Development Of Biophysical Markers That Quantify Metastatic Potentials Of Prostate Cancer Cells Using Tsunami Microscope

Dysregulated trafficking of receptor tyrosine kinases has been linked to oncogenesis. Here we study the trafficking patterns and dynamics of epidermal growth factor receptor (EGFR) trafficking of benign (BPH1), non-invasive malignant (LNCaP), and highly invasive malignant (PC-3) prostate cells using an advanced 3D single-particle tracking technique termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination). As a feedback-control tracking system, TSUNAMI is capable of tracking fluorescent nanoparticle-tagged EGFR for up to 10 minutes and in the z-direction for up to ±50 microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, a trajectory analysis algorithm is developed to classify trajectories and extract the dynamic parameters, such as diffusivity, inward movement, and internalization duration. These parameters can be used to quantify the metastatic potentials of prostate cancer cell lines. For instance, the diffusivities of EGFRs on the highly invasive malignant PC-3 cells (0.010 ± 0.014 µm2/s) are around one-quarter of those estimated from the benign BPH1 cells (0.036 ± 0.058 µm2/s), possibly due to the abnormally high expression of EGFRs on the PC-3 cells. The highly invasive PC-3 cells also exhibit longer (2.83 ± 0.23 µm vs. 1.45 ± 0.16 µm) and faster (0.021 ± 0.016 µm/s vs. 0.005 ± 0.002 µm/s) inward movement as compared with the non-invasive LNCaP prostate cancer cells, which could be due to the high endocytotic activity associated with the invasive PC-3 cells. In addition, the dynamics parameters extracted from the EGFR trajectories are correlated with the expression levels of a number of epithelial-mesenchymal-transition (EMT)-related genes. The high EGFR expression is related to the decrease of EGFR diffusivity, and the increase of dynamins coheres with more active inward movement. After EMT induction, the non-invasive LNCaP prostate cancer cells also exhibit gene expression profiles and EGFR trafficking patterns similar to those shown in invasive PC-3 cells. Our work demonstrates that EGFR trajectory-derived dynamics parameters are linked to metastatic potentials. A new class of biophysical markers can be established based on the analysis of membrane protein trajectories.