ORIGINAL RESEARCH article
Front. Oncol.
Sec. Molecular and Cellular Oncology
Volume 15 - 2025 | doi: 10.3389/fonc.2025.1632245
This article is part of the Research TopicCirculating Cancer, Cancer-Associated, Atypical and Stromal Cells in Neoplastic and Preneoplastic ConditionsView all articles
Complementary detection strategies for circulating tumor cells in breast cancer: Clinical implications of combining immunofluorescence and cytopathological stainingA comparative analysis of immunofluorescence and routine cytological staining for optimization of breast cancer circulating tumor cells detection
Provisionally accepted
- Institute of Oncology Ljubljana, Ljubljana, Slovenia
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Background: Circulating Tumor Cells (CTCs) serve as important biomarkers for disease monitoring and treatment response in patients with metastatic breast cancer. Their detection remains challenging because of their low abundance, phenotypic diversity and non-standardized mode of detection. Cytopathological Giemsa and Immunofluorescence (IF) staining can offer complementary approaches for CTC characterization. Giemsa staining enables assessment of cellular morphology, while IF allows for marker-specific identification, together providing a more comprehensive and accurate evaluation of CTCs. Methods: We developed an IF staining protocol with antibodies against Cytokeratin (CK), vimentin (VIM), and Cluster of Differentiation 45 (CD45) to distinguish epithelial, mesenchymal, hybrid and hematopoietic cells for CTC detection and characterization and compared it with cytopathologic method of detection via Giemsa staining with regard to CTC detection rates and morphological detail. Results: Study was performed on the samples of 29 heavily pretreated patients with metastatic breast cancer (median duration of metastatic disease 19.4 months). Giemsa staining enabled the detection of a higher number of CTCs compared to our IF protocol. Lower detection rate was potentially due to the loss of fragile or loosely adherent cells during methanol fixation and IF staining. Additionally, in IF-stained samples, some CTCs presented faint nuclear signals, potentially impairing their recognition. The IF staining supported the identity of CTCs detected on Giemsa-stained slides by employing a three-color antibody panel-based approach and allowed detailed phenotypic discrimination and structural analysis of CTCs, including the identification of a distinctive CK polarization pattern suggestive of a transitional state during intravasation. Conclusion: Giemsa and IF may thus be complementary rather than mutually exclusive and relying on a single detection approach could underestimate the true CTC burden. An integrative strategy combining both techniques may offer a more comprehensive view of CTC populations in metastatic breast cancer, thereby enhancing diagnostic precision.
Keywords: biomarker, breast cancer, circulating tumor cells, Cytopathological detection, Immunofluorescence-based detectioncirculating tumor cells, morphology, Staining protocol, immunofluorescence
Received: 20 May 2025; Accepted: 09 Jul 2025.
Copyright: © 2025 Jesenko, Grašič Kuhar, Pisljar, Miceska, Kloboves-Prevodnik and Cemazar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Tanja Jesenko, Institute of Oncology Ljubljana, Ljubljana, Slovenia
Maja Cemazar, Institute of Oncology Ljubljana, Ljubljana, Slovenia
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