Novel Digital Pathology Pipeline May Reduce Costs of Circulating Tumor Cell Characterization

by Christos Evangelou, MSc, PhD – Medical Writer and Editor


Despite the crucial role of circulating tumor cells (CTCs) in metastasis and tumor recurrence, their clinical utility is limited to monitoring disease relapse and treatment response among patients with breast, colorectal, and prostate cancer. The high cost of current CTC isolation and characterization assays is a key factor contributing to the limited cost-effectiveness and clinical adoption of CTCs for cancer screening.

In a recent proof-of-concept study, Trinity College Dublin researchers at Coombe Women’s and Infants University Hospital and St. James’s Hospital, Dublin, Ireland, developed and tested a low-cost CTC isolation and characterization pipeline based on digital pathology methods.1

Using open-source software to combine morphological and biomarker information from histochemical and multiparametric immunofluorescence stains, this method allows for in-depth analysis of CTCs without the need for expensive imaging hardware.

“While the work published in this paper will likely not have direct clinical implications, it creates a robust and accessible basic research tool that may help accelerate the realization of the clinical utility of liquid biopsy across multiple malignancies,” said Brendan Ffrench PhD, researcher at Trinity College Dublin and the first author of the study.

Dr. Ffrench noted that liquid biopsy platforms closest to the clinic do not appear to be best suited for characterizing novel CTC phenotypes. “In our opinion, the approach we have developed is one of the strongest in terms of ability to characterize new CTC biomarkers and cell states,” he explained.

“Although this method is, perhaps, not best placed to be used in the clinic, it empowers the basic research tier to identify new clinically relevant biomarkers. Such biomarkers could then be ported closer to the clinic,” he added.

The report was published in the journal Heliyon.


Study Rationale: Developing a Low-cost CTC Isolation and Characterization Method

CTCs are a heterogeneous subset of tumor cells that disseminate from the tumor and travel through the circulatory system to generate metastatic lesions at distant sites.1 Although only a very small proportion of CTCs survive at distant organs and develop new tumors, distant metastasis is often incurable and contributes to poor prognosis.

“We originally set out to apply our cellular characterization ethos to the study of CTCs, with the aim of understanding if certain cell types or clusters of cell types conveyed advantages to CTCs to survive and form distant metastases. However, we quickly found that there were no accessible tools to carry out the type of CTC characterization studies we were interested in,” said Dr. Ffrench.

This gap in accessible CTC characterization tools and the need for a protein biomarker-independent method for capturing and enriching CTCs drove the research team to conduct this study.

“We believe that further understanding of the basic CTC biology of other malignancies is required to achieve wider clinical utility of CTCs in cancers other than breast, prostate, and colorectal cancer,” Dr. Ffrench noted.


Development of a Biomarker-independent Method of Capturing and Enriching CTCs

The team developed and tested a low-cost CTC isolation and characterization pipeline based on an ‘isolation by size’ approach. To this end, they used the ScreenCell Cyto platform to isolate CTCs. Subsequently, they combined information from histochemical morphology stains and multiparametric immunofluorescence to enrich and characterize the isolated CTCs.1

“Our approach is based on the ScreenCell Cyto platform, a protein biomarker-independent method of capturing and enriching CTCs. It uses a combination of modified Giemsa staining and immunofluorescence to characterize the isolated CTCs,” Dr. Ffrench explained.

The modified Giemsa stain served as a redundant backup for CTC detection. Giemsa stains all cells and allows CTCs to be identified through cytological features independent of protein biomarkers. These cytological features include a high nuclear-to-cytoplasmic ratio and the presence of a hyperchromatic nucleus with coarse chromatin.

This approach enables the isolation and characterization of single CTCs and CTC clusters without the need for staining individual biomarkers. The immunofluorescence panel can be easily adapted to include different biomarkers, allowing them to be tailored depending on the malignancy type or characterization work being carried out.


Development of an Automated Image Registration Algorithm

Although the combination of modified Giemsa staining and multiparametric immunofluorescence allows the isolation and characterization of CTCs independent of biomarkers, it is technically challenging to align stained images from multiple microscope sources.

To overcome challenges associated with image alignment, the research team developed open-source software that facilitates the synchronization of microscopy images from multiple sources.1 The fact that the algorithm is freely available provides the opportunity for multiparameter histochemical and immunofluorescent analysis of CTCs with existing microscopes without the need for CTC-specific imaging hardware.

“We created an automated image registration algorithm and developed accompanying software to automate this image alignment process, making the approach, as a whole, highly accessible to other research groups. By developing the wet-work methods around the ScreenCell Cyto CTC capture device, we did not need large capital investment into a CTC isolation platform,” Dr. Ffrench said.


Looking Ahead

Although this cell isolation and characterization pipeline is geared toward CTC research, the principles are highly applicable to the study of any rare cell population. Combining data from multiple microscopy sources can add immense value to the study of rare cell events. With some relatively minor adjustments, this alignment approach can be applied to virtually any cytology specimen.

“To date, the alignment of tissue sections is widely achievable, but such approaches often fail when applied to cytology specimens due to the lack of ‘macro’ features in the digital slide. Our approach overcomes such limitations,” said Dr. Ffrench.

Dr. Ffrench also noted that the CTC isolation and characterization pipeline needs to be tested in a larger cohort of patients to validate its performance. “With time, this will hopefully allow for the robust characterization of novel CTC biomarkers to progress CTCs from additional malignancies toward clinical utility,” he said.

“We have created wet-bench and in silico tools and made them freely available to help reduce the financial barrier for CTC research and to allow others to adapt and develop them to meet their needs. We feel that the more researchers in this space, the faster we will understand the nuanced cell biology that drives distant metastasis,” he concluded.


  1. Ffrench B, Kashdan E, Huang Y, et al. CTC-5: A novel digital pathology approach to characterise circulating tumour cell biodiversity. Heliyon. 2023;9(1):e13044. doi:10.1016/j.heliyon.2023.e13044

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