Circulating tumor cells (CTCs) carried by the patient's bloodstream are known to lead to the metastatic spread of cancer. It is becoming increasingly clear that an understanding of the nanomechanical characteristics of CTCs, such as elasticity and adhesiveness, represents advancements in tracking and monitoring cancer progression and metastasis. In the present work, we describe a combined microfluidic-atomic force microscopy (AFM) platform that uses antibody-antigen capture to routinely isolate and nanomechanically characterize CTCs present in blood samples from prostate cancer patients. We introduce the reversible assembly of a microfluidic device and apply refined and robust chemistry to covalently bond antibodies onto its glass substrate with high density and the desired orientation. As a result, we show that the device can efficiently capture CTCs from patients with localized and metastatic prostate cancer through anti-EpCAM, anti-PSA, and anti-PSMA antibodies, and it is suitable for AFM measurements of captured intact CTCs. When nanomechanically characterized, CTCs originating from metastatic cancer demonstrate decreased elasticity and increased deformability compared to those originating from localized cancer. While the average adhesion of CTCs to the AFM tip surface remained the same in both the groups, there were fewer multiple adhesion events in metastatic CTCs than there were in their counterparts. The developed platform is simple, robust, and reliable and can be useful in the diagnosis and prognosis of prostate cancer as well as other forms of cancer.
In this work, for first time, circulating tumor cells (CTCs) are captured on an open biofunctionalized substrate with multiplexing capability. This is achieved by developing a new microfluidic probe (MFP) integrated with radially staggered herringbone (HB) elements for microvortex generation. The new tool, named as herringbone microfluidic probe (HB‐MFP), is a channel‐less microfluidic system with physically separated bottom capture substrate and top fluidics delivery system. The concept allows for functionalizing the capture substrate with multiple biorecognition ligands (in this work, stripes of different capture antibodies) and scanning the fluidics delivery system across the substrate in a 2D printing‐like movement. Using the HB‐MFP, CTCs are efficiently captured from prostate cancer blood samples through their specific EpCAM, PSMA, and PSA antigens in a single run, with counts ranging from as low as 6 CTCs mL‐1 (localized cancer patients) to as high as 280 CTCs mL‐1 (metastatic cancer patients). In the process, CTC clusters with sizes of as high as 40–50 cells are also successfully captured. The results indicate that multiplex profiles of CTCs could reveal certain cellular phenotypes based on PSMA and PSA expression levels. The developed HB‐MFP is simple and robust to use, allows for high throughput sample processing, and provides seamless access to captured CTCs for further downstream characterization.
Schematic representation of the methodology developed for creating cryopreservable high throughput paper-based arrays of 3D tumor models for drug screening applications.
The continuous development of simple and practical cell cryopreservation methods is of great importance to a variety of sectors, especially when considering the efficient short‐ and long‐term storage of cells and their transportation. Although the overall success of such methods has been increased in recent years, there is still need for a unified platform that is highly suitable for efficient cryogenic storage of cells in addition to their easy‐to‐manage retrieval. Here, a paper‐based cell cryopreservation method as an alternative to conventional cryopreservation methods is presented. The method is space‐saving, cost‐effective, simple and easy to manage, and requires no additional fine‐tuning to conventional freezing and thawing procedures to yield comparable recovery of viable cells. It is shown that treating papers with fibronectin solution enhances the release of viable cells post thawing as compared to untreated paper platforms. Additionally, upon release, the remaining cells within the paper lead to the formation and growth of spheroid‐like structures. Moreover, it is demonstrated that the developed method works with paper‐based 3D cultures, where preformed 3D cultures can be efficiently cryopreserved.
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