Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow.

Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays. We provide a brief overview of successful, perspective, and hopeful attempts at using genetically encoded fluorescent sensors in HTS of modulators of ion channels, Ca2+ homeostasis, GPCR activity, and for screening cytotoxic, anticancer, and anti-parasitic compounds. We discuss the advantages of sensors in whole organism drug screening models and the perspectives of the combination of human disease modeling by CRISPR techniques with genetically encoded fluorescent sensors for drug screening.

Unveiling the Power of Anticancer Drug Screening: A Clinical Case Study Comparing the Effectiveness of Hollow Fiber Assay Microtube Array Membrane (MTAM-HFA) in Breast Cancer Patients.

Breast cancer is a severe public health problem, and early treatment with powerful anticancer drugs is critical for success. The researchers investigated the clinical results of a novel screening tool termed Microtube Array Membrane Hollow Fiber Assay (MTAM-HFA) in breast cancer patients in this clinical investigation. In all trial participants, the MTAM-HFA was utilized to identify active medicines for the treatment of breast cancer. The MTAM-HFA was shown to be extremely useful in predicting patient response to anticancer medication therapy in this study. Furthermore, the substantial association between the MTAM-HFA screening outcome and the clinical outcome of the respective patients emphasizes the promise of this unique screening technology in discovering effective anticancer medication combinations for the treatment of breast cancer. These findings indicate that the MTAM-HFA has clinical significance and might be a valuable tool in the development of tailored therapy for cancer care. This study provides helpful information for physicians and scientists working on breast cancer therapy research. The potential benefits of employing MTAM-HFA to find accurate therapies for breast cancer patients might lead to enhanced personalized medicine approaches to cancer care, resulting in better patient outcomes. Overall, the MTAM-HFA screening approach has the potential to revolutionize customized cancer therapy, providing hope to both patients and physicians.

Establishment and characterization of two novel patient-derived cell lines from giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a rare bone tumor with osteolytic features, composed of stromal cells with a monotonous appearance, macrophages, and osteoclast-like giant cells. GCTB is commonly associated with a pathogenic mutation in the H3-3A gene. While complete surgical resection is the standard cure for GCTB, it often results in local recurrence and, rarely, metastasis. Thus, an effective multidisciplinary treatment approach is necessary. Although patient-derived cell lines is an essential tool for investigating novel treatment strategies, there are only four GCTB cell lines available in public cell banks. Therefore, this study aimed to establish novel GCTB cell lines and successfully created NCC-GCTB6-C1 and NCC-GCTB7-C1 cell lines from two patients' surgically removed tumor tissues. These cell lines exhibited H3-3A gene mutations, consistent proliferation, and invasive properties. After characterizing their behaviors, we performed high-throughput screening of 214 anti-cancer drugs for NCC-GCTB6-C1 and NCC-GCTB7-C1 and integrated their screening data with those of NCC-GCTB1-C1, NCC-GCTB2-C1, NCC-GCTB3-C1, NCC-GCTB4-C1, and NCC-GCTB5-C1 that we previously established. We identified histone deacetylase inhibitor romidepsin as a possible treatment for GCTB. These findings suggest that NCC-GCTB6-C1 and NCC-GCTB7-C1 could be valuable tools for preclinical and basic research on GCTB.

Layer-by-Layer Cellulose Nanofibrils: A New Coating Strategy for Development and Characterization of Tumor Spheroids as a Model for In Vitro Anticancer Drug Screening.

Three-dimensional multicellular spheroids (MCSs) are complex structure of cellular aggregates and cell-to-matrix interaction that emulates the in-vivo microenvironment. This research field has grown to develop and improve spheroid generation techniques. Here, we present a new platform for spheroid generation using Layer-by-Layer (LbL) technology. Layer-by-Layer (LbL) containing cellulose nanofibrils (CNF) assemble on a standard 96 well plate. Various bi-layer numbers, multiple cell seeding concentration, and two tumor cell lines (HEK 293 T, HCT 116) are utilized to generate and characterize spheroids. The number and proliferation of generated spheroids, the viability, and the response to the anti-cancer drug are examined. The spheroids are formed and proliferated on the LbL-CNF coated wells with no significant difference in connection to the number of LbL-CNF bi-layers; however, the number of formed spheroids correlates positively with the cell seeding concentration (122 ± 17) and (42 ± 8) for HCT 116 and HEK 293T respectively at 700 cells ml-1 . The spheroids proliferate progressively up to (309, 663) µm of HCT 116 and HEK 293T respectively on 5 bi-layers coated wells with maintaining viability. The (HCT 116) spheroids react to the anti-cancer drug. We demonstrate a new (LbL-CNF) coating strategy for spheroids generation, with high performance and efficiency to test anti-cancer drugs.

3D Multicellular Tumor Spheroids in a Microfluidic Droplet System for Investigation of Drug Resistance.

A three-dimensional (3D) tumor spheroid model plays a critical role in mimicking tumor microenvironments in vivo. However, the conventional culture methods lack the ability to manipulate the 3D tumor spheroids in a homogeneous manner. To address this limitation, we developed a microfluidic-based droplet system for drug screening applications. We used a tree-shaped gradient generator to control the cell density and encapsulate the cells within uniform-sized droplets to generate a 3D gradient-sized tumor spheroid. Using this microfluidic-based droplet system, we demonstrated the high-throughput generation of uniform 3D tumor spheroids containing various cellular ratios for the analysis of the anti-cancer drug cytotoxicity. Consequently, this microfluidic-based gradient droplet generator could be a potentially powerful tool for anti-cancer drug screening applications.

Establishment and characterization of NCC-UPS4-C1: a novel cell line of undifferentiated pleomorphic sarcoma from a patient with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome caused by a germline mutation of the TP53. The lifetime risk of cancer in individuals with LFS is ≥ 70% for men and ≥ 90% for women. Undifferentiated pleomorphic sarcoma (UPS) is one of the core cancers associated with LFS. UPS is a subtype of undifferentiated soft tissue sarcoma that shows no identifiable line of differentiation. The standard curative treatment for UPS is complete surgical resection. However, local recurrence and distant metastasis to the lung can usually be found after resection of the UPS. Therefore, a novel treatment strategy for patients with UPS is required. Although well characterized, patient-derived tumor cell lines facilitate the high-throughput screening of a large number of drugs, and no sarcoma cell lines derived from a patient with LFS have been registered in public cell banks. Thus, this study aimed to establish a novel, well-characterized UPS cell line from a patient with LFS. From surgically resected UPS tumor tissues, we established the first UPS cell line from a patient with LFS and named it NCC-UPS4-C1. NCC-UPS4-C1 harbored copy number alterations and had the TP53 tumor suppressor gene mutation. The cells exhibited constant cell growth and invasive ability. This well-characterized NCC-UPS4-C1 cell line was then utilized for high-throughput screening of 214 anti-cancer drugs, and two effective drugs were identified. One of the two drugs, romidepsin, was commonly effective for the NCC-UPS1-C1, NCC-UPS2-C1, and NCC-UPS3-C1 cell lines that we previously reported; a potential drug for the treatment of UPS was suggested using well-characterized UPS cell lines. These data indicate that NCC-UPS4-C1, which is the first sarcoma cell line established from a patient with LFS, enables researchers to conduct vigorous preclinical research on UPS.

Immunosuppressive mesenchymal stem cells aggregates incorporating hydrogel microspheres promote an in vitro invasion of cancer cells.

INTRODUCTION: The objective of this study is to design a co-culture system of cancer cells and three-dimensional (3D) mesenchymal stem cells (MSC) aggregates for the in vitro evaluation of cancer invasion. METHODS: First, the MSC of an immunosuppressive phenotype (MSC2) were prepared by the MSC stimulation of polyriboinosinic polyribocytidylic acid. By simple mixing MSC2 and gelatin hydrogel microspheres (GM) in a U-bottomed well of 96 well plates which had been pre-coated with poly (vinyl alcohol), 3D MSC2 aggregates incorporating GM were obtained. The amount of chemokine (C-C motif) ligand 5 (CCL5) secreted from the MSC2 aggregates incorporating GM. Finally, an invasion assay was performed to evaluate the cancer invasion rate by co-cultured cancer cells and the 3D MSC2 incorporating GM. RESULTS: The amount of CCL5 secreted for the 3D MSC2 aggregates incorporating GM was significantly higher than that of two-dimensional (2D) MSC, 2D MSC2, and 3D MSC aggregates incorporating GM. When MDA-MB-231 human breast cancer cells were co-cultured with the 3D MSC2 aggregates incorporating GM, the invasion rate of cancer cells was significantly high compared with that of 2D MSC or 2D MSC2 and 3D MSC aggregates incorporating GM. In addition, high secretion of matrix metalloproteinase-2 was observed for the 3D MSC2 aggregates/cancer cells system. CONCLUSIONS: It is concluded that the co-culture system of 3D MSC2 aggregates incorporating GM and cancer cells is promising to evaluate the invasion of cancer cells in vitro.

A cancer invasion model of cancer-associated fibroblasts aggregates combined with TGF-ß1 release system.

INTRODUCTION: The objective of this study is to design a cancer invasion model where the cancer invasion rate can be regulated in vitro. METHODS: Cancer-associated fibroblasts (CAF) aggregates incorporating gelatin hydrogel microspheres (GM) containing various concentrations of transforming growth factor-ß1 (TGF-ß1) (CAF-GM-TGF-ß1) were prepared. Alpha-smooth muscle actin (α-SMA) for the CAF aggregates was measured to investigate the CAF activation level by changing the concentration of TGF-ß1. An invasion assay was performed to evaluate the cancer invasion rate by co-cultured of cancer cells with various CAF-GM-TGF-ß1. RESULTS: The expression level of α-SMA for CAF increased with an increased in the TGF-ß1 concentration. When co-cultured with various types of CAF-GM-TGF-ß1, the cancer invasion rate was well correlated with the α-SMA level. It is conceivable that the TGF-ß1 concentration could modify the level of CAF activation, leading to the invasion rate of cancer cells. In addition, at the high concentrations of TGF-ß1, the effect of a matrix metalloproteinase (MMP) inhibitor on the cancer invasion rate was observed. The higher invasion rate would be achieved through the higher MMP production. CONCLUSIONS: The present model is promising to realize the cancer invasion whose rate can be modified by changing the TGF-ß1 concentration.

3D cell-based biosensor for cell viability and drug assessment by 3D electric cell/matrigel-substrate impedance sensing.

Preclinical efficacy and toxicity assessment of drug candidates plays a significant role in drug discovery and development. Traditional planar cell culture is a common way to perform the preclinical drug test, but it is difficult to correctly predict the drug efficacy and toxicity due to the simple two-dimensional (2D) extracellular microenvironment. Compared to the planar cell culture, three-dimensional (3D) cell culture system can better mimic the complex extracellular microenvironment where cells reside in the 3D tissues/organs in vivo. However, the conventional imaging techniques are difficult to achieve the dynamic and label-free monitoring of cellular behavior in thick sample by 3D cell culture. Here, 3D electric cell/matrigel-substrate impedance sensing (3D-ECMIS) is developed for real-time and non-invasive monitoring of 3D cell viability and drug susceptibility. In this study, human hepatoma cells (HepG2) are encapsulated in the matrigel scaffold and cultured in a 3D ECMIS chip which involves a pair of vertical golden electrodes on the opposite sidewalls of the culture chamber for the in-situ impedance measurement. Moreover, a portable multichannel system is developed to monitor the 3D cell/matrigel construct. The number of 3D-cultured cells was inversely proportional to the impedance magnitude of the entire cell/matrigel construct. Furthermore, anti-cancer drug screening will be conducted on the 3D-cultured cells when the cell proliferation reaches to a plateau phase. To validate the performance of 3D-ECMIS for the cell viability and drug susceptibility, the cell live/dead staining are utilized to confirm the results of drug susceptibility by this 3D-cell-based biosensor system. It is demonstrated that the 3D cell-based biosensor and 3D-ECMIS system will be a promising platform to improve the accuracy of cell-based anti-cancer drug screening in vitro.

Development of an in vitro cell-sheet cancer model for chemotherapeutic screening.

Epithelial cancer grows in vivo in a microenvironment that comprises tumour, stroma, and immune cells. A three-dimensional (3D) culture model might be able to mimic the tumour microenvironment in vivo; therefore, we developed a new 3D epithelial cancer model using in vitro cell-sheet engineering and compared the results of treatment with several chemotherapeutic drugs among the 3D cell-sheet model, spheroid culture, and 2D cell culture. Methods: The cell sheet comprised keratinocytes and a plasma fibrin matrix containing fibroblasts. Cancer spheroids with or without cancer-associated fibroblasts (CAFs) were interposed between the keratinocytes and fibrin layer. Cell growth, viability, and hypoxia were measured using the cell counting kit-8, LIVE/DEAD assay, and propidium iodide and LOX-1 staining. The morphology, invasion, and mRNA and protein expression were compared among the different cell culture models. Results: Enhanced resistance to sorafenib and cisplatin by cancer spheroids and CAFs was more easily observed in the 3D than in the 2D model. Invasion by cancer-CAF spheroids into the fibrin matrix was more clearly observed in the 3D cell sheet. The expansion of viable cancer cells increased in the 3D cell sheet, particularly in those with CAFs, which were significantly inhibited by treatment with 10 µM sorafenib or 20 µM cisplatin (P < 0.05). TGF-ß1, N-cadherin, and vimentin mRNA and protein levels were higher in the 3D cell-sheet model. Conclusions: The 3D cell sheet-based cancer model could be applied to in vitro observation of epithelial cancer growth and invasion and to anticancer drug testing.

Quantification of cell viability and rapid screening anti-cancer drug utilizing nanomechanical fluctuation.

Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs.

Optimization of Aqueous Biphasic Tumor Spheroid Microtechnology for Anti-Cancer Drug Testing in 3D Culture.

Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories.

An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance.