Emulation and evaluation of tumor cell combined chemotherapy in isotropic/anisotropic collagen fiber microenvironments.

The rapid emergence of anisotropic collagen fibers in the tissue microenvironment is a critical transition point in late-stage breast cancer. Specifically, the fiber orientation facilitates the likelihood of high-speed tumor cell invasion and metastasis, which pose lethal threats to patients. Thus, based on this transition point, one key issue is how to determine and evaluate efficient combination chemotherapy treatments in late-stage cancer. In this study, we designed a collagen microarray chip containing 241 high-throughput microchambers with embedded metastatic breast cancer cell MDA-MB-231-RFP. By utilizing collagen's unique structure and hydromechanical properties, the chip constructed three-dimensional isotropic and anisotropic collagen fiber structures to emulate the tumor cell microenvironment at early and late stages. We injected different chemotherapeutic drugs into its four channels and obtained composite biochemical concentration profiles. Our results demonstrate that anisotropic collagen fibers promote cell proliferation and migration more than isotropic collagen fibers, suggesting that the geometric arrangement of fibers plays an important role in regulating cell behavior. Moreover, the presence of anisotropic collagen fibers may be a potential factor leading to the poor efficacy of combined chemotherapy in late-stage breast cancer. We investigated the efficacy of various chemotherapy drugs using cell proliferation inhibitors paclitaxel and gemcitabine and tumor cell migration inhibitors 7rh and PP2. To ensure the validity of our findings, we followed a systematic approach that involved testing the inhibitory effects of these drugs. According to our results, the drug combinations' effectiveness could be ordered as follows: paclitaxel + gemcitabine > gemcitabine + 7rh > PP2 + paclitaxel > 7rh + PP2. This study shows that the biomimetic chip system not only facilitates the creation of a realistic in vitro model for examining the cell migration mechanism in late-stage breast cancer but also has the potential to function as an effective tool for future chemotherapy assessment and personalized medicine.

3D collagen microchamber arrays for combined chemotherapy effect evaluation on cancer cell numbers and migration.

Breast cancer metastasis involves complex mechanisms, particularly when patients are undergoing chemotherapy. In tissues, tumor cells encounter cell-cell interactions, cell-microenvironment interactions, complex nutrient, and drug gradients. Currently, two-dimensional cell culture systems and animal models are challenging to observe and analyze cell responses to microenvironments with various physical and bio-chemical conditions, and microfluidic technology has been systematically developed to address this dilemma. In this study, we have constructed a combined chemotherapy evaluation chip (CCEC) based on microfluidic technology. The chip possesses 192 diamond-shaped microchambers containing MDA-MB-231-RFP cells, and each microchamber is composed of collagen to mimic breast cancer and its surrounding microenvironment. In addition, by adding medium containing different drugs to the medium channels of CCEC, composite drug (paclitaxel+gemcitabine+7rh and paclitaxel+fluorouracil+PP2) concentration gradients, and single drug (paclitaxel, gemcitabine, 7rh, fluorouracil, PP2) concentration gradients have been established in the five collagen regions, respectively, so that each localized microchamber in the regions has a unique drug microenvironment. In this way, we evaluated the composite and single chemotherapy efficacy on the same chip by statistically analyzing their effects on the numbers and migration of the cell. The quantitative results in CCECs reveal that the inhibition effects on the numbers and migration of MDA-MB-231-RFP cell under the composite drug gradients are more optimal than those of the single drugs. Besides, the cancer cell inhibition effect between the groups composed of two drugs has also been compared, that is the paclitaxel+gemcitabine, paclitaxel+fluorouracil, and paclitaxel+PP2 have better cell numbers and migration inhibition effects than paclitaxel+7rh. The results indicate that the bio-mimetic and high-throughput combined chemotherapy evaluation platform can serve as a more efficient and accurate tool for preclinical drug development and screening.

Microfluidic Chip for Detection of Drug Resistance at the Single-cell Level.

Drug-resistant bacterial strains seriously threaten human health. Rapid screening of antibiotics is urgently required to improve clinical treatment. Conventional methods of antimicrobial susceptibility testing rely on turbidimetry that is evident only after several days of incubation. The lengthy time of the assay can delay clinical treatment. Here, we proposed a single-cell level rapid system based on a microfluidic chip. The detection period of 30 min to 2 h was significantly shorter than the conventional turbidity-based method. To promote detection efficiency, 16 independent channels were designed, permitting the simultaneous screening of 16 drugs in the microfluidic chip. Prepositioning of drugs in the chip permitted prolonged transportation and storage. This may allow for the widespread use of the novel system, particularly in the regions where medical facilities are scarce. The growth curves were reported rapidly through a custom code in Matlab after tracking and photographing the bacteria during microscopy examination. The capability of the proposed system was validated by antimicrobial susceptibility testing trials with standard strains. The system provides a potentially useful detection tool for drug-resistant bacteria.

Biological gel-based microchamber array for tumor cell proliferation and migration studies in well-controlled biochemical gradients.

Breast cancer metastasis is a complex process controlled by multiple factors, including various cell-cell interactions, cell-environment coupling, and oxygen, nutrient and drug gradients that are intimately related to the heterogeneous breast tissue structure. In this study, we constructed a high-throughput in vitro biochip system containing an array of 642 microchambers arranged in a checkerboard configuration, with each chamber embedded in a composite extracellular matrix (ECM) composed of engineered collagen and Matrigel to mimic local heterogeneous environment in vivo. In addition, a controllable complex tetragonal chemical concentration profile can be achieved by imposing chemical compounds at the four boundaries of the chip, leading to distinct local nutrient and/or drug gradients in the individual microchambers. Here, the microchamber array with composite ECM (MACECM) device aims to simulate multiple tumor cell niches composed of both breast epithelial cells (MCF-10A-GFP) and metastatic breast cancer cells (MDA-MB-231-RFP), which enables systematic studies of cell responses to a variety of biochemical conditions. The results obtained from the MACECM studies indicate that discoidin domain receptor 1 (DDR1) inhibitor 7rh and matrix metalloproteinase inhibitor batimastat, in association with epidermal growth factor (EGF) had no significant effects on the growth of MCF-10A-GFP cells, but had significant effects on DDR1 expression and the related migratory behavior of MDA-MB-231-RFP cells. The MACECM design not only enables the construction of a more realistic in vitro model for investigating cancer cell migration mechanisms but also has considerable potential for further development as a platform for next-generation high-throughput and therapeutic screening (e.g., anti-cancer drug evaluation) and personalized medicine.

Shannon entropy for time-varying persistence of cell migration.

Cell migration, which can be significantly affected by intracellular signaling pathways and extracellular matrix, plays a crucial role in many physiological and pathological processes. Cell migration is typically modeled as a persistent random walk, which depends on two critical motility parameters, i.e., migration speed and persistence time. It is generally very challenging to efficiently and accurately quantify the migration dynamics from noisy experimental data. Here, we introduce the normalized Shannon entropy (SE) based on the FPS of cellular velocity autocovariance function to quantify migration dynamics. The SE introduced here possesses a similar physical interpretation as the Gibbs entropy for thermal systems in that SE naturally reflects the degree of order or randomness of cellular migration, attaining the maximal value of unity for purely diffusive migration (i.e., SE = 1 for the most "random" dynamics) and the minimal value of 0 for purely ballistic dynamics (i.e., SE = 0 for the most "ordered" dynamics). We also find that SE is strongly correlated with the migration persistence but is less sensitive to the migration speed. Moreover, we introduce the time-varying SE based on the WPS of cellular dynamics and demonstrate its superior utility to characterize the time-dependent persistence of cell migration, which typically results from complex and time-varying intra- or extracellular mechanisms. We employ our approach to analyze experimental data of in vitro cell migration regulated by distinct intracellular and extracellular mechanisms, exhibiting a rich spectrum of dynamic characteristics. Our analysis indicates that the SE and wavelet transform (i.e., SE-based approach) offers a simple and efficient tool to quantify cell migration dynamics in complex microenvironment.

Deriving time-varying cellular motility parameters via wavelet analysis.

Cell migration, which is regulated by intracellular signaling pathways (ICSP) and extracellular matrix (ECM), plays an indispensable role in many physiological and pathological process such as normal tissue development and cancer metastasis. However, there is a lack of rigorous and quantitative tools for analyzing the time-varying characteristics of cell migration in heterogeneous microenvironment, resulted from, e.g. the time-dependent local stiffness due to microstructural remodeling by migrating cells. Here, we develop a wavelet-analysis approach to derive the time-dependent motility parameters from cell migration trajectories, based on the time-varying persistent random walk model. In particular, the wavelet denoising and wavelet transform are employed to analyze migration velocities and obtain the wavelet power spectrum. Subsequently, the time-dependent motility parameters are derived via Lorentzian power spectrum. Our results based on synthetic data indicate the superiority of the method for estimating the intrinsic transient motility parameters, robust against a variety of stochastic noises. We also carry out a systematic parameter study and elaborate the effects of parameter selection on the performance of the method. Moreover, we demonstrate the utility of our approach via analyzing experimental data ofin vitrocell migration in distinct microenvironments, including the migration of MDA-MB-231 cells in confined micro-channel arrays and correlated migration of MCF-10A cells due to ECM-mediated mechanical coupling. Our analysis shows that our approach can be as a powerful tool to accurately derive the time-dependent motility parameters, and further analyze the time-dependent characteristics of cell migration regulated by complex microenvironment.

Development of elastic artificial vessels with a digital pulse flow system to investigate the risk of restenosis and vasospasm.

The postoperative risk of stenosis is a complex issue, with risk factors including the status of human umbilical vein endothelial cells, the shear stress of dynamic blood flow, and blood physiology. Current research would benefit from in vitro models that can mimic the microenvironment of living vessels, to study the response of endothelial cells to stent placement. In this study, we constructed a digital pulse flow system based on a group of programmable solenoid valves, to mimic dynamic blood flows in the left coronary artery. Elastic artificial vessels, with internally cultured endothelial cells, were used to simulate vessel function and physiology. Based on this novel platform, we systematically explored cell proliferation and function in artificial vessels implanted with bare metal stents or drug-eluting stents, using unstented vessels as controls, under static and pulse flow conditions. The results indicate that the natural shear stresses of dynamic blood flow actually benefit endothelial cell attachment and proliferation. And drug-eluting stents showed stronger inhibition of cell proliferation than bare metal stents, but had a more negative effect on the synthesis of nitric oxide synthase (NOS), suggesting that drug elution might reduce the postoperative risk of restenosis, while increasing the risk of vasospasm. The results suggest that stent evaluation should include both the risk of restenosis and the effect on endothelial cells. Our simulation establishes a realistic in vitro model for pathological studies of restenosis and vasospasm, shows potential for evaluation of new stent designs, and could help develop individualised therapies for patients with atherosclerosis.