Publications

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12 Publications visible to you, out of a total of 12
Utilizing convolutional neural networks for discriminating cancer and stromal cells in three-dimensional cell culture images with nuclei counterstain.

Abstract (Expand)

SIGNIFICANCE: Accurate cell segmentation and classification in three-dimensional (3D) images are vital for studying live cell behavior and drug responses in 3D tissue culture. Evaluating diverse cell … populations in 3D cell culture over time necessitates non-toxic staining methods, as specific fluorescent tags may not be suitable, and immunofluorescence staining can be cytotoxic for prolonged live cell cultures. AIM: We aim to perform machine learning-based cell classification within a live heterogeneous cell culture population grown in a 3D tissue culture relying only on reflectance, transmittance, and nuclei counterstained images obtained by confocal microscopy. APPROACH: In this study, we employed a supervised convolutional neural network (CNN) to classify tumor cells and fibroblasts within 3D-grown spheroids. These cells are first segmented using the marker-controlled watershed image processing method. Training data included nuclei counterstaining, reflectance, and transmitted light images, with stained fibroblast and tumor cells as ground-truth labels. RESULTS: Our results demonstrate the successful marker-controlled watershed segmentation of 84% of spheroid cells into single cells. We achieved a median accuracy of 67% (95% confidence interval of the median is 65-71%) in identifying cell types. We also recapitulate the original 3D images using the CNN-classified cells to visualize the original 3D-stained image's cell distribution. CONCLUSION: This study introduces a non-invasive toxicity-free approach to 3D cell culture evaluation, combining machine learning with confocal microscopy, opening avenues for advanced cell studies.

Authors: H. T. Nguyen, N. Pietraszek, S. E. Shelton, K. Arthur, R. D. Kamm

Date Published: 26th Aug 2024

Publication Type: Journal Article

Polyploidy of MDA-MB-231 cells drives increased extravasation with enhanced cell-matrix adhesion.

Abstract (Expand)

Metastasis, the leading cause of cancer-related deaths, involves a complex cascade of events, including extravasation. Despite extensive research into metastasis, the mechanisms underlying extravasation … remain unclear. Molecular targeted therapies have advanced cancer treatment, yet their efficacy is limited, prompting exploration into novel therapeutic targets. Here, we showed the association of polyploidy in MDA-MB-231 breast cancer cells and their extravasation, using microfluidic systems to reproduce the in vivo microvascular environment. We observed enhanced extravasation in polyploid cells alongside upregulated expression of genes involved in cell-substrate adhesion and cell mechanical dynamics. These findings offer insights into the relationship between polyploidy and extravasation, highlighting potential targets for cancer therapy.

Authors: S. Hirose, T. Osaki, R. D. Kamm

Date Published: 2nd Jul 2024

Publication Type: Journal Article

Impact of Fibrinogen, Fibrin Thrombi, and Thrombin on Cancer Cell Extravasation Using In Vitro Microvascular Networks.

Abstract (Expand)

A bidirectional association exists between metastatic dissemination and the hypercoagulable state associated with many types of cancer. As such, clinical studies have provided evidence that markers … associated with elevated levels of coagulation and fibrinolysis correlate with decreased patient survival. However, elucidating the mechanisms underpinning the effects of different components of the coagulation system on metastasis formation is challenging both in animal models and 2D models lacking the complex cellular interactions necessary to model both thrombosis and metastasis. Here, an in vitro, 3D, microvascular model for observing the formation of fibrin thrombi is described, which is in turn used to study how different aspects of the hypercoagulable state associated with cancer affect the endothelium. Using this platform, cancer cells expressing ICAM-1 are shown to form a fibrinogen-dependent bridge and transmigrate through the endothelium more effectively. Cancer cells are also demonstrated to interact with fibrin thrombi, using them to adhere, spread, and enhance their extravasation efficiency. Finally, thrombin is also shown to enhance cancer cell extravasation. This system presents a physiologically relevant model of fibrin clot formation in the human microvasculature, enabling in-depth investigation of the cellular interactions between cancer cells and the coagulation system affecting cancer cell extravasation.

Authors: E. Angelidakis, S. Chen, S. Zhang, Z. Wan, R. D. Kamm, S. E. Shelton

Date Published: 28th Jul 2023

Publication Type: Journal Article

Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes.

Abstract (Expand)

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes … probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Authors: Z. Gong, K. van den Dries, R. A. Migueles-Ramirez, P. W. Wiseman, A. Cambi, V. B. Shenoy

Date Published: 22nd May 2023

Publication Type: Journal Article

Interstitial flow promotes the formation of functional microvascular networks in vitro through upregulation of matrix metalloproteinase-2.

Abstract (Expand)

Self-organized microvascular networks (MVNs) have become key to the development of many microphysiological models. However, the self-organizing nature of this process combined with variations between … types or batches of endothelial cells (ECs) often lead to inconsistency or failure to form functional MVNs. Since interstitial flow (IF) has been reported to play a beneficial role in angiogenesis, vasculogenesis, and 3D capillary morphogenesis, we systematically investigated the role IF plays during neovessel formation in a customized single channel microfluidic chip for which IF has been fully characterized. Compared to static conditions, MVNs formed under IF have higher vessel density and diameters and greater network perfusability. Through a series of inhibitory experiments, we demonstrated that IF treatment improves vasculogenesis by ECs through upregulation of matrix metalloproteinase-2 (MMP-2). We then successfully implemented a novel strategy involving the interplay between IF and MMP-2 inhibitor to regulate morphological parameters of the self-organized MVNs, with vascular permeability and perfusability well maintained. The revealed mechanism and proposed methodology were further validated with a brain MVN model. Our findings and methods have the potential to be widely utilized to boost the development of various organotypic MVNs and could be incorporated into related bioengineering applications where perfusable vasculature is desired.

Authors: S. Zhang, Z. Wan, G. Pavlou, A. X. Zhong, L. Xu, R. D. Kamm

Date Published: 21st Oct 2022

Publication Type: Journal Article

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