基于CTC的生物标志物检测方法开发与工作流程优化，用于预测HR+/HER2-乳腺癌对CDK4/6抑制剂的应答

Introduction: CDK4/6 inhibitors (CDK4/6i) are the standard of care for hormone receptor-positive/HER2-negative breast cancer (HR+/HER2- BC). Yet resistance is common and often characterized by loss or dysfunction of the Retinoblastoma (Rb) protein or inadequate suppression of its phosphorylation. Circulating tumor cells (CTCs) offer minimally invasive means to monitor tumor biology. Here, we describe the analytical performance of a multiplex immunofluorescence (mIF) assay integrated with an image-analysis pipeline to quantify Rb and phospho-Rb (pRb) expression on CTCs and evaluation of cell recovery efficiency by CTCeptor, a microfluidic CTC isolation platform. Methods: The mIF assay utilized antibodies against Rb (AF595), pRb (AF488), cytokeratin (CK, AF647), and CD45 (AF555), with DAPI for nuclear staining. Antibody specificity and minimal non-specific binding were verified using single-marker staining and isotype controls. Assay performance (sensitivity, specificity, signal-to-noise ratio (SNR), and dynamic range) was evaluated using positive (MCF7, T47D) and negative (T47D-PalboR, MDA-MB-468, or leukocytes) control cell lines. Healthy donor leukocytes were spiked with control cell lines to simulate patient-derived matrices. Various blocking buffers, wash buffers, and cell attachment methods were compared. Cell recovery was evaluated after processing blood spiked with MCF7 cells using CTCeptor followed by staining with anti-CK-AF647, anti-CD45-AF555, and DAPI in HyPICC chamber. Imaging was performed on Leica STED SP8 or Nikon N-STORM microscopes. Quantitative analysis of mean fluorescence intensity (MFI) was conducted using FIJI, and statistical analysis was performed using GraphPad Prism v10. Results: The optimized mIF with integrated image analysis pipeline demonstrated high analytical sensitivity (Rb: 84%, pRb: 83%) and specificity (Rb: 96%, pRb: 100%). SNRs were 5 for Rb and 50 for pRb, with dynamic range exceeding two log units for both markers. Single-marker staining and isotype controls confirmed antibody specificity and minimal non-specific binding. Based on comparative analyses, 1% BSA + 0.5% goat serum was selected as the blocking buffer, and Triton-based buffer as the wash buffer. RareCyte® cell attachment method yielded superior cell retention (79%). Recovery rate after processing the spiked blood with MCF7 cells using CTCeptor followed by staining in HyPICC chamber ranged from 40-60%. Conclusion: We have developed and optimized a quantitative mIF assay and image analysis workflow to detect Rb and pRb expression on CTCs. Future studies will assess the predictive biomarker utility of the assay by measuring Rb and pRb dynamics on CTCs in metastatic HR+/HER2- BC patients before and after CDK4/6i therapy. Acknowledgement: This research was funded by CPRIT (RP210148) and NCATS CTSA pilot grant (1UM1TR004539-01A1).