Three-dimensional Growth Conditions Research Articles

Abstract 4989: 3D spheroid screen yields SCD1 pathway inhibitors for the treatment of cancer

Abstract With three-dimensional growth conditions, multicellular tumor spheroids reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients, characteristic of poorly vascularized tumor regions. 3D high content screening (HCS) identified compounds that selectively kill tumor cells in the inner core of tumor cell spheroids by targeting the Stearoyl CoA Desaturase 1 (SCD1) pathway. SCD1 catalyzes the rate-limiting step in the production of mono-unsaturated fatty acids (MUFAs). Cancer cells are dependent on higher levels of MUFAs compared to normal cells and SCD1 is highly expressed in multiple tumor types. Changes in the MUFA / SFA (saturated fatty acid) ratio alters lipid biosynthesis and thus triggers cellular (ER) stress and induces the Unfolded Protein Response. Although the lead compound was very effective in vitro, it had unfavorable PK and physical chemistry properties, including low permeability and solubility and very high lipophilicity. This led to insufficient oral bioavailability, which could be overcome by optimization of PK and physical chemistry properties. Here, we report on the in vitro/in vivo effects of our 3D HCS compounds which showed high potency in the 3D spheroid inner core death assay with T47D breast cancer cells. In this in vitro model compound-induced inner core cell death is enhanced by SCD1 substrates palmitic or stearic acid and rescued by the SCD1 products palmitoleic or oleic acid. Furthermore, the effects can be reproduced in 2D cultures, which become increasingly sensitive to inhibition by our 3D HCS compounds with decreasing FBS concentration in the culture medium and this effect can also be rescued by addition of MUFAs but not of palmitic acid. Mode of action analysis showed that our compounds reduced palmitoleoyl- or oleoyl-CoA levels and simultaneously increased saturated fatty acyl-CoAs of palmitate or stearate in several cell lines as well as in vivo. In the sensitive T47D cells, the compounds induced expression of stress response genes and genes related to lipid metabolism. While these results support the SCD1 pathway as target for our 3D HCS compounds, we also observed striking differences to published SCD1 inhibitors suggesting a new cancer target beyond SCD1. Thus, further validation of our inhibitors in vitro and in vivo will be required, but these results suggest that 3D spheroid cultures may be a valuable tool for elucidation of new drug targets for cancer therapy. Citation Format: Sylvia Gruenewald, Carolyn Sperl, Patrick Steigemann, Alexander Walter, Sylvia Zacharias, Uwe Eberspaecher, Roland Neuhaus, Ludwig Zorn, Wolfgang Schwede, Kai Thede, Sven Christian. 3D spheroid screen yields SCD1 pathway inhibitors for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4989. doi:10.1158/1538-7445.AM2017-4989

Abstract 317: 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

Abstract Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions and therefore represent a promising model system for discovery of phenotypes specifically expressed in stressed, hypoxic and nutrient-depleted conditions. We here show the setup of a 3D cell culture compatible high-content screening system for the identification of substances that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. This setup was used in a pilot screen to identify tool compounds and subsequently in a larger screening approach covering approximately 500000 compounds from the Bayer substance library. The resulting hit list could be subdivided into two different categories based on the identification of two distinct phenotypes. Mode of action studies identified the first category as inhibitors of the mitochondrial respiratory chain, being critically dependent on extracellular glucose concentrations. On the other hand the second hit category could be identified as modifiers of fatty acid metabolism. The identified hits offer the opportunity for further characterization in vivo, particularly in combination with drugs targeting the well perfused, proliferating tumor areas, such as chemotherapy. Taken together the data presented here show for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. Citation Format: Carsten Wenzel, Sven Christian, Carolyn Algire, Wolfgang Schwede, Roland Neuhaus, Judith Guenther, Ningshu Liu, Sebastian Raese, Karsten Parczyk, Stefan Prechtl, Patrick Steigemann. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. [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 317. doi:10.1158/1538-7445.AM2015-317

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors.With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions.We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

Ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Abstract 486: The novel tumor suppressor DIAPH3 governs transition to an amoeboid phenotype

Abstract Introduction: We previously reported that loss of DIAPH3, proximal to RB1 on chromosome 13q and encoding the cytoskeletal regulator Diaphanous-related Formin-3 (DRF3), is associated with metastatic prostate cancer. Here we examine further the impact of genomic DIAPH3 loss on motility and metastasis, in multiple cancers. Methods: We employed publically available genomic data sets (including the Genomic Identification of Significant Targets in Cancer platform), Oncomine expression profiles, Affymetrix Human Genome-wide SNP arrays, and tissue microarrays to determine DIAPH3 copy number and DRF3 expression in carcinomas and with disease progression. In cultured cancer cells, biochemical and cell biological approaches in two- and three-dimensional growth conditions were used to assess mechanisms underlying phenotypic changes associated with DRF3 deficiency. The in vivo impact of DIAPH3 loss was investigated with a murine experimental metastasis model. Results: Analysis of somatic copy number alterations along chromosome 13q revealed a region of significant focal deletion, in which the DIAPH3 locus was central, common to hepatocellular, breast, and prostate carcinomas. Employing prostate cancer as a disease-relevant model, we detected reduced DIAPH3 copy number in multiple, independent patient cohorts. DIAPH3 deletions accumulated during disease progression, were strongly associated with metastatic disease, and were prevalent in disseminated tumor cells from patient bone marrow aspirates. Tissue microarrays confirmed reduced expression of DRF3 in metastases relative to normal tissue and primary tumor. In vitro, stable silencing of DRF3 in prostate cancer and HRAS-transformed human mammary epithelial cells dramatically altered cell morphology, evoking an ‘amoeboid’ morphological phenotype. Accordingly, biochemical hallmarks of amoeboid behavior, as well as enhanced migration, invasion, and chemotaxis, were associated with DRF3 loss. Consistent with its role as a regulator of cytoskeletal dynamics, DRF3 depletion destabilized microtubules, thereby perturbing endocytic receptor trafficking. EGFR was observed to accumulate in endosomes and was hyperactive, coinciding with persistent signaling through the MEK-ERK pathway. Inhibition of MEK reversed the amoeboid phenotype stimulated by DRF3 loss. Enforced expression of DRF3 accelerated down-regulation of EGFR and suppressed amoeboid morphology. Phosphorylation was observed to modulate DRF3 function. In a murine metastasis model, the formation of pulmonary lesions was markedly potentiated by DRF3 silencing, supporting a role for the protein in mitigation of tumor cell dissemination. Conclusions: Genomic deletions at the DIAPH3 locus are prevalent in multiple carcinomas and are strongly associated with metastasis. DRF3 is a metastasis suppressor that prevents transition to an amoeboid tumor cell phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 486. doi:1538-7445.AM2012-486

Nuclear Factor-κB Enhances ErbB2-Induced Mammary Tumorigenesis and Neoangiogenesis in Vivo

The (HER2/Neu) ErbB2 oncogene is commonly overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in transgenic mice. Nuclear factor (NF)-kappaB activity is increased in both human and murine breast tumors. The immune response to mammary tumorigenesis may regulate tumor progression. The role of endogenous mammary epithelial cell NF-kappaB had not previously been determined in immune-competent animals. Furthermore, the role of the NF-kappaB components, p50 and p65, in tumor growth was not known. Herein, the expression of a stabilized form of the NF-kappaB-inhibiting IkappaBalpha protein (IkappaBalphaSR) in breast tumor cell lines that express oncogenic ErbB2 inhibited DNA synthesis and growth in both two- and three-dimensional cultures. Either NF-kappaB inhibition or selective silencing of p50 or p65 led to a loss of contact-independent tumor growth in vitro. IkappaBalphaSR reversed the features of the oncogene-induced phenotype under three-dimensional growth conditions. The NF-kappaB blockade inhibited ErbB2-induced mammary tumor growth in both immune-competent and immune-deficient mice. These findings were associated with both reduced tumor microvascular density and a reduction in the amount of vascular endothelial growth factor. The expression of IkappaBalphaSR in breast cancer tumors inhibited angiogenesis. Thus, mammary epithelial cell NF-kappaB activity enhances ErbB2-mediated mammary tumorigenesis in vivo by promoting both growth and survival signaling via the promotion of tumor vasculogenesis.

Hollow‐Spheres: A New Model for Analyses of Differentiation of Pancreatic Duct Epithelial Cells

We discovered a unique feature of a subclone of the pancreatic carcinoma cell line A818. A818-1-derived hollow-spheres developed under three-dimensional growth conditions. Hollow-spheres consist of a single layer of 50-200 epithelial cells surrounding an inner lumen. In contrast to A818-1, the subclone A818-4 and all other pancreatic tumor cell lines tested (n = 5), formed spheroids as the only three-dimensional phenotype. A dramatically reduced proliferation rate compared to the corresponding monolayer was observed in hollow-spheres when bromodeoxyuridine (BrdU) incorporation was measured. This finding was confirmed by immunostaining using the MIB-1 antibody. Mechanically disrupted hollow-spheres not only attached but also grew as monolayer with the same doubling time as the founder cells. Hollow-spheres developed in fetal calf serum (FCS) containing RPMI 1640 medium without additionally added cytokines. A818-1 hollow-sphere formation and integrity was influenced by interferon-gamma. Tumor necrosis factor-alpha (TNF-alpha) led to cell death. Exogenously added hepatocyte growth factor (HGF) showed no effect neither on hollow-sphere formation nor on the integrity of completely developed hollow-spheres. Moreover, no changes were observed when cells were treated with a neutralizing antibody for HGF. Interestingly, hollow-spheres showed intensive immunoreactivity for the HGF-receptor (c-met) and its ligand (HGF). Immunostaining for the biliary glycoprotein (BGP), the non-specific cross-reacting antigen 95 (NCA95) and beta-catenin revealed a polar organization of hollow-spheres. Immunhistochemically, hollow-spheres were negative for the carcinoembryonic antigen (CEA). When hollow-spheres were embedded into matrigel, duct-like tubes grew out. Taken together, A818-1 hollow-spheres resemble normally differentiated duct-like structures and will serve as an excellent model to study differentiation of human pancreatic epithelial cells.

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression.

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three-dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)

Critical thickness during two-dimensional and three-dimensional epitaxial growth in semiconductor heterostructures

Abstract The homogeneous nucleation of misfit dislocations in two-dimensional and three-dimensional epitaxial structures on rigid substrates was analyzed. It is shown that nucleation and growth of a dislocation in the epilayer involves an activation energy barrier. We propose that this activation energy barrier can be overcome from the residual coherent strain energy in the film. A critical thickness of the epilayer is defined at which a dislocation is nucleated. The activation energy and the coherent strain energy were determined for several different configurations of 60° glide and 90° climb dislocations. The discrepancies that are associated with different formulations are pointed out. Specific numerical calculations were performed for dislocation nucleation in the GaAs/Si system. The results are given in terms of the various energy contributions responsible for nucleation of misfit dislocations, and the critical thickness of the epilayer is evaluated. The critical thickness of the epilayer under the two-dimensional and three-dimensional growth conditions are compared and the results described in terms of the mechanisms of dislocation nucleation.

How culture conditions modulate the morphofunctional differentiation of the human estradiol-sensitive mammary cell line (MCF-7)

The MCF-7 cell line grown on plastic surfaces is widely accepted as a model for hormone sensitivity in molecular biology. However, in vitro results concerning estrogen sensitivity remain controversial. In search of culture conditions most closely simulating the in vivo microenvironment we cultured MCF-7 cells on diverse substrates and in suspension culture. The different factors of the contact environment: (A) influence of diffusive medium, (B) influence of cell to cell contacts, and (C) influence of cell to substrate contacts were considered. Using morphological criteria:phase contrast microscopy, scanning and transmission electron microscopy we observed MCF-7 morphofunctional differentiation under the different culture conditions. Plastic, corneal endothelial cell extracellular matrix, and attached collagen gels imposed a planar medium-aggregate interface. The impermeability of the free surface and the intense basal tension antagonized epithelial polarization. Only at post-confluence did domes and clusters appear above the monolayer. On floating collagen gels and in suspension culture the cells established intimate cell-cell contacts over large surfaces and reconstituted tissular architecture. Three-dimensional growth conditions which approach the in vivo contact environment of epithelial cells should be used instead of the traditional monolayer cultures for assessing hormonal and pharmacological responses of human breast carcinomas.