Advancing circulating tumor cell isolation and analysis: Development of optimized immunomagnetic techniques and molecular characterization approaches for early cancer detection

Circulating tumor cells (CTCs) are critical to cancer metastasis and offer valuable insights for improving cancer diagnostics and personalized treatment strategies. This thesis addresses the main challenges in CTC isolation and analysis—low antigen expression, tumor heterogeneity, and molecular degradation—by developing innovative approaches to enhance capture efficiency, broaden detection range, and preserve cellular integrity for downstream applications.

The research begins with a systematic evaluation of existing CTC isolation techniques and highlights their limitations, particularly in capturing heterogeneous CTC populations. To improve capture efficiency, various commercial streptavidin-coated magnetic beads are tested, identifying those around 100–150 nm as optimal. Building on this, custom-designed silica-coated magnetic nanobeads (NC@silica-SA) are synthesized, offering superior magnetic responsiveness and reduced non-specific binding. These advances significantly improve the isolation of CTCs, even those with low EpCAM expression.

To address tumor heterogeneity, the study introduces a dual-targeting strategy combining recombinant VAR2CSA protein (rVAR2), which binds broadly to cancer cells, with anti-EpCAM antibodies. This approach increases the capture of diverse CTC populations across non-small-cell lung cancer lines. Additionally, fixation-free staining and isolation methods are developed to maintain RNA integrity, enabling reliable single-cell gene expression analyses. Together, these advancements enhance CTC detection, support more accurate molecular profiling, and open new avenues for personalized cancer care.