize the in vitro study, analyzing the cellular activity or viability noninvasively in advance of the screening is essential. The aim of the current study is to establish a method that can evaluate the cellular activity depending on spheroid sizes by means of oxygen consumption and to determine the valid diameter of hepatocyte spheroids. To measure the respiratory activity of the spheroids, which were formed on a nanopillar sheet, we applied scanning electrochemical microscopy (SECM). From the viewpoint of high respiratory activity and its small variation, we determined that spheroids with 70 μm in diameter were adequate. We then performed a gene expression analysis by using a real-time PCR to evaluate the correlation with respiratory activity. As a result, a higher expression level of Hnf4α, which is essential for hepatocytes to fulfill many liver functions and is the indicator of well-differentiated hepatocytes, showed relatively higher respiratory activity. We concluded that the noninvasive SECM technique could evaluate the cellular activity of a single spheroid. Noninvasively measuring cellular activity by SECM makes it possible to evaluate the cellular activity prior to a nonclinical test and enables the continued monitoring of the drug response by using single spheroid. SECM becomes a powerful tool to satisfying the increasing demand for an in vitro system in the course of new drug development.

Necrotic and apoptotic features of cell death in response to Foscan ® photosensitization of HT29 monolayer and multicell spheroids

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan ®. Kinetics of apoptosis and necrosis after Foscan ®-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan ® was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Δ ψ m) addressed by flow cytometry technique at different time points post-Foscan ®-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan ®-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.

Cytochrome P450 activity in control and induced long-term cultures of rat hepatocyte spheroids

Long-term events such as enzyme induction or chronic toxicity require long-term liver culture models that maintain activity of xenobiotic metabolising enzymes. The levels of these enzyme activities and their responsiveness to chemical induction was studied in rat hepatocyte spheroids, a potential long-term hepatocyte culture model. In comparison with other long-term liver culture models, the basal metabolic activity of spheroids has not been well studied. Additionally, no existing data on the induction of CYP3A activity in spheroids could be found. The basal xenobiotic metabolising activity of rat hepatocyte spheroids was monitored over 14 days in culture, using testosterone as a probe substrate. When spheroids from days 2–14 in culture were compared to 24-h control spheroids, there was a differential maintenance of basal CYP activity. CYP2A and CYP3A activities were maintained over the culture period, while there were time-related decreases in CYP2C11 and CYP2C/CYP2B1/2 activities. The responsiveness of rat hepatocyte spheroids to chemical induction was studied following treatment with phenobarbitone (PB) or dexamethasone (DEX). PB treatment induced CYP2A, CYP2C, CYP2B1/2 and CYP3A activities. DEX treatment resulted in an induction of CYP3A and CYP2C11 activities. The results demonstrate that rat hepatocyte spheroids retained some of the liver-specific functions essential in a long-term hepatocyte culture model, thus making spheroids comparable to other long-term culture models available.

Intercellular communication, three-dimensional cell contact and radiosensitivity.

When electrically coupled mammalian cells are cultured as spherical clones (spheroids) and exposed to ionizing radiation they are less radiosensitive than monolayers of the same cell line. Investigations into the possible role of coupling (gap junctions) and three-dimensional contact in the expression of this phenomenon revealed 1) a correlation between cell coupling and the activity of adenylate cyclase in monolayers, 2) a sharp drop of cyclase activity in spheroids of coupled cells compared to monolayers, and 3) a decrease of coupling with age ("maturation") of the spheroids. These results suggest profound physiological alterations in communicating cells induced under conditions of tight three-dimensional contact as a possible cause for the reduced radiosensitivity of spheroids.