Deciphering fibroblast-induced drug resistance in non-small cell lung carcinoma through patient-derived organoids in agarose microwells.

Patient-derived organoids (PDOs) serve as invaluable 3D tumor models, retaining the histological complexity and genetic heterogeneity found in primary tumors. However, the limitation of small sample volumes and the lack of tailored platforms have hindered the research using PDOs. Within the tumor microenvironment, cancer-associated fibroblasts play a pivotal role in influencing drug sensitivity. In this study, we introduce an agarose microwell platform designed for PDO-based tumor and tumor microenvironment models, enabling rapid drug screening and resistance studies with small sample volumes. These microwells, constructed using 3D printing molds, feature a U-shaped bottom and 200 µm diameter. We successfully generated co-culture spheroids of non-small cell lung carcinoma (NSCLC) cells, including NCI-H358 or A549, and NSCLC PDOs F231 or F671 with fibroblast cell line, WI-38. Our results demonstrate the production of uniformly-sized spheroids (coefficient of variation <30%), high viability (>80% after 1 week), and fibroblast-induced drug resistance. The PDOs maintained their viability (>81% after 2 weeks) and continued to proliferate. Notably, when exposed to adagrasib, a KRASG12C inhibitor, we observed reduced cytotoxicity in KRASG12C-mutant spheroids when co-cultured with fibroblasts or their supernatant. The fibroblast supernatant sustained proliferative signals in tumor models. Taking into account the physical features, viability, and drug resistance acquired through supernatants from the fibroblasts, our platform emerges as a suitable platform for in vitro tumor modeling and the evaluation of drug efficacy using patient-derived tissues.

Numerical Modeling of Physical Cell Trapping in Microfluidic Chips.

Microfluidic methods have proven to be effective in separation and isolation of cells for a wide range of biomedical applications. Among these methods, physical trapping is a label-free isolation approach that relies on cell size as the selective phenotype to retain target cells on-chip for follow-up analysis and imaging. In silico models have been used to optimize the design of such hydrodynamic traps and to investigate cancer cell transmigration through narrow constrictions. While most studies focus on computational fluid dynamics (CFD) analysis of flow over cells and/or pillar traps, a quantitative analysis of mechanical interaction between cells and trapping units is missing. The existing literature centers on longitudinally extended geometries (e.g., micro-vessels) to understand the biological phenomenon rather than designing an effective cell trap. In this work, we aim to make an experimentally informed prediction of the critical pressure for a cell to pass through a trapping unit as a function of cell morphology and trapping unit geometry. Our findings show that a hyperelastic material model accurately captures the stress-related softening behavior observed in cancer cells passing through micro-constrictions. These findings are used to develop a model capable of predicting and extrapolating critical pressure values. The validity of the model is assessed with experimental data. Regression analysis is used to derive a mathematical framework for critical pressure. Coupled with CFD analysis, one can use this formulation to design efficient microfluidic devices for cell trapping and potentially perform downstream analysis of trapped cells.

Single Cell Analysis of Inertial Migration by Circulating Tumor Cells and Clusters.

Single-cell analysis provides a wealth of information regarding the molecular landscape of the tumor cells responding to extracellular stimulations, which has greatly advanced the research in cancer biology. In this work, we adapt such a concept for the analysis of inertial migration of cells and clusters, which is promising for cancer liquid biopsy, by isolation and detection of circulating tumor cells (CTCs) and CTC clusters. Using high-speed camera tracking live individual tumor cells and cell clusters, the behavior of inertial migration was profiled in unprecedented detail. We found that inertial migration is heterogeneous spatially, depending on the initial cross-sectional location. The lateral migration velocity peaks at about 25% of the channel width away from the sidewalls for both single cells and clusters. More importantly, while the doublets of the cell clusters migrate significantly faster than single cells (~two times faster), cell triplets unexpectedly have similar migration velocities to doublets, which seemingly disagrees with the size-dependent nature of inertial migration. Further analysis indicates that the cluster shape or format (for example, triplets can be in string format or triangle format) plays a significant role in the migration of more complex cell clusters. We found that the migration velocity of a string triplet is statistically comparable to that of a single cell while the triangle triplets can migrate slightly faster than doublets, suggesting that size-based sorting of cells and clusters can be challenging depending on the cluster format. Undoubtedly, these new findings need to be considered in the translation of inertial microfluidic technology for CTC cluster detection.

Three-dimensional lab-on-a-foil device for dielectrophoretic separation of cancer cells.

A simple, low-cost, three-dimensional (3D) lab-on-a-foil microfluidic device for dielectrophoretic separation of circulating tumor cells (CTCs) is designed and constructed. Disposable thin films are cut by xurography and microelectrode array are made with rapid inkjet printing. The multilayer device design allows the studying of spatial movements of CTCs and red blood cells (RBCs) under dielectrophoresis (DEP). A numerical simulation was performed to find the optimum driving frequency of RBCs and the crossover frequency for CTCs. At the optimum frequency, RBCs were lifted 120 µm in z-axis direction by DEP force, and CTCs were not affected due to negligible DEP force. By utilizing the displacement difference, the separation of CTCs (modeled with A549 lung carcinoma cells) from RBCs in z-axis direction was achieved. With the nonuniform electric field at optimized driving frequency, the RBCs were trapped in the cavities above the microchannel, whereas the A549 cells were separated with a high capture rate of 86.3% ± 0.2%. The device opens not only the possibility for 3D high-throughput cell separation but also for future developments in 3D cell manipulation through rapid and low-cost fabrication.

Microfluidic techniques for isolation, formation, and characterization of circulating tumor cells and clusters.

Circulating tumor cell (CTC) clusters that are shed from the primary tumor into the bloodstream are associated with a poor prognosis, elevated metastatic potential, higher proliferation rate, and distinct molecular features compared to single CTCs. Studying CTC clusters may give us information on the differences in the genetic profiles, somatic mutations, and epigenetic changes in circulating cells compared to the primary tumor and metastatic sites. Microfluidic systems offer the means of studying CTC clusters through the ability to efficiently isolate these rare cells from the whole blood of patients in a liquid biopsy. Microfluidics can also be used to develop in vitro models of CTC clusters and make possible their characterization and analysis. Ultimately, microfluidic systems can offer the means to gather insight on the complexities of the metastatic process, the biology of cancer, and the potential for developing novel or personalized therapies. In this review, we aim to discuss the advantages and challenges of the existing microfluidic systems for working with CTC clusters. We hope that an improved understanding of the role microfluidics can play in isolation, formation, and characterization of CTC clusters, which can lead to increased sophistication of microfluidic platforms in cancer research.

Non-small cell lung carcinoma spheroid models in agarose microwells for drug response studies.

There is a growing interest in developing personalized treatment strategies for each cancer patient, especially those with non-small cell lung carcinoma (NSCLC) which annually accounts for the majority of cancer related deaths in the US. Yet identifying the optimal NSCLC treatment strategy for each cancer patient is critical due to a multitude of mutations, some of which develop following initial therapy and can result in drug resistance. A key difficulty in developing personalized therapies in NSCLC is the lack of clinically relevant assay systems that are suitable to evaluate drug sensitivity using a minuscule amount of patient-derived material available following biopsies. Herein we leverage 3D printing to demonstrate a platform based on miniature microwells in agarose to culture cancer cell spheroids. The agarose wells were shaped by 3D printing molds with 1000 microwells with a U-shaped bottom. Three NSCLC cell lines (HCC4006, H1975 and A549) were used to demonstrate size uniformity, spheroid viability, biomarker expressions and drug response in 3D agarose microwells. Results show that our approach yielded spheroids of uniform size (coefficient of variation <22%) and high viability (>83% after 1 week-culture). Studies using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKIs) drugs gefitinib and osimertinib showed clinically relevant responses. Based on the physical features, cell phenotypes, and responses to therapy of our spheroid models, we conclude that our platform is suitable for in vitro culture and drug evaluation, especially in cases when tumor sample is limited.

Acoustic bubble for spheroid trapping, rotation, and culture: a tumor-on-a-chip platform (ABSTRACT platform).

Cancer is the leading cause of death globally, with 90% of deaths being caused by cancer metastasis. Circulating tumor cells (CTCs) play an important role in early diagnosis of cancer metastasis and in monitoring of therapeutic response. Therefore, reliable methods to isolate, collect and culture CTCs are required to obtain information on metastasis status and therapeutic treatment. In this work, we present a CTC-processing system: acoustic bubble for spheroid trapping, rotation, and culture: a tumor-on-a-chip platform (ABSTRACT). The platform consists of a main channel, several parallel sub-microchannels with microcavities and culture chambers. The microcavity is designed to trap a bubble with desired shape at the entrance of the sub-microchannel. Under the acoustic actuation, the trapped bubble oscillates and creates a secondary radiation force to trap and rotate CTCs at a desired location. By controlling the acoustic bubble, CTCs can be continuously trapped from the blood flow, rotated to form a spheroid, and released to the microchamber for culture. We systematically investigated the effects of device geometry, flow parameters, and input voltage on trapping of CTCs to optimize the performance. Additionally, the successful on-chip spheroid culture demonstrates the biocompatibility and the simplicity of this platform. Besides simplifying conventional complex CTC processing procedures, this ABSTRACT platform also shows great potential for downstream analysis of tumor cells, such as monitoring the progression of metastasis and personalized drug testing.

Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFß Signaling via Activation of Calcineurin.

Control of ionic gradients is critical to maintain cellular homeostasis in both physiological and pathological conditions, but the role of ion channels in cancer cells has not been studied thoroughly. In this work we demonstrated that activity of the Kv11.1 potassium channel plays a vital role in controlling the migration of colon cancer cells by reversing the epithelial-to-mesenchymal transition (EMT) into the mesenchymal-to-epithelial transition (MET). We discovered that pharmacological stimulation of the Kv11.1 channel with the activator molecule NS1643 produces a strong inhibition of colon cancer cell motility. In agreement with the reversal of EMT, NS1643 treatment leads to a depletion of mesenchymal markers such as SNAIL1, SLUG, TWIST, ZEB, N-cadherin, and c-Myc, while the epithelial marker E-cadherin was strongly upregulated. Investigating the mechanism linking Kv11.1 activity to reversal of EMT into MET revealed that stimulation of Kv11.1 produced a strong and fast inhibition of the TGFß signaling. Application of NS1643 resulted in de-phosphorylation of the TGFß downstream effectors R-SMADs by activation of the serine/threonine phosphatase PP2B (calcineurin). Consistent with the role of TGFß in controlling cancer stemness, NS1643 also produced a strong inhibition of NANOG, SOX2, and OCT4 while arresting the cell cycle in G0/G1. Our data demonstrate that activation of the Kv11.1 channel reprograms EMT into MET by inhibiting TGFß signaling, which results in inhibition of motility in colon cancer cells.

Microfluidic systems for hydrodynamic trapping of cells and clusters.

Microfluidic devices have been widely applied to trapping and isolation of cells and clusters for controllable intercellular environments and high-throughput analysis, triggering numerous advances in disease diagnosis and single-cell analysis. Passive hydrodynamic cell trapping is one of the simple and effective methods that has been gaining attention in recent years. Our aim here is to review the existing passive microfluidic trapping approaches, including microposts, microfiltration, microwells, and trapping chambers, with emphasis on design principles and performance. We summarize the remarkable advances that hydrodynamic trapping methods offer, as well as the existing challenges and prospects for development. Finally, we hope that an improved understanding of hydrodynamic trapping approaches can lead to sophisticated and useful platforms to advance medical and biological research.

Label-free microfluidic sorting of microparticles.

Massive growth of the microfluidics field has triggered numerous advances in focusing, separating, ordering, concentrating, and mixing of microparticles. Microfluidic systems capable of performing these functions are rapidly finding applications in industrial, environmental, and biomedical fields. Passive and label-free methods are one of the major categories of such systems that have received enormous attention owing to device operational simplicity and low costs. With new platforms continuously being proposed, our aim here is to provide an updated overview of the state of the art for passive label-free microparticle separation, with emphasis on performance and operational conditions. In addition to the now common separation approaches using Newtonian flows, such as deterministic lateral displacement, pinched flow fractionation, cross-flow filtration, hydrodynamic filtration, and inertial microfluidics, we also discuss separation approaches using non-Newtonian, viscoelastic flow. We then highlight the newly emerging approach based on shear-induced diffusion, which enables direct processing of complex samples such as untreated whole blood. Finally, we hope that an improved understanding of label-free passive sorting approaches can lead to sophisticated and useful platforms toward automation in industrial, environmental, and biomedical fields.

A microfluidic in-line ELISA for measuring secreted protein under perfusion.

Recent progress in the development of microfluidic microphysiological systems such as 'organs-on-chips' and microfabricated cell culture is geared to simulate organ-level physiology. These tissue models leverage microengineering technologies that provide capabilities of presenting cultured cells with input signals in a more physiologically relevant context such as perfused flow. Proteins that are secreted from cells have important information about the health of the cells. Techniques to quantify cellular proteins include mass spectrometry to ELISA (enzyme-linked immunosorbent assay). Although our capability to perturb the cells in the microphysiological systems with varying inputs is well established, we lack the tools to monitor in-line the cellular responses. User intervention for sample collection and off-site is cumbersome, causes delays in obtaining results, and is especially expensive because of collection, storage, and offline processing of the samples, and in many case, technically impractical to carry out because of limitated sample volumes. To address these shortcomings, we report the development of an ELISA that is carried out in-line under perfusion within a microfluidic device. Using this assay, we measured the albumin secreted from perfused hepatocytes without and under stimulation by IL-6. Since the method is based on a sandwich ELISA, we envision broad application of this technology to not just organs-on-chips but also to characterizing the temporal release and measurement of soluble factors and response to drugs.