Tumor Microenvironment Responsive TPZ-Loaded Core-Shell Polymeric Nanoparticles for Selective Cancer Bioreductive Therapy

PurposeTwo-dimensional (2D) cell culture is a valuable method for cell-based research but can provide unpredictable, misleading data about in vivo responses. In this study, we created a three-dimensional (3D) cell culture environment to mimic tumor characteristics and cell-cell interactions to better characterize the tumor formation response to chemotherapy.Materials and methodsWe fabricated the 3D cell culture samples using a 3D cell bio printer and the bladder cancer cell line 5637. T24 cells were used for 2D cell culture. Then, rapamycin and Bacillus Calmette-Guérin (BCG) were used to examine their cancer inhibition effects using the two bladder cancer cell lines. Cell-cell interaction was measured by measuring e-cadherin and n-cadherin secreted via the epithelial-mesenchymal transition (EMT).ResultsWe constructed a 3D cell scaffold using gelatin methacryloyl (GelMA) and compared cell survival in 3D and 2D cell cultures. 3D cell cultures showed higher cancer cell proliferation rates than 2D cell cultures, and the 3D cell culture environment showed higher cell-to-cell interactions through the secretion of E-cadherin and N-cadherin. Assessment of the effects of drugs for bladder cancer such as rapamycin and BCG showed that the effect in the 2D cell culture environment was more exaggerated than that in the 3D cell culture environment.ConclusionsWe fabricated 3D scaffolds with bladder cancer cells using a 3D bio printer, and the 3D scaffolds were similar to bladder cancer tissue. This technique can be used to create a cancer cell-like environment for a drug screening platform.

Background Radiofrequency ablation (RFA) is a common procedure for the management of colorectal liver metastases. RFA-generated lesions are surrounded by a rim of hypoxia that is associated with aggressive outgrowth of intrahepatic micrometastases. Hypoxia-activated pro-drugs such as tirapazamine (TPZ) are designed to selectively kill tumour cells under hypoxic conditions. Therefore we hypothesised that TPZ may have therapeutic value in limiting hypoxia-associated tumour outgrowth following RFA. Methods Murine C26 and MC38 colorectal cancer cells were grown under hypoxia and normoxia in vitro and were treated with different concentrations of TPZ. Apoptosis and cell cycle distribution were assessed by western blot and FACS analysis. Proliferative capacity was tested by colony formation assays. Mice harbouring microscopic colorectal liver metastases were treated with RFA, followed by a single injection of TPZ (60 mg/kg) or saline. Tumour load was assessed morphometrically 7 days later. Results TPZ induced apoptosis of colorectal tumour cells under hypoxia in vitro, which completely abolished clone-forming capacity. Under normoxia, TPZ caused a G2 cell cycle arrest from which cells recovered partly. This reduced, but did not abolish, colony-forming capacity. A single dose of TPZ largely prevented accelerated outgrowth of hypoxic micrometastases following RFA. TPZ administration was associated with minimal toxicity. Conclusions TPZ kills colorectal cancer cells in a hypoxia-dependent manner and potently suppresses hypoxia-associated outgrowth of liver metastases with limited toxicity. This warrants further studies to assess the potential value of TPZ, or other hypoxia-activated pro-drugs, as adjuvant therapeutics following RFA treatment of colorectal liver metastases. 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 3676. doi:1538-7445.AM2012-3676

Three-dimensional (3D) cell culture has developed rapidly over the past 5-10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.

The effect of IFNα-2b on the migration, proliferation, and expression of epithelial and mesenchymal markers of MCF-7 tumor adenocarcinoma cells in 2D and 3D cell cultures was examined. A significant cytostatic effect of IFNα-2b on the tumor population was detected. It was found that changes in the expression of epithelial (CKs and EpCAM) and mesenchymal markers were caused by changing the growth type of the tumor population. IFNα-2b inhibited migration of tumor cells to the suspension fraction and promoted an increase in expression of CK and EpCAM in 2D and 3D cell cultures, but only in the 3D culture was expression of vimentin increased. IFNα-2b caused an increase in CK and EpCAM expression by 50.5% and 47.8%, respectively, compared with the control in the 2D cell culture. In the 3D cell culture this increase was 33% and 34%, respectively, compared with the control. IFNα-2b stimulated the differentiation and inhibited the migrational ability of tumor cells in the early stages of breast cancer development.

Artonin E is a naturally isolated compound from Artocarpus elasticus and was evaluated for their anticancer property in the present study. This study aimed to develop a 3D ovarian cell culture model and to examine the cytotoxicity of artonin E in 2D and 3D cell culture models. The 3D cell culture was performed using BD™ Puramatrix™ Peptide Hydrogel. Alamar blue assay, selectivity index analysis, morphological apoptotic double staining and immunofluorescence study were conducted to study the antiproliferative and apoptotic induction mechanism of artonin E in 3D SKOV-3 spheroid culture. SKOV-3 cells encapsulated in BD™ Puramatrix™ Peptide Hydrogel clearly demonstrated 3D-spheroid formation. The Alamar blue assay result showed that artonin E inhibited the growth of SKOV-3 cells in 2D and 3D culture with the IC50 values at 72 h treatment of 6.0 ± 0.8 µg/mL and 25.0 ± 0.8 µg/mL, respectively. This compound was found to be less toxic to the normal human ovarian cell lines, T1074, with IC50 values at 72 h of 28.0 ± 0.8 µg/mL in 2D culture and 85.0 ± 0.5 µg/mL in 3D culture, respectively. The selectivity index analysis more than 2 indicated that artonin E was selective against cancer cells compared to normal cells. Artonin E treatment caused apoptotic morphological changes in 3D SKOV-3 spheroids. In a 3D immunofluorescence study, elevated levels of cleaved caspase-3, cleaved caspase-9, apoptotic proteins bax, and decreased levels of antiapoptotic proteins bcl-2, Hsp70, and survivin were observed. In conclusion, artonin E increases chemoresistance and induces cell death in a 3D SKOV-3 spheroids culture via a pro- and anti-apoptotic protein pathway. These findings demonstrate that 3D spheroid culture is an effective platform for testing artonin E therapeutic candidates in an in vivo mimic microenvironment.

A combination of three-dimensional (3D) cell culturing and non-viral gene transfection is promising in improving outcomes of cell transplantation therapy. Herein, gene transfection profiles in 3D cell culture were compared between plasmid DNA (pDNA) and messenger RNA (mRNA) introduction, using mesenchymal stem cell (MSC) 3D spheroids. Green fluorescence protein (GFP) mRNA induced GFP protein expression in 77% of the cells in the spheroids, whereas only 34% of the cells became GFP positive following pDNA introduction. In mechanistic analyses, most of the cells in MSC spheroids were non-dividing, and pDNA failed to induce GFP expression in most of the non-dividing cells. In contrast, both dividing and non-dividing cells became GFP-positive after mRNA introduction, which led to a high overall percentage of GFP-positive cells in the spheroids. Consequently, mRNA encoding an osteogenic factor, runt-related transcription factor 2 (Runx2), allowed in vitro osteogenic differentiation of MSCs in spheroids more efficiently compared to Runx2 pDNA. Conclusively, mRNA exhibits high potential in gene transfection in 3D cell culture, in which the cell division rate is lower than that in monolayer culture, and the combination of mRNA introduction and 3D cell culture is a promising approach to improve outcomes of cell transplantation in future regenerative therapy.

BackgroundThree-dimensional (3D) cell cultures are the new frontier for reproducing the tumor micro-environment in vitro. The aims of the study were (1) to establish primary 3D cell cultures from canine spontaneous neoplasms and (2) to demonstrate the morphological, phenotypic and genotypic similarities between the primary canine neoplasms and the corresponding 3D cultures, through the expression of tumor differentiation markers.ResultsSeven primary tumors were collected, including 4 carcinomas and 3 soft tissue sarcomas. 3D cell cultures reproduced the morphological features of the primary tumors and showed an overlapping immunophenotype of the primary epithelial tumors. Immunohistochemistry demonstrated the growth of stromal cells and macrophages admixed with the neoplastic epithelial component, reproducing the tumor microenvironment. Mesenchymal 3D cultures reproduced the immunophenotype of the primary tumor completely in 2 out of 3 examined cases while a discordant expression was documented for a single marker in one case. No single nucleotide variants or small indel were detected in TP53 or MDM2 genes, both in primary tumors and in 3D cell cultures specimens. In one sample, MDM2 amplicons were preferentially increased in number compared to TP53 ones, indicating amplification of MDM2, detectable both in the primary tumor and in the corresponding cell culture specimen.ConclusionHere we demonstrate a good cell morphology, phenotype and genetic profile overlap between primary tumors and the corresponding 3D cultures grown in a repeatable system.

BackgroundDespite the success of immunotherapy in ‘hot’ tumors like melanoma with pre-existing immune infiltrates, ‘cold’ tumors like prostate cancer remain unresponsive.1–3 We identified that these tumors harbor regions of hypoxia...

In Vitro three-dimensional (3D) cell culture tools for spheroid and organoid models.

WITHDRAWN: In Vitro three-dimensional (3D) cell culture tools for spheroid and organoid models

Recently, the 3D spheroid cell culture application has been extensively used in the treatment of bone defects. A wide variety of methodologies have been used, which has made the comparison of results complex. Therefore, this systematic review has two aims: (i) to perform an analysis focused on the role of 3D spheroid cell culture in bone regeneration strategies; and (ii) address the main challenges in clinical application. A search of the following keywords "3D cell culture", "spheroid", and "bone regeneration" was carried out in the PubMed, Scopus, and ScienceDirect databases and limited to the years 2010-2020. Studies were included if their primary objective was the behavior of cell aggregates to formed spheroids structures by different 3D cell culture techniques focused on the regeneration of bone tissue. To address the risk of bias for in vitro studies, the United States national toxicology program tool was applied, and descriptive statistics of the data were performed, with the SPSS V.22 program. A total of 16 studies were included, which met the established criteria corresponding to in vitro and in vitro/in vivo studies; most of these studies used stem cells for the 3D cell spheroids. The most often methods used for the 3D formation were low adherence surface and rotational methods, moreover, mesenchymal stem cells were the cell line most frequently used because of their regenerative potential in the field of bone tissue engineering. Although the advances in research on the potential use of 3D spheroids in bone regeneration have made great strides, the constant innovation in cell spheroid formation methodologies means that clinical application remains in the future as strategy for 3D tissue bioprinting.

Cancer is one of the leading causes of death in the world. A great deal of effort has been made to discover new agents for cancer treatment. Xao tam phan (Paramignya trimera) is a traditional medicine of Vietnam used in cancer treatment for a long time, yet there is not much scientific evidence proving its anticancer potency. The study aimed to evaluate the toxicity of Paramignya trimera extract (PTE) on multicellular tumor spheres (MCTS) of MCF-7 cells using hanging drop technique. Firstly, MCF-7 cells were seeded on hanging drop plates, spheroid size was tracked, and growth curve was measured by MTT assay and AlamarBlue® assay. The necrotic core of MCTS was evaluated by propidium iodide (PI) staining. Toxicity of doxorubicin (DOX) and tirapazamine (TPZ) was then tested on 3D model compared to 2D culture condition. The results showed that the IC50 of DOX on 3D MCF-7 cells was nearly 50 times greater than monolayer MCF-7 cells. In contrast, TPZ (an agent which is specifically toxic under hypoxic conditions) had significantly lower IC50 in 3D condition than in 2D. The toxicity tests for PTE showed that PTE strongly inhibited MCF-7 cells in both 2D and 3D conditions. Interestingly, the IC50 of PTE in 3D model was remarkably lower than in 2D (IC50 value was 168.9±11.65μg/ml compared to 260.8±16.54μg/ml, respectively). The invasion assay showed that PTE completely inhibited invasion of MCF-7 cells at 250μg/mL concentration. Also, flow cytometry results indicated that PTE effectively induced apoptosis in MCF-7 spheroids in 3D condition at 250μg/mL concentration. The results from this study emphasize the promise of PTE in cancer therapy.

This study aims to evaluate the bioeffects of glutathione-responsive β-cyclodextrin-based nanosponges (GSH-NSs) on two- (2D) and three-dimensional (3D) cell cultures. The bioeffects of two types of GSH-NS formulations, with low (GSH-NS B) and high (GSH-NS D) disulfide-bond content, were evaluated on 2D colorectal (HCT116 and HT-29) and prostatic (DU-145 and PC3) cancer cell cultures. In particular, the cellular uptake of GSH-NS was evaluated, as their effects on cell growth, mitochondrial activity, membrane integrity, cell cycle distribution, mRNA expression, and reactive oxygen species production. The effect of GSH-NSs on cell growth was also evaluated on multicellular spheroids (MCS) and a comparison of the GSH-NS cell growth inhibitory activity, in terms of inhibition concentration (IC)50 values, was performed between 2D and 3D cell cultures. A significant decrease in 2D cell growth was observed at high GSH-NS concentrations, with the formulation with a low disulfide-bond content, GSH-NS B, being more cytotoxic than the formulation with a high disulfide-bond content, GSH-NS D. The cell growth decrease induced by GSH-NS was owing to G1 cell cycle arrest. Moreover, a significant down-regulation of mRNA expression of the cyclin genes CDK1, CDK2, and CDK4 and up-regulation of mRNA expression of the cyclin inhibitor genes CDKN1A and CDKN2A were observed. On the other hand, a significant decrease in MCS growth was also observed at high GSH-NS concentrations, but not influenced by the nanosponge disulfide-bond content, with the MCS IC50 values being significantly higher than those obtained on 2D cell cultures. GSH-NSs are suitable nanocarries as they provoke limited cellular effects, as cell cycle arrest only occurred at concentrations significantly higher than those used for drug delivery.

Tirapazamine (TPZ) is an anticancer drug with highly selective cytotoxicity toward hypoxic cells. TPZ is converted to a radical intermediate under hypoxic conditions, and this intermediate interacts with intracellular macromolecules, including DNA. TPZ has been reported to indirectly induce DNA double-strand breaks (DSBs) through the formation of various intermediate DNA lesions under hypoxic conditions. Although the topoisomerase II-DNA complex has been identified as one of these intermediates, other lesions have not yet been defined. In order to obtain a deeper understanding of the mechanisms responsible for the selective cytotoxicity of TPZ toward hypoxic cells, its cellular sensitivity was systematically examined with genetically isogenic DNA-repair-deficient mutant DT40 cell lines. Our results showed that tdp1-/-, tdp2-/-, parp1-/-, and aptx1-/- cells displayed hypersensitivity to TPZ only under hypoxic conditions. These results strongly suggest that the accumulation of the topoisomerase I-trapped DNA complex, topoisomerase II-trapped DNA complex, and abortive ligation products with 5'-AMP are the potential causes of TPZ-induced hypoxic cell death. Furthermore, our genetic analysis revealed that under normoxic conditions (as well as hypoxic conditions), TPZ exhibited significant cytotoxicity toward cell lines deficient in homologous recombination, nonhomologous end joining, base excision repair, and translesion synthesis. Ascorbic acid, a radical scavenger, suppressed TPZ-induced cytotoxicity toward normoxic cells. These results suggest the involvement of oxidative DNA damage and DSBs produced by reactive oxygen species generated from superoxide, a byproduct of the oxidation of TPZ radical intermediates in normoxic cells. Collectively, our results demonstrate that TPZ induces oxidative DNA damage under normoxic and hypoxic conditions and selectively introduces abortive topoisomerase-DNA complexes and unligatable DNA ends under hypoxic conditions.

Traditionally neuroblastoma research has predominantly used 2D cell lines, which, despite their simplicity, often fall short in mirroring the physiological complexity of actual tumors. Recently, 3D models like patient-derived tumor organoids (PDO) and patient-derived xenografts (PDX) have emerged as superior alternatives, offering more realistic predictions of patient treatment responses. This study aims to discern the differences in drug response between conventional 2D cell lines and the more advanced 3D neuroblastoma models - specifically, patient-derived organoids (nPDO) and patient-derived xenograft organoids (nPDXO). Employing high-throughput drug screening of 528 cancer drugs, we evaluated the drug responses across traditional 2D cell lines, nPDOs, and nPDXOs. Additionally, focused experiments with 29 selected drugs were conducted to determine the influence of culture topology on drug efficacy. The findings were compared to a study by Hansson et al.1 that used nPDXO. Our study discovered a pronounced difference in drug sensitivity between 2D cell lines and 3D models, with 3D models showing heightened sensitivity. Interestingly, 2D cell cultures had a more heterogenous drug response compared to 3D models. The drug response correlation between nPDOs and nPDXOs was relatively high (Spearman ~0.8), indicating consistent responses across these models. Our comparison underscores the impact of model system choice on drug response studies in neuroblastoma. The study highlights important differences in drug response between neuroblastoma 2D and 3D cell culture models. However, only subtle differences were found between PDO and PDX-derived organoid lines, despite differences in treatment centers and media compositions. This suggests that while the choice of cell culture topology greatly impacts drug response, PDO and PDX derived lines can largely be used interchangeably for drug screening experiments.