ABSTRACT
Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.
Subject(s)
Biomarkers, Tumor/genetics , Cell Proliferation , Knowledge Bases , Neoplasms/pathology , Software , Spheroids, Cellular/pathology , Tumor Microenvironment , Cell Culture Techniques/methods , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/classification , Neoplasms/metabolism , RNA-Seq , Reproducibility of Results , Spheroids, Cellular/immunology , Spheroids, Cellular/metabolism , Tumor Cells, CulturedABSTRACT
Colorectal cancer (CRC) is the second deadliest cancer in the world. Besides APC and p53 alterations, the PI3K/AKT/MTOR and MAPK pathway are most commonly mutated in CRC. So far, no treatment options targeting these pathways are available in routine clinics for CRC patients. We systematically analyzed the response of CRC cells to the combination of small molecular inhibitors targeting the PI3K and MAPK pathways. We used CRC cells in 2D, 3D spheroid, collagen gel cultures and freshly isolated organoids for drug response studies. Readout for drug response was spheroid or organoid growth, spheroid outgrowth, metabolic activity, Western blotting and immunofluorescence. We found profound tumor cell destruction under treatment with a combination of Torin 1 (inhibiting mTOR), MK2206 (targeting AKT) and selumetinib (inhibiting MEK) in 3D but not in 2D. Induction of cell death was due to apoptosis. Western blot analysis revealed efficient drug action. Gedatolisib, a dual PI3K/mTOR inhibitor, could replace Torin1/MK2206 with similar efficiency. The presence of PI3K and/or RAS-RAF-MAPK pathway mutations accounted for treatment responsiveness. Here, we identified a novel, efficient therapy, which induced proliferation stop and tumor cell destruction in vitro based on the genetic background. These preclinical findings show promise to further test this combi-treatment in vivo in mice and to potentially develop a mutation specific targeted therapy for CRC patients.
Subject(s)
Colonic Neoplasms , Mitogen-Activated Protein Kinase Kinases , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , TOR Serine-Threonine Kinases , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Mutation , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , HumansABSTRACT
BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.
Subject(s)
Methyltransferases , Prostatic Neoplasms , Animals , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , DNA-Binding Proteins/physiology , Humans , Male , Methyltransferases/genetics , Mice , Mutation , Prostatic Neoplasms/metabolism , Exome SequencingABSTRACT
WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.
Subject(s)
Cancer-Associated Fibroblasts/metabolism , Colonic Neoplasms/blood supply , Colonic Neoplasms/pathology , Neovascularization, Pathologic/chemically induced , Wnt2 Protein/metabolism , Wnt2 Protein/pharmacology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Coculture Techniques , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Culture Media, Conditioned/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/physiology , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Tumor Microenvironment/physiologyABSTRACT
Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo These 3D models displayed augmented anti-tumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.
Subject(s)
Cell Culture Techniques/methods , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Shape/drug effects , Humans , MAP Kinase Signaling System/drug effects , Phenotype , Phosphorylation/drug effects , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathologyABSTRACT
Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca2+-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.
Subject(s)
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Cancer-Associated Fibroblasts/pathology , Colon/pathology , Colorectal Neoplasms/pathology , Rectum/pathology , Signal Transduction , Calcium/metabolism , Cancer-Associated Fibroblasts/metabolism , Cardiac Myosins/metabolism , Cell Line, Tumor , Cell Movement , Colon/metabolism , Colorectal Neoplasms/metabolism , Humans , Myosin Light Chains/metabolism , Neoplasm Invasiveness/pathology , Rectum/metabolism , rho-Associated Kinases/metabolismABSTRACT
Invasive colorectal cancer is associated with poor prognosis requiring treatment with systemic chemotherapies usually including 5-fluorouracil. A consequence of prolonged treatment is the acquisition of resistance eventually resulting in the recurrence of highly metastatic cancer cells. To address the relationship between drug resistance and increased lymphatic metastatic potential, we used a 3D co-culture model of colon tumour cell spheroids of parent CCL227 cells and subclones with gradually increasing resistance against 5-fluorouracil. From each investigated cell line, homogeneous tumour spheroids were generated in the presence of methylcellulose yielding emboli of â¼700 µm diameter. When invasive, tumour spheroids disrupt the continuous lymphendothelial cell (LEC) layer and generate a 'circular chemorepellent-induced defect' (CCID), reminiscent of the entry gates through which tumour emboli intravasate lymphatic vasculature. Here we provide evidence that increasingly chemoresistant colon cancer spheroids were strongly associated with enhanced intravasative properties. In naïve CCL227 spheroids, miR-200 family members were released into exosomes thereby repressing the epithelial to mesenchymal transition-regulating transcription factors ZEB1 and SLUG in LEC. As a consequence of attenuated plasticity and migration of LEC, CCID formation was impaired. Loss of exosomal transferred miR-200c in resistant colon cells rendered LEC more susceptible to pro-migratory signals that were generated and directly transmitted by colon cancer spheroids. This observation indicates a common molecular axis in colon cancer and LEC where miR-200 family members act as regulators of ZEB proteins. The data support the notion that horizontal miR-200 signalling prevents the permeation of cells into adjacent epithelia and contributes to organ integrity.
Subject(s)
Colonic Neoplasms/metabolism , Endothelial Cells/metabolism , Fluorouracil/pharmacology , MicroRNAs/metabolism , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Drug Resistance, Neoplasm , Endothelial Cells/drug effects , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Lymphatic Metastasis , MicroRNAs/genetics , Multigene Family , Neoplasm Invasiveness , Snail Family Transcription Factors , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, Cultured , Zinc Finger E-box-Binding Homeobox 1ABSTRACT
Solid cancers are not simple accumulations of malignant tumor cells but rather represent complex organ-like structures. Despite a more chaotic general appearance as compared to the highly organized setup of healthy tissues, cancers still show highly differentiated structures and a close interaction with and dependency on the interwoven connective tissue. This complexity within cancers is not known in detail at the molecular level so far. The first part of this article will shortly describe the technology and strategies to quantify and dissect the heterogeneity in human solid cancers. Moreover, there is urgent need to better understand human cancer biology since the development of novel anti-cancer drugs is far from being efficient, predominantly due to the scarcity of predictive preclinical models. Hence, in vivo and in vitro models were developed, which better recapitulate the complexity of human cancers, by their intrinsic three-dimensional nature and the cellular heterogeneity and allow functional intervention for hypothesis testing. Therefore, in the second part 3D in vitro cancer models are presented that analyze and depict the heterogeneity in human cancers. Advantages and drawbacks of each model are highlighted and their suitability to preclinical drug testing is discussed.
Subject(s)
Carcinoma/metabolism , Carcinoma/pathology , Cell Communication , Models, Biological , Tumor Microenvironment , Animals , Carcinoma/etiology , Cell Communication/genetics , Disease Models, Animal , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Spheroids, Cellular , Stromal Cells/metabolism , Stromal Cells/pathology , Tissue Culture Techniques , Tumor Cells, Cultured , Tumor Microenvironment/geneticsABSTRACT
BACKGROUND: The arachidonic acid metabolite 12(S)-HETE is suspected to enhance metastatic spread by inducing cancer cell- and lymph endothelial cell (LEC) motility. However, the molecular mechanisms leading to 12(S)-HETE-triggered cell migration are still elusive. METHODS: To delineate the signalling pathways involved in 12(S)-HETE-mediated migration, inhibitors against RHO and ROCK, and specific siRNAs downregulating 12(S)-HETE receptor (12-HETER) and myosin light chain 2 (MLC2) were used. The breaching of the endothelial barrier was investigated by an assay measuring tumour spheroid-triggered 'circular chemorepellent-induced defects' (CCIDs), and respective signal transduction was elucidated by western blotting. RESULTS: We provide evidence that 12(S)-HETE phosphorylated (and activated) MLC2, which regulates actin/myosin-based contraction. MLC2 activation was found to be essential for LEC retraction and CCID formation. Furthermore, we show that 12(S)-HETE activated a 12-HETER-RHO-ROCK-MYPT signalling cascade to induce MLC2 function. CONCLUSIONS: Signalling via this pathway is described for this metabolite for the first time. This may provide potential targets for the intervention of metastatic colonisation.
Subject(s)
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Endothelium, Lymphatic/metabolism , Myosin Light Chains/metabolism , Receptors, Eicosanoid/metabolism , Rho Factor/metabolism , Signal Transduction , rho-Associated Kinases/metabolism , Cell Line, Tumor , Humans , Permeability , PhosphorylationABSTRACT
Exposure to chemicals and environmental pollutants among them cadmium, lead, and mercury can harm reproduction. The metals cross the placenta, accumulate in placental tissue, and pass onto fetal blood and fetal organs to variable amounts. Still, the mechanisms underlying their transplacental passage are largely unknown and the human placenta is the most poorly understood organ in terms of reproduction toxicology. The genetic factors modulating placental toxicokinetics remain unclear just as well. From a genetic perspective, three aspects, which influence capacities of the human placenta to metabolize and transport toxicants, need to be considered. These are 1/presence and interplay of two genotypes, 2/chromosomal aberrations including aneuploidies and sequence variations, and 3/epigenetics and genetic imprinting. In this review, we summarize the current state of knowledge on how genetics and epigenetics affect placental (patho)physiology and thus fetal development and health.
Subject(s)
Cadmium/toxicity , Environmental Pollutants/toxicity , Epigenesis, Genetic , Lead/toxicity , Mercury/toxicity , Placenta/drug effects , Polymorphism, Genetic , Aneuploidy , Cadmium/metabolism , Chromosome Aberrations/chemically induced , Chromosome Aberrations/embryology , Drug Resistance , Environmental Pollutants/metabolism , Epigenesis, Genetic/drug effects , Female , Fetal Development/drug effects , Humans , Lead/metabolism , Maternal-Fetal Exchange/drug effects , Mercury/metabolism , Placenta/metabolism , Placentation/drug effects , Pregnancy , Tissue Distribution , ToxicokineticsABSTRACT
BACKGROUND: Hypoxic and necrotic regions that accrue within solid tumors in vivo are known to be associated with metastasis formation, radio- and chemotherapy resistance, and drug metabolism. Therefore, integration of these tumor characteristics into in vitro drug screening models is advantageous for any reliable investigation of the anticancer activity of novel drug candidates. In general, usage of cell culture models with in vivo like characteristics has become essential in preclinical drug studies and allows evaluation of complex problems such as tumor selectivity and anti-invasive properties of the drug candidates. MATERIALS AND METHODS: In this study, we investigated the anticancer activity of clinically approved, investigational and experimental drugs based on platinum (cisplatin, oxaliplatin and KP1537), gallium (KP46), ruthenium (KP1339) and lanthanum (KP772) in different cell culture models such as monolayers, multicellular spheroids, as well as invasion and metastasis models. Results Application of the Alamar Blue assay to multicellular spheroids and a spheroid-based invasion assay resulted in an altered rating of compounds with regard to their cytotoxicity and ability to inhibit invasion when compared with monolayer-based cytotoxicity and transwell assays. For example, the gallium-based drug candidate KP46 showed in spheroid cultures significantly enhanced properties to inhibit protrusion formation and fibroblast mediated invasiveness, and improved cancer cell selectivity. CONCLUSION: Taken together, our results demonstrate the advantages of spheroid-based assays and underline the necessity of using different experimental models for reliable preclinical investigations assessing and better predicting the anticancer potential of new compounds.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Cell Cycle/drug effects , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Cisplatin/pharmacology , Coculture Techniques , Humans , Hypoxia , Organometallic Compounds/pharmacology , Organoplatinum Compounds/pharmacology , Oxaliplatin , Oxyquinoline/analogs & derivatives , Oxyquinoline/pharmacology , Phenanthrolines/pharmacology , Spheroids, Cellular/drug effects , Spheroids, Cellular/physiology , Tumor Cells, CulturedABSTRACT
Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Amniotic Fluid/cytology , Apoptosis , Cell Differentiation , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/physiology , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cell Line , Cell Line, Tumor , Down-Regulation , Embryoid Bodies/cytology , Embryoid Bodies/physiology , Germ Layers/cytology , Humans , RNA, Small Interfering , TOR Serine-Threonine Kinases/metabolism , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/geneticsABSTRACT
Metastatic breast cancer is linked to an undesired prognosis. One early and crucial metastatic step is the interaction of cancer emboli with adjacent stroma or endothelial cells, and understanding the mechanisms of this interaction provides the basis to define new targets as well as drugs for therapy and disease management. A three-dimensional (3D) co-culture model allowing the examination of lymphogenic dissemination of breast cancer cells was recently developed which facilitates not only the study of metastatic processes but also the testing of therapeutic concepts. This 3D setting consists of MCF-7 breast cancer cell spheroids (representing a ductal and hormone-dependent subtype) and of hTERT-immortalised lymph endothelial cell (LEC; derived from foreskin) monolayers. Tumour spheroids repel the continuous LEC layer, thereby generating "circular chemorepellent-induced defects" (CCIDs) that are reminiscent to the entry gates through which tumour emboli intravasate lymphatics. We found that the ion channel blocker carbamazepine (which is clinically used to treat epilepsy, schizophrenia and other neurological disorders) inhibited CCID formation significantly. This effect correlated with the inhibition of the activities of NF-κB, which contributes to cell motility, and with the inactivation of the mobility proteins MLC2, MYPT1 and FAK which are necessary for LEC migration. NF-κB activity and cell movement are prerequisites of CCID formation. On the other hand, the expression of the motility protein paxillin and of the NF-κB-dependent adhesion mediator ICAM-1 was unchanged. Also the activity of ALOX12 was unaffected. ALOX12 is the main enzyme synthesising 12(S)-HETE, which then triggers CCID formation. The relevance of the inhibition of CYP1A1, which is also involved in the generation of mid-chain HETEs such as 12(S)-HETE, by carbamazepine remains to be established, because the constitutive level of 12(S)-HETE did not change upon carbamazepine treatment. Nevertheless, the effect of carbamazepine on the inhibition of CCID formation as an early step of breast cancer metastasis was significant and substantial (~30 %) and achieved at concentrations that are found in the plasma of carbamazepine-treated adults (40-60 µM). The fact that carbamazepine is a drug approved by the US Food and Drug Administration facilitates a "from-bench-to-bedside" perspective. Therefore, the here presented data should undergo scrutiny in vivo.
Subject(s)
Carbamazepine/pharmacology , Cell Culture Techniques/methods , Drug Screening Assays, Antitumor/methods , Endothelial Cells/drug effects , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Arachidonate 12-Lipoxygenase/metabolism , Cardiac Myosins/metabolism , Coculture Techniques , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1A1/metabolism , Endothelial Cells/cytology , Focal Adhesion Kinase 1/metabolism , Humans , MCF-7 Cells/drug effects , MCF-7 Cells/pathology , Myosin Light Chains/metabolism , Myosin-Light-Chain Phosphatase/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Phosphorylation/drug effects , Spheroids, Cellular/drug effectsABSTRACT
Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.
Subject(s)
Cell Migration Assays , Neoplasm Invasiveness , Cell Migration Assays, Leukocyte , Cell Movement , Humans , Spheroids, CellularABSTRACT
Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Cell Movement/drug effects , Endothelial Cells/drug effects , Flavonoids/pharmacology , Propiophenones/pharmacology , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Biomarkers, Tumor/metabolism , Blotting, Western , Breast Neoplasms/metabolism , Cell Adhesion/drug effects , Coculture Techniques , Cytochrome P-450 CYP1A1/metabolism , Dose-Response Relationship, Drug , E-Selectin/metabolism , Endothelial Cells/metabolism , Endothelial Cells/pathology , Epithelial-Mesenchymal Transition/drug effects , Female , HEK293 Cells , Humans , Intercellular Adhesion Molecule-1/metabolism , MCF-7 Cells , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Invasiveness , Spheroids, Cellular , TransfectionABSTRACT
Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.
Subject(s)
Breast Neoplasms/drug therapy , Endothelial Cells/drug effects , Lymphatic Metastasis/prevention & control , Terpenes/pharmacology , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Blotting, Western , Breast Neoplasms/pathology , Cell Movement , Coculture Techniques , Cytochrome P-450 CYP1A1/metabolism , Endothelial Cells/metabolism , Female , Humans , MCF-7 Cells , NF-kappa B/metabolism , Paxillin/metabolismABSTRACT
More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are present in 40% of colorectal cancer (CRC) in the two main CRC subgroups, MSS (Microsatellite Stable) and MSI (Microsatellite Instable). Studies in RAS-driven tumors have shown essential roles of the RAS effectors RAF and specifically of RAF1, which can be dependent or independent of RAF's ability to activate the MEK/ERK module. In this study, we demonstrate that RAF1, but not its kinase activity, plays a crucial role in the proliferation of both MSI and MSS CRC cell line-derived spheroids and patient-derived organoids, and independently of KRAS mutation status. Moreover, we could define a RAF1 transcriptomic signature which includes genes that contribute to STAT3 activation, and could demonstrate that RAF1 ablation decreases STAT3 phosphorylation in all CRC spheroids tested. The genes involved in STAT3 activation as well as STAT3 targets promoting angiogenesis were also downregulated in human primary tumors expressing low levels of RAF1. These results indicate that RAF1 could be an attractive therapeutic target in both MSI and MSS CRC regardless of their KRAS status and support the development of selective RAF1 degraders rather than RAF1 inhibitors for clinical use in combination therapies.
Subject(s)
Colorectal Neoplasms , Humans , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins B-raf/genetics , Microsatellite Repeats , Mutation , Microsatellite Instability , Cell Proliferation/genetics , STAT3 Transcription Factor/geneticsABSTRACT
BACKGROUND: Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Unlike many other cancers, PM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several malignancies. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on PM cells. METHODS: Meso-CAFs were isolated from surgical specimens of PM patients and analyzed by array comparative genomic hybridization, next generation sequencing, transcriptomics and proteomics. Human PM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, as well as the response to small molecule inhibitors, cisplatin and pemetrexed treatment was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis. RESULTS: Meso-CAFs show a normal diploid genotype without gene copy number aberrations typical for PM cells. They express CAF markers and lack PM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in significantly increased proliferation and migration of PM cells. A similar effect on PM cell growth and migration was induced by Meso-CAF-conditioned medium. Inhibition of c-Met with crizotinib, PI3K with LY-2940002 or WNT signaling with WNT-C59 significantly impaired the Meso-CAF-mediated growth stimulation of PM cells in co-culture at concentrations not affecting the PM cells alone. Meso-CAFs did not provide protection of PM cells against cisplatin but showed significant protection against the EGFR inhibitor erlotinib. CONCLUSIONS: Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on PM cell growth and migration, two key characteristics of PM aggressiveness, indicating a major role of Meso-CAFs in driving PM progression. Moreover, we identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for novel therapeutic strategies against PM.
Subject(s)
Mesothelioma, Malignant , Mesothelioma , Humans , Cisplatin/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Wnt Signaling Pathway , Comparative Genomic Hybridization , Mesothelioma/drug therapy , Mesothelioma/genetics , Mesothelioma/metabolism , Mesothelioma, Malignant/metabolism , Fibroblasts/metabolism , Cell Proliferation , Cell Line, Tumor , Tumor MicroenvironmentABSTRACT
BACKGROUND & AIMS: Patient-derived organoid cancer models are generated from epithelial tumor cells and reflect tumor characteristics. However, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we developed a colorectal cancer organoid model that incorporates matched epithelial cells and stromal fibroblasts. METHODS: Primary fibroblasts and tumor cells were isolated from colorectal cancer specimens. Fibroblasts were characterized for their proteome, secretome, and gene expression signatures. Fibroblast/organoid co-cultures were analyzed by immunohistochemistry and compared with their tissue of origin, as well as on gene expression levels compared with standard organoid models. Bioinformatics deconvolution was used to calculate cellular proportions of cell subsets in organoids based on single-cell RNA sequencing data. RESULTS: Normal primary fibroblasts, isolated from tumor adjacent tissue, and cancer associated fibroblasts retained their molecular characteristics in vitro, including higher motility of cancer associated compared with normal fibroblasts. Importantly, both cancer-associated fibroblasts and normal fibroblasts supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. Organoids grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared with mono-cultures and closely resembled the in vivo tumor morphology. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by considerably deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. Thrombospondin-1 was identified as a critical factor for fibroblast invasiveness. CONCLUSION: We developed a physiological tumor/stroma model, which will be vital as a personalized tumor model to study disease mechanisms and therapy response in colorectal cancer.
Subject(s)
Cancer-Associated Fibroblasts , Colorectal Neoplasms , Humans , Fibroblasts/metabolism , Coculture Techniques , Organoids/metabolism , Cancer-Associated Fibroblasts/metabolism , Colorectal Neoplasms/pathology , Tumor MicroenvironmentABSTRACT
Human amniotic fluid stem cells (hAFSCs) can be grown in large quantities, have a low risk for tumour development and harbour a high differentiation potential. They are a very promising new fetal stem cell type for cell-based therapy approaches and for studying differentiation processes without raising the ethical concerns associated with embryonic stem cells. Recently, a protocol for studies on renal development has been established in which murine embryonic kidneys are dissociated into single-cell suspension and then reaggregated to form organotypic renal structures. Using this approach, we formed chimeric renal structures via mixing murine embryonic kidney cells with monoclonal hAFSCs. We demonstrate here that hAFSCs harbour the potential to contribute to renal tissue formation accompanied by induction of specific renal marker expression. As part of the two kinase complexes mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signalling pathway, which is involved in the regulation of differentiation and in the development of a wide variety of human genetic diseases many with characteristic kidney symptoms. Modulating endogenous mTOR activity via specific siRNA approaches revealed that contribution of hAFSCs to renal tissue formation is regulated by mTORC1 and mTORC2. These findings (i) demonstrate renal differentiation potential of hAFSCs, (ii) prove chimeric cultures of mixtures of murine embryonic kidney cells and hAFSCs to be a powerful tool to study the effects of gene knockdowns for renal structure formation and (iii) provide new insights into the role of the mTOR pathway for renal development.