Drug testing of monodisperse arrays of live microdissected tumors using a valved multiwell microfluidic platform.

Cancer drug testing in animals is an extremely poor predictor of the drug's safety and efficacy observed in humans. Hence there is a pressing need for functional testing platforms that better predict traditional and immunotherapy responses in human, live tumor tissue or tissue constructs, and at the same time are compatible with the use of mouse tumor tissue to facilitate building more accurate disease models. Since many cancer drug actions rely on mechanisms that depend on the tumor microenvironment (TME), such platforms should also retain as much of the native TME as possible. Additionally, platforms based on miniaturization technologies are desirable to reduce animal use and sensitivity to human tissue scarcity. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, microdissected tumors (µDTs) or "spheroids" that retain an intact TME have shown promising responses to immunomodulators acting on native immune cells. However, difficult tissue handling after microdissection has reduced the throughput of drug testing on µDTs, thereby constraining the inherent advantages of producing numerous TME-preserving units of tissue for drug testing. Here we demonstrate a microfluidic 96-well platform designed for drug treatment of hundreds of similarly-sized, cuboidal µDTs ("cuboids") produced from a single tumor sample. The platform organizes a monodisperse array of four cuboids per well in 384 hydrodynamic traps. The microfluidic device, entirely fabricated in thermoplastics, features 96 microvalves that fluidically isolate each well after the cuboid loading step for straightforward multi-drug testing. Since our platform makes the most of scarce tumor tissue, it can potentially be applied to human biopsies that preserve the human TME while minimizing animal testing.

A multidimensional biosensor system to guide LUAD individualized treatment.

Lung cancer, mainly non-small cell lung cancer (NSCLC), has been a global health problem, leading to maximum cancer death. Across adenocarcinoma patients, significant genetic and phenotypic heterogeneity was identified as responsible for individual cancer drug resistance, driving an urgent need for individualized treatment. High expectation has been set on individualized treatment for better responses and extended survival. There are pressing needs for and significant advantages of testing dosages and drugs directly on patient-specific cancer cells for preclinical drug testing and personalized drug selection. Monitoring the drug response based on patient-derived cells (PDCs) is a step toward effective drug development and individualized treatment. Despite the dependence on optical labels, optical equipment, and other complex manual operation, we here report a multidimensional biosensor system to guide adenocarcinoma individualized treatment by integrating 2D and 3D PDC models and cellular impedance biosensors. The cellular impedance biosensors were applied to quantitate drug response in 2D and 3D environments. Compared with 2D plate culture, 3D cultured cells were found to show higher resistance to anti-cancer drugs. Cell-cell, cell-ECM, and mechanical interactions in the 3D environment led to stronger drug resistance. The in vivo results demonstrated the reliability of the multidimensional biosensor system. Cellular impedance biosensors allow a fast, non-invasive, and quantitative manner for preselected drug screening in individualized treatment. Considering the potential for good distinguishment of different anti-cancer drugs, our newly developed strategy may contribute to drug response prediction in individualized treatment and new drug development.

Lung cancer organoids analyzed on microwell arrays predict drug responses of patients within a week.

While the potential of patient-derived organoids (PDOs) to predict patients' responses to anti-cancer treatments has been well recognized, the lengthy time and the low efficiency in establishing PDOs hamper the implementation of PDO-based drug sensitivity tests in clinics. We first adapt a mechanical sample processing method to generate lung cancer organoids (LCOs) from surgically resected and biopsy tumor tissues. The LCOs recapitulate the histological and genetic features of the parental tumors and have the potential to expand indefinitely. By employing an integrated superhydrophobic microwell array chip (InSMAR-chip), we demonstrate hundreds of LCOs, a number that can be generated from most of the samples at passage 0, are sufficient to produce clinically meaningful drug responses within a week. The results prove our one-week drug tests are in good agreement with patient-derived xenografts, genetic mutations of tumors, and clinical outcomes. The LCO model coupled with the microwell device provides a technically feasible means for predicting patient-specific drug responses in clinical settings.

Acoustic Droplet-Assisted Superhydrophilic-Superhydrophobic Microarray Platform for High-Throughput Screening of Patient-Derived Tumor Spheroids.

Cell-based high-throughput screening is a key step in the current disease-based research, drug development, and precision medicine. However, it is challenging to establish a rapid culture and screening platform for rare cells (patient-derived) due to the obvious differences between the traditional 2D cell model and the tumor microenvironment, as well as the lack of a low-consumption screening platform for low numbers of cells. Here, we developed an acoustic drop-assisted superhydrophilic-superhydrophobic microarray platform for the rapid culture and screening of a few cells. By employing hydrophilic and hydrophobic microarrays, we can automatically distribute the cell suspension into uniform droplets, and these cells can spontaneously form compact 3D cell spheroids within 36 h (similar to the microenvironment of tumors in vivo). By using the acoustic droplet ejection device, we can accurately inject a drug solution with a volume of ∼pL to ∼nL into the droplet, and the whole process can be completed within 20 ms (one print). By using three different cell lines (Caco-2, MCF-7, and HeLa) to optimize the platform, the culture and screening of five patients' colon cancer were subsequently realized. Using three conventional chemotherapeutics (5-fluorouracil, cetuximab, and panitumumab) of various concentrations, the best treatment was screened out and compared with the actual treatment effect of the patients, and the results were extremely similar. As a proof-of-concept application, we have proved that our platform can quickly cultivate patient samples and effectively screen the best treatment methods, highlighting its wide application in precision medicine, basic tumor research, and drug development.

Polysaccharide hydrogel based 3D printed tumor models for chemotherapeutic drug screening.

A series of stable and ready-to-use bioinks have been developed based on the xeno-free and tunable hydrogel (VitroGel) system. Cell laden scaffold fabrication with optimized polysaccharide-based inks demonstrated that Ink H4 and RGD modified Ink H4-RGD had excellent rheological properties. Both bioinks were printable with 25-40 kPa extrusion pressure, showed 90% cell viability, shear-thinning and rapid shear recovery properties making them feasible for extrusion bioprinting without UV curing or temperature adjustment. Ink H4-RGD showed printability between 20 and 37 °C and the scaffolds remained stable for 15 days at temperature of 37 °C. 3D printed non-small-cell lung cancer (NSCLC) patient derived xenograft cells (PDCs) showed rapid spheroid growth of size around 500 µm in diameter and tumor microenvironment formation within 7 days. IC50 values demonstrated higher resistance of 3D spheroids to docetaxel (DTX), doxorubicin (DOX) and erlotinib compared to 2D monolayers of NSCLC-PDX, wild type triple negative breast cancer (MDA-MB-231 WT) and lung adenocarcinoma (HCC-827) cells. Results of flow property, shape fidelity, scaffold stability and biocompatibility of H4-RGD suggest that this hydrogel could be considered for 3D cell bioprinting and also for in-vitro tumor microenvironment development for high throughput screening of various anti-cancer drugs.

Ex vivo cultures and drug testing of primary acute myeloid leukemia samples: Current techniques and implications for experimental design and outcome.

New treatment options of acute myeloid leukemia (AML) are rapidly emerging. Pre-clinical models such as ex vivo cultures are extensively used towards the development of novel drugs and to study synergistic drug combinations, as well as to discover biomarkers for both drug response and anti-cancer drug resistance. Although these approaches empower efficient investigation of multiple drugs in a multitude of primary AML samples, their translational value and reproducibility are hampered by the lack of standardized methodologies and by culture system-specific behavior of AML cells and chemotherapeutic drugs. Moreover, distinct research questions require specific methods which rely on specific technical knowledge and skills. To address these aspects, we herein review commonly used culture techniques in light of diverse research questions. In addition, culture-dependent effects on drug resistance towards commonly used drugs in the treatment of AML are summarized including several pitfalls that may arise because of culture technique artifacts. The primary aim of the current review is to provide practical guidelines for ex vivo primary AML culture experimental design.

Automated microfluidic platform for dynamic and combinatorial drug screening of tumor organoids.

Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.

Microfluidic-enabled self-organized tumor model for in vitro cytotoxicity assessment of doxorubicin.

The advent of microfluidic technologies has enabled a better recapitulation of in vitro tumor model with higher biological relevance over conventional monolayer assays. This work built upon a microfluidic system that supported the spontaneous aggregate formation of tumoral cells under flow-induced dynamic physical forces in a confined microchamber without additional matrix materials. Our findings indicated that fluidic streams significantly modulated the biological and architectural features of human breast adenocarcinoma cell (MCF-7), human hepatocarcinoma cell (HepG2), and human cervix adenocarcinoma cell (HeLa) with cell-type-dependent variation. The microfluidic platform was further integrated with a fluorescence detection and imaging system, allowing for non-invasive monitoring of cellular accumulation and spatial distribution of a chemotherapeutic agent, doxorubicin (DOX). The cytotoxic effects of DOX of various concentrations were determined and compared in MCF-7 cells in conventional two-dimensional (2D) static and microfluidic culture conditions. Dose-dependent response to DOX was noticed in both cultures, whereas tumor micronodules grown in microfluidic devices demonstrated significantly lower sensitivity to DOX at increased concentration. Our platform owns promising potentials as a universal modality for bridging traditional 2D cell cultures and in vivo experimentation for preclinical anticancer drug screening.

Application of microfluidic technology in cancer research and therapy.

Cancer is a heterogeneous disease that requires a multimodal approach to diagnose, manage and treat. A better understanding of the disease biology can lead to identification of novel diagnostic/prognostic biomarkers and the discovery of the novel therapeutics with the goal of improving patient outcomes. Employing advanced technologies can facilitate this, enabling better diagnostic and treatment for cancer patients. In this regard, microfluidic technology has emerged as a promising tool in the studies of cancer, including single cancer cell analysis, modeling angiogenesis and metastasis, drug screening and liquid biopsy. Microfluidic technologies have opened new ways to study tumors in the preclinical and clinical settings. In this chapter, we highlight novel application of this technology in area of fundamental, translational and clinical cancer research.

Three-Dimensional Cell Cultures as an In Vitro Tool for Prostate Cancer Modeling and Drug Discovery.

In the last decade, three-dimensional (3D) cell culture technology has gained a lot of interest due to its ability to better recapitulate the in vivo organization and microenvironment of in vitro cultured cancer cells. In particular, 3D tumor models have demonstrated several different characteristics compared with traditional two-dimensional (2D) cultures and have provided an interesting link between the latter and animal experiments. Indeed, 3D cell cultures represent a useful platform for the identification of the biological features of cancer cells as well as for the screening of novel antitumor agents. The present review is aimed at summarizing the most common 3D cell culture methods and applications, with a focus on prostate cancer modeling and drug discovery.

Novel N-cadherin antagonist causes glioblastoma cell death in a 3D bioprinted co-culture model.

Glioblastoma multiforme (GBM) is a deadly type of brain cancer. There is a need to identify novel therapies for GBM as current treatments only marginally increase survival. Modelling the complexity of cancerous tissues using 3D bioprinted constructs serves as a novel approach for preclinical testing of anticancer drugs. A novel small molecule antagonist of the cell adhesion molecule, N-cadherin (NCAD), (S)-1-(3,4-Dichlorophenoxy)-3-(4-((S)-2-hydroxy-3-(4-methoxyphenoxy)propylamino)piperidin-1-yl)propan-2-ol has shown promise as an anticancer agent. This study investigated the influence of this antagonist on GBM cells bioprinted with astrocytes into 3D constructs. The NCAD antagonist prevented spheroid formation and induced cell death in the 3D model. This is the first demonstration that an NCAD antagonist can cause GBM cell death.

Three-dimensional culture models to study drug resistance in breast cancer.

Despite recent advances in breast cancer treatment, drug resistance frequently presents as a challenge, contributing to a higher risk of relapse and decreased overall survival rate. It is now generally recognized that the extracellular matrix and cellular heterogeneity of the tumor microenvironment influences the cancer cells' ultimate fate. Therefore, strategies employed to examine mechanisms of drug resistance must take microenvironmental influences, as well as genetic mutations, into account. This review discusses three-dimensional (3D) in vitro model systems which incorporate microenvironmental influences to study mechanisms of drug resistance in breast cancer. These bioengineered models include spheroid-based models, biomaterial-based models such as polymeric scaffolds and hydrogels, and microfluidic chip-based models. The advantages of these model systems over traditionally studied two-dimensional tissue culture polystyrene are examined. Additionally, the applicability of such 3D models for studying the impact of tumor microenvironment signals on drug response and/or resistance is discussed. Finally, the potential of such models for use in the development of strategies to combat drug resistance and determine the most promising treatment regimen is explored.

High-throughput analysis of cell-cell crosstalk in ad hoc designed microfluidic chips for oncoimmunology applications.

Understanding the interactions between immune and cancer cells occurring within the tumor microenvironment is a prerequisite for successful and personalized anti-cancer therapies. Microfluidic devices, coupled to advanced microscopy systems and automated analytical tools, can represent an innovative approach for high-throughput investigations on immune cell-cancer interactions. In order to study such interactions and to evaluate how therapeutic agents can affect this crosstalk, we employed two ad hoc fabricated microfluidic platforms reproducing advanced 2D or 3D tumor immune microenvironments. In the first type of chip, we confronted the capacity of tumor cells embedded in Matrigel containing one drug or Matrigel containing a combination of two drugs to attract differentially immune cells, by fluorescence microscopy analyses. In the second chip, we investigated the migratory/interaction response of naïve immune cells to danger signals emanated from tumor cells treated with an immunogenic drug, by time-lapse microscopy and automated tracking analysis. We demonstrate that microfluidic platforms and their associated high-throughput computed analyses can represent versatile and smart systems to: (i) monitor and quantify the recruitment and interactions of the immune cells with cancer in a controlled environment, (ii) evaluate the immunogenic effects of anti-cancer therapeutic agents and (iii) evaluate the immunogenic efficacy of combinatorial regimens with respect to single agents.

Microfluidics-Based Single-Cell Protrusion Analysis for Screening Drugs Targeting Subcellular Mitochondrial Trafficking in Cancer Progression.

Cancer cell migration is often guided by cell protrusions, whose formation and activity involve subcellular localization of mitochondria. However, the role of subcellular mitochondrial trafficking during cell protrusion generation is not well-understood amidst a lack of quantitative data. Here, we present a high-throughput microfluidic platform that enables the quantitative, single-cell precision analysis of cell protrusion formation during cell migration that is regulated by subcellular mitochondrial trafficking. Gene expression profiling of the isolated cell protrusions suggested that mitochondria were found in high numbers within cell protrusions, a finding validated by mitochondrial staining. Quantitative analysis revealed that the formation of cell protrusions could be effectively suppressed by inhibiting subcellular mitochondrial trafficking. We further demonstrated that rapid screening of mitochondria-specific therapeutic drugs to evaluate their effects on cell protrusion formation with single-cell precision could be achieved in the microfluidic platform, which could have clinical utility in the development of new anticancer agents.

A growth model of neuroendocrine tumor surrogates and the efficacy of a novel somatostatin-receptor-guided antibody-drug conjugate: Perspectives on clinical response?

BACKGROUND: As patient-derived xenografts and other preclinical models of neuroendocrine tumors for testing personalized therapeutics are lacking, we have developed a perfused, 3D bioreactor model to culture tumor surrogates from patient-derived neuroendocrine tumors. This work evaluates the duration of surrogate culture and surrogate response to a novel antibody-drug conjugate. METHODS: Twenty-seven patient-derived neuroendocrine tumors were cultured. Histologic sections of a pancreatic neuroendocrine tumor xenograft (BON-1) tumor were assessed for SSTR2 expression before tumor implantation into 2 bioreactors. One surrogate was treated with an antibody-drug conjugate composed of an anti-mitotic Monomethyl auristatin-E linked to a somatostatin receptor 2 antibody. Viability and therapeutic response were assessed by pre-imaging incubation with IR-783 and the RealTime-Glo AnnexinV Apoptosis and Necrosis Assay (Promega Corporation, Madison, WI) over 6 days. A primary human pancreatic neuroendocrine tumor was evaluated similarly. RESULTS: Mean surrogate growth duration was 34.8 days. Treated BON-1 surrogates exhibited less proliferation (1.2 vs 1.9-fold) and greater apoptosis (1.5 vs 1.1-fold) than controls, whereas treated patient-derived neuroendocrine tumor bioreactors exhibited greater degrees of apoptosis (13- vs 9-fold) and necrosis (2.5- vs 1.6-fold). CONCLUSION: Patient-derived neuroendocrine tumor surrogates can be cultured reliably within the bioreactor. This model can be used to evaluate the efficacy of antibody-guided chemotherapy ex vivo and may be useful for predicting clinical responses.

Development and verification of a three-dimensional (3D) breast cancer tumor model composed of circulating tumor cell (CTC) subsets.

Breast cancer is one of the most common cancer types among women in which early tumor invasion leads to metastases and death. EpCAM (epithelial cellular adhesion molecule) and HER2 (human epidermal growth factor receptor 2) are two main circulating tumor cell (CTC) subsets in HER2+ breast cancer patients. In this regard, the main aim of this study is to develop and characterize a three-dimensional (3D) breast cancer tumor model composed of CTC subsets to evaluate new therapeutic strategies and drugs. For this reason, EpCAM(+) and HER2(+) sub-populations were isolated from different cell lines to establish 3D tumor model that mimics in situ (in vivo) more closely than two-dimensional (2D) models. EpCAM(+)/HER2(+) cells had a high proliferation rate and low tendency to attach to the surface in comparison with parental MDA-MB-453 cells as CTC subsets. Aggressive breast cancer subpopulations cultured in 3D porous chitosan scaffold had enhanced cell-cell and cell-matrix interactions compared to 2D cultured cells and these 3D models showed more aggressive morphology and behavior, expressed higher levels of pluripotency marker genes, Nanog, Sox2 and Oct4. For the verification of the 3D model, the effects of doxorubicin which is a chemotherapeutic agent used in breast cancer treatment were examined and increased drug resistance was determined in 3D cultures. The 3D tumor model comprising EpCAM(+)/HER2(+) CTC subsets developed in this study has a promising potential to be used for investigation of an aggressive CTC microenvironment in vitro that mimics in vivo characteristics to test new drug candidates against CTCs.

Typography-Like 3D-Printed Templates for the Lithography-Free Fabrication of Microfluidic Chips.

Typography-like templates for polydimethylsiloxane (PDMS) microfluidic chips using a fused deposition modeling (FDM) three-dimensional (3D) printer are presented. This rapid and fast proposed scheme did not require complicated photolithographic fabrication facilities and could deliver resolutions of ~100 µm. Polylactic acid (PLA) was adopted as the material to generate the 3D-printed units, which were then carefully assembled on a glass substrate using a heat-melt-curd strategy. This craft of bonding offers a cost-effective way to design and modify the templates of microfluidic channels, thus reducing the processing time of microfluidic chips. Finally, a flexible microfluidic chip to be employed for cell-based drug screening was developed based on the modularized 3D-printed templates. The lithography-free, typography-like, 3D-printed templates create a modularized fabrication process and promote the prevalence of integrated microfluidic systems with minimal requirements and improved efficiency.

In vitro assay for the development of small molecule inhibitors targeting PD-1/PD-L1.

Cancer immunotherapy has recently emerged as one of the hot research field since clinical successes achieved by antibody drugs of immune checkpoints, among which PD-1 and its ligand PD-L1 are the well established molecules. PD-1/PD-L1 pathway induces immune tolerance and immune evasion, especially in tumor microenvironment, cancer cell is capable to escape the immune surveillance by up-regulating the expression level of PD-1 or PD-L1. Blockade of PD-1/PD-L1 can unleash the anti-tumor activity, and the strategy shows great successes in the treatment of various cancer types in the late stage. Beside antibody drugs, many other molecules such as peptides, high affinity PD(L)-1 mutants, chemical compounds, and DNA aptamers are designed for inhibitors of PD-1/PD-L1 pathway. Each modulators show their pros and cons based on their own physiochemical properties. Here we introduced the methods for identifying low molecular weight inhibitors of PD-1/PD-L1 and mainly discussed the cell-based blocking test.

The Isolation and Characterization of Circulating Tumor Cells from Head and Neck Cancer Patient Blood Samples Using Spiral Microfluidic Technology.

Metastasis is responsible for 90% of cancer-related deaths. The study of circulating tumor cells (CTCs) enables the study of the units of disease responsible for the process of metastasis. While the biology of the primary tissue is relatively known, little is understood about the cells en route to distant sites. Here we describe the isolation of CTCs using the spiral microfluidic technology for the efficient sorting of CTCs from head and neck cancer (HNC) patient blood samples. Furthermore, the molecular characterization of CTCs can aid in stratifying patients for targeted therapy such as immunotherapy, which is having a profound impact in the treatment of metastatic HNC.

Digital Holographic Imaging as a Method for Quantitative, Live Cell Imaging of Drug Response to Novel Targeted Cancer Therapies.

Digital holographic imaging (DHI) is a noninvasive, live cell imaging technique that enables long-term quantitative visualization of cells in culture. DHI uses phase-shift imaging to monitor and quantify cellular events such as cell division, cell death, cell migration, and drug responses. In recent years, the application of DHI has expanded from its use in the laboratory to the clinical setting, and currently it is being developed for use in theranostics. Here, we describe the use of the DHI platform HoloMonitorM4 to evaluate the effects of novel, targeted cancer therapies on cell viability and proliferation using the HeLa cancer cell line as a model. We present single cell tracking and population-wide analysis of multiple cell morphology parameters.