Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues

Area of Science:

• Oncology
• Biomedical Engineering
• Pathology

Background:

• The spatial heterogeneity of the tumor microenvironment poses challenges for traditional 2D histology.
• Accurate characterization of tumor heterogeneity is crucial for effective cancer treatment strategies.

Purpose of the Study:

• To evaluate the feasibility of CLARITY tissue processing for analyzing breast cancer patient biopsies.
• To compare the insights gained from 3D CLARITY analysis with conventional 2D FFPE histology.

Main Methods:

• Formalin-fixed breast cancer biopsies underwent CLARITY processing, including lipid clearing and multiplexed immunostaining for biomarkers (pan-cytokeratin, Ki67, CD3).
• Specimens were imaged using confocal microscopy after refractive index matching.
• CLARITY-processed data were quantitatively compared to standard FFPE histology.

Main Results:

• CLARITY preserved tissue morphology and achieved high optical transparency, with minor limitations in fibrotic areas.
• Immunostaining for key markers was successful with optimized antibody conditions.
• 3D CLARITY analysis revealed significant intra-tumoral heterogeneity in Ki67 expression, which was not apparent in 2D FFPE sections, while composite Ki67 scores agreed with histology.

Conclusions:

• CLARITY processing is feasible for archived FFPE breast cancer specimens, enabling 3D analysis.
• CLARITY offers a more accurate and unbiased approach to tumor sample analysis compared to conventional histology.
• This technique enhances the visualization of intra-tumoral heterogeneity, potentially leading to improved diagnostic accuracy and treatment planning.