Circulating tumor cells (CTCs) invade blood vessels in solid tumors and promote metastases by circulating in the blood. CTCs are thus recognized as targets for liquid biopsy and can provide useful information for design of treatments. This diagnostic approach must consider not only the number of CTCs but also their molecular and genetic characteristics. For this purpose, use of devices that enrich CTCs independent of these characteristics and detectors that recognize various CTC characteristics is essential. In the present study, we developed a CTC detection system comprising ClearCell FX and ImageStream Mark II. We clarified the analytical performance of this system by evaluating recovery rate, lower limits of detection, and linearity. These parameters are critical for detecting rare cells, such as CTCs. We tested these parameters using three cell lines with different expression levels of the epithelial marker-epithelial cell adhesion molecule (EpCAM) and spiked these cells into whole-blood samples from healthy donors. The average recovery rate and lower limit of detection were approximately 40% and five cells/7.5 mL of whole blood, respectively. High linearity was observed for all evaluated samples. We also evaluated the ability of the system to distinguish between normal and abnormal cells based on protein expression levels and gene amplification and found that the system can identify abnormal cells using these characteristics. The CTC detection system thus displays the ability to distinguish specific characteristics of CTC, thereby providing valuable information for cancer treatment.

T cells play a pivotal role in the immune system's response to various conditions. They are activated by antigen‐presenting cells (APCs) via T‐cell surface receptors, resulting in cytokine production and T‐cell proliferation. These interactions occur through the formation of immunological synapses. The advent of imaging flow cytometry has enabled detailed statistical analyses of these cellular interactions. However, the dynamics of T‐cell receptors in response to in vitro stimulation are yet to receive attention, despite it being a crucial aspect of understanding T‐cell behavior. In this article, we explore the responses of T cells to in vitro antibody‐based stimulation without APCs. Specifically, we established a Th1 cell clone, subjected it to a combination of centrifugation‐induced mechanical stress and anti–human CD3 and anti–human CD28 antibody stimulation as the in vitro antibody‐based stimulation, and captured and analyzed bright‐field and fluorescence images of single cells various hours after stimulation using an imaging flow cytometer. Our results indicate distinct temporal dynamics of CD3 and CD28. Notably, CD3 and CD28 relocated on the T‐cell surface immediately after stimulation, with CD3 receptors dispersing after 3.5 h, whereas CD28 remained clustered for 7.5 h. These receptor morphological changes precede cytokine production, suggesting their potential as early indicators of T‐cell activation.

Background/Objectives: Liquid biopsy methods have gained prominence as minimally invasive tools to improve cancer treatment outcomes. Circulating tumor cells (CTCs) offer valuable insights into both primary and metastatic lesions. However, validating the CTC test results requires confirmation that the detected cells originate from cancer tissue. While studies have identified CTCs in colorectal cancer (CRC) patients using molecular markers, simultaneous validation of their cancer tissue origin remains unexplored. Methods: This study introduces a simple approach to detect adenomatous polyposis coli (APC) gene abnormalities alongside established CTC markers using a molecular imaging flow cytometer (MI-FCM). Given that APC gene abnormalities occur in 60–70% of CRC patients, their detection serves as strong evidence of cancer origin. Results: Our method achieved 92% concordance with DNA sequence analysis of tumor-derived cells. In a proof-of-concept study using 5 mL of whole blood from CRC patients, we observed a high frequency of cells exhibiting APC abnormalities, cytokeratin (CK), and vimentin (Vim) expression. Extending the study to 80 CRC patients across pathological stages I–IV confirmed CK and Vim as valid CTC markers. Three distinct cell populations were identified in blood: CK+/Vim−, CK+/Vim+, and CK−/Vim+. CTC number and frequency increased progressively with cancer stage. Conclusions: This is the first report demonstrating CK and Vim as effective markers for direct CTC detection in CRC patients. Our findings provide evidence-based validation of CTC markers, offering new insights and advancing approaches for patient care.

We integrated in vitro antibody-based stimulation, imaging flow cytometry, and deep learning to evaluate the morphological characteristics associated with T cell activation.