Abstract
Immunohistochemistry (IHC) is one of the main clinical techniques for biomarker assessment on tissue biopsies. It consists in chromogenic labeling with specific antibodies, followed by optical imaging, and it is used for diagnosis and therapeutic targeting. A well-known drawback of IHC is its limited robustness, which often precludes quantitative biomarker assessment. We combine microfluidic immunostaining, fluorescence imaging, and image-based cell segmentation to create an ultrafast procedure for accurate biomarker assessment via IHC. The experimental protocol is very simple and based on fast delivery of reagents in a microfluidic chamber created by clamping a half-chamber patterned in a silicon chip on top of a tumor tissue section. Also, the imaging procedure simply requires a standard fluorescence microscope, already widely used in clinical practice. The image processing is based on local-contrast enhancement and thresholding of the obtained fluorescence image, with subsequent Voronoi segmentation. To assess the experimental and analytical procedure on robust biological controls, we apply our method to well-characterized cell lines, which guarantee higher reproducibility than whole-tissue samples and therefore enable to disentangle the technical variability from the biological variability. To increase the potential translationality, we address the detection and quantification of the human epidermal growth factor receptor 2 (HER2) protein, which is a biomarker for HER2-type breast carcinoma diagnosis and therapy. We report both ultrafast immunofluorescence staining (5min per sample) of two breast cancer biomarkers and ultrafast cell segmentation (1min per sample = processing of thousands of cells). This provides a quantitative, cell-based immunofluorescent signal, with which we propose a potential diagnostic criterion to separate HER2-positive and HER2-negative breast cancer cells at high sensitivity and specificity.
Highlights
Assessment of Breast Cancer Status in ClinicsIn the field of breast cancer research and diagnostics, the human epidermal growth factor receptor 2 (HER2) receives major clinical interest since this membrane protein is targeted with an FDA-approved drug, namely Trastuzumab.[1]
To test the opportunity to characterize clinical controls at the cell level with a rapid and robust procedure, we applied a protocol of immunofluorescence staining to breast cancer cell lines with the microfluidic tissue processor and, subsequently, analyzed the fluorescence images with an automatic processing algorithm
Based on previous results reporting the importance of both HER2 and CK assessment for potential breast cancer diagnostics,[10] we stained and quantified both biomarkers
Summary
Assessment of Breast Cancer Status in Clinics. In the field of breast cancer research and diagnostics, the human epidermal growth factor receptor 2 (HER2) receives major clinical interest since this membrane protein is targeted with an FDA-approved drug, namely Trastuzumab.[1] To assess the status of the HER2 protein and to decide about the application of an HER2-targeted therapy, clinical guidelines have been set.[2] They are based on the outcome of a standard experimental technique for the evaluation of protein overexpression, which is called immunohistochemistry (IHC).[3] It consists of using antibodies and enzymatic reactions to stain a tissue slice obtained from a tumor biopsy of the patient. The optical readout consists in colored membranes of which the intensity indicates the overexpression level, scored as 0, 1þ (negative), 2þ (ambiguous), and 3þ (positive). For which 3þ patients can benefit from HER2-targeted therapy,[2] a unique score is attributed to the entire tissue, but previous work was proposed to score each cell individually,[4] which is advantageous to study intratumoral heterogeneity and its consequences in disease prognosis.[5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
