3D-cultured Cancer Cells Research Articles

DNA templated Click Chemistry via 5-vinyl-2′-deoxyuridine and an acridine-tetrazine conjugate induces DNA damage and apoptosis in cancer cells

AimsClick Chemistry is providing valuable tools to biomedical research, but its direct use in therapies remains nearly unexplored. For cancer treatment, nucleoside analogues (NA) such as 5-vinyl-2′-deoxyuridine (VdU) can be metabolically incorporated into cancer cell DNA and subsequently “clicked” to form a toxic product. The inverse electron-demand Diels-Alder (IEDDA) reaction between VdU and an acridine-tetrazine conjugate (PINK) has previously been used to label cell nuclei of cultured cells. Here, we report tandem usage of VdU and PINK to induce cytotoxicity. Main methodsCell lines were subsequently treated with VdU and PINK, and cell viability was measured via well confluency and 3D tumor spheroid assays. DNA damage and apoptosis were evaluated using Western Blotting and cell cycle analysis by flow cytometry. Double stranded DNA break (DSB) formation was measured using the comet assay. Apoptosis was assessed by fluorescent detection of externalized phosphatidylserine residues. Key findingsWe report that the combination of VdU and PINK synergistically induces cytotoxicity in cultured human cells. The combination of VdU and PINK strongly reduced cell viability in 2D and 3D cultured cancer cells. Mechanistically, the compounds induced DNA damage through DSB formation, which leads to S-phase accumulation and apoptosis. SignificanceThe combination of VdU and PINK represents a novel and promising DNA-templated “click” approach for cancer treatment via selective induction of DNA damage.

13C metabolic flux analysis clarifies distinct metabolic phenotypes of cancer cell spheroid mimicking tumor hypoxia

Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by 13C-metabolic flux analysis (13C-MFA). Principal component analysis of 13C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. 13C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism.

Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells.

Simple SummaryThe EGFR is a tyrosine kinase receptor that responds to different stresses such as UV irradiation, hypoxia and drug treatment by internalizing into endosomal compartments. Receptor trafficking and degradation due to tyrosine kinase inhibitors has been widely studied in two- dimensional (2D) cell culture systems, but little is known about how cells respond to these types of drugs in more physiologically relevant models such as three-dimensional (3D) cultures, whose nanostructured properties allow cells to grow, proliferate, migrate and extend cellular processes in their 3D space. In this study, we show that EGFR suffers degradation in response to erlotinib treatment in 3D-cultured cancer cells but not in classic 2D culture systems, demonstrating that dimensionality strongly affects cell drug response. This 3D model may pave the way for the development of more physiological culture platforms to obtain mechanistic insights into how cells respond to chemotherapy.The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.

Generation of Rat Monoclonal Antibody for Human IQGAP1 by Immunization of Three-Dimensional-Cultured Cancer Cells.

The scaffold protein IQ motif containing GTPase activating protein 1 (IQGAP1) is an adherens junction component in the epithelial tissue that binds many signaling and structural molecules to regulate biological processes. It is known that IQGAP1 is overexpressed in some tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from three-dimensional (3D)-cultured DLD-1 cells to elucidate a characteristic feature of a tumor. In cancer research, 3D-cultured cancer cells are used as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. Our results showed that mAb 7E11 recognized increasing antigen in the lysate of 3D-cultured cells comparing with two-dimensional-cultured cells, and its antigen is the human IQGAP1. Furthermore, we indicated that mAb 7E11 was used in immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful in the analysis of human cancer.

G-quadruplex-forming nucleic acids interact with splicing factor 3B subunit 2 and suppress innate immune gene expression.

G‐quadruplex (G4), a non‐canonical higher‐order structure formed by guanine‐rich nucleic acid sequences, affects various genetic events in cis, including replication, transcription and translation. Whereas up‐regulation of innate immune/interferon‐stimulated genes (ISGs) is implicated in cancer progression, G4‐forming oligonucleotides that mimic telomeric repeat‐containing RNA suppress ISG induction in three‐dimensional (3D) culture of cancer cells. However, it is unclear how G4 suppresses ISG expression in trans. In this study, we found that G4 binding to splicing factor 3B subunit 2 (SF3B2) down‐regulated STAT1 phosphorylation and ISG expression in 3D‐cultured cancer cells. Liquid chromatography‐tandem mass spectrometry analysis identified SF3B2 as a G4‐binding protein. Either G4‐forming oligonucleotides or SF3B2 knockdown suppressed ISG induction, whereas Phen‐DC3, a G4‐stabilizing compound, reversed the inhibitory effect of G4‐forming oligonucleotides on ISG induction. Phen‐DC3 inhibited SF3B2 binding to G4 in vitro. SF3B2‐mediated ISG induction appeared to occur independently of RNA splicing because SF3B2 knockdown did not affect pre‐mRNA splicing under the experimental conditions, and pharmacological inhibition of splicing by pladienolide B did not repress ISG induction. These observations suggest that G4 disrupts the ability of SF3B2 to induce ISGs in cancer. We propose a new mode for gene regulation, which employs G4 as an inhibitory trans‐element.

Microencapsulation of low-passage poorly-differentiated human mucoepidermoid carcinoma cells by alginate microcapsules: in vitro profiling of angiogenesis-related molecules

BackgroundHuman mucoepidermoid carcinoma (MEC) is regarded as the most common primary salivary malignancy. High-grade MEC has a high risk of recurrence and poor prognosis. Tumor angiogenesis, induced by poorly differentiated cancer cells of high-grade MEC, contributes to tumor growth and metastasis. Therefore, elucidating molecular mechanisms underlying the pro-angiogenic ability of poorly differentiated MEC cells is critical for the understanding of high-grade MEC progression. It is well known that three-dimensional (3D) cell culture, in contrast with conventional two-dimensional (2D) culture, provides a better approach to in vitro recapitulation of in vivo characteristics of cancer cells and their surrounding microenvironment. The purpose of this study was to model a 3D environment for in vitro gene expression profiling of key molecules in poorly differentiated MEC cells for cancer neovascularization and compared them with traditional 2D cell culture.MethodsLow-passage poorly differentiated MEC cells, derived from human patient samples of high-grade MEC, were microencapsulated in sodium alginate gel microcapsules (3D culture) and compared with cells grown in 2D culture. Cancer cell proliferation was determined by MTT assays for 1 week, and gene expression of VEGF-A, bFGF and TSP-1 was analyzed by western blotting or ELISA. The hypoxic environment in 3D versus 2D culture were assessed by western blotting or immunofluorescence for HIF1α, and the effect of hypoxia on VEGF-A gene expression in 3D cultured cancer cells was assessed by western blotting with the use of the HIF1α inhibitor, 2-methoxyestradiol (2-MeOE2).ResultsWhen encapsulated in alginate gel microcapsules, low-passage poorly differentiated human MEC cells grew in blocks and demonstrated stronger and relatively unlimited proliferation activities. Moreover, significant differences were found in gene expression, with 3D-grown cancer cells a significant increment of VEGF-A and bFGF and a drastic reduction of TSP-1. Consistently, 3D-grown cancer cells secreted significantly more VEGF-A than 2D culture cancer cells. Furthermore, 3D-grown cancer cells showed significantly higher expression of HIF1α, a molecular indicator of hypoxia; the increased expression of VEGF-A in 3D cultured cancer cells was shown to be dependent on the HIF1α activities.ConclusionsThe present work shows the effects of 3D culture model by alginate microencapsulation on the proangiogenic potentials of low-passage poorly differentiated human MEC cells. Cancer cells in this 3D system demonstrate significant intensification of key molecular processes for tumor angiogenesis. This is due to a better modeling of the hypoxic tumor microenvironment during 3D culture.

A photo-polymerized poly(Nε-acryloyl l-lysine) hydrogel for 3D culture of MCF-7 breast cancer cells.

The most common in vitro cell culture platform, standard two-dimensional (2D) monolayer cell culture, often fails to mimic the tumor microenvironment, while animal models complicate research on the effect of individual factors on cell behaviors. Both are unsatisfactory in the research of molecular mechanisms of tumor development and progression and the discovery and development of anticancer drugs. In vitro three-dimensional (3D) cell culture can partially simulate in vivo conditions and 3D-cultured cancer cells can recapture many essential features of native tumor tissues. In this study, to mimic the in vivo breast tumor microenvironment, novel reduction-responsive poly(Nε-acryloyl l-lysine) (pLysAAm) hydrogels were synthesized by rapid photo-polymerization of Nε-acryloyl l-lysine and using N,N'-bis(acryloyl)-(l)-cystine as a crosslinker, and their physicochemical properties were characterized systemically. The results showed that the pLysAAm hydrogels were formed within 93 s under UV irradiation and exhibited almost total elastic recovery from compressions as high as 75%. The lyophilized hydrogel samples displayed a highly porous structure with interconnected pores, had an equilibrium swelling ratio of about 20, and were degraded faster in a glutathione-containing solution than in PBS solution. The biological versatility of the pLysAAm hydrogels was demonstrated by both in vitro MCF-7 cell culture and in vivo tumor formation. Compared to cells cultured as 2D monolayers, the 3D-cultured cells presented 3D cell morphology, exhibited better cell viability, expressed higher levels of pro-angiogenic factors, and showed significantly greater migration and invasion abilities. The results from assay of tumorigenicity in nude mice and histologic analysis demonstrated the enhanced tumorigenic and angiogenic capabilities of the MCF-7 cells pre-cultured in pLysAAm hydrogels. These findings suggest that pLysAAm hydrogels may be used to bridge the gap between standard in vitro cell cultures and living tissues, aid breast cancer research, and help researchers to develop novel anticancer therapeutics.

3D Tumor Models and Time-Lapse Analysis by Multidimensional Microscopy.

The 3D culture is advantageous in reflecting the in vivo condition compared to the 2D culture; however, imaging 3D-cultured cells may be a challenge due to technical restrictions. Recent development of confocal spinning disc microscope system as well as sophisticated software has enabled us to monitor dynamism of cell movement in multiple dimensions. Here we describe the method for time-lapse imaging of 3D-cultured cancer cells co-cultured with non-cancerous cells and discuss current limitations and future perspectives.

Mitochondrial DNA reduced by hypoxic conditions in three-dimensional (3D) spheroid cell cultures.

Three-dimensional (3D) cell culture reflects many of the important properties of solid tumors, such as the inadequate diffusion of oxygen that results in hypoxia. To understand the mitochondrial states in cancer, we performed comparisons of the levels of mitochondrial DNA (mtDNA), fusion- and fission-related mitochondrial messenger RNA (mRNA), and mitochondrial protein expression between monolayer (2D)- and 3D-cultured cancer cells. The mtDNA levels were observed to be significantly lower in the 3D cells compared with the monolayer cells. In contrast, the differences in expression of the mitochondrial fusion- and fission-related mRNAs and mitochondrial proteins between 2D- and 3D-cultured cancer cells were not significant, as shown by real-time PCR and immunoblot analysis. Therefore, although mtDNA levels decrease as a whole during 3D culture, this does not appear to affect the fusion and fission of individual mitochondria. Indeed, the factors regulating mitochondrial dynamics during 3D cell culture remain unclear. This study provides the basis for future, more detailed studies on the regulation of mtDNA.