• circulating tumor cell
• PAINT imaging

Circulating tumor cells (CTCs) play a crucial role in cancer research, as they are cells that
detach from the primary tumor site and contribute to metastasis throughout the body.
Isolating and analyzing CTCs presents significant challenges due to their scarcity in blood
and heterogeneity. Nevertheless, they could contribute to cancer diagnostics,
understanding cancer progression, and exploiting therapeutic markers involved in
metastasis.
The main objective of my thesis is to develop an effective, high throughput method to
isolate and capture circulating tumor cells for live cell imaging. This includes isolation of
CTCs from the rest of blood components (platelets, red and white blood cells), capture the
cells into a glass slide for imaging and identification of CTCs among impurities, while
preserving cell viability.
The isolation process is based on a density gradient separation and negative selection of
white blood cells. To test this, cell models such as MDA-468 and A431 cells were employed
as standards in technique development. The results indicate an effective isolation of cancer
cells and a strong linearity within broad range of cell concentrations, both using Jurkat cells,
as a model for white blood cells, and full blood.
The capturing method consists of functionalizing a glass slide with EpCAM (CTC marker)
antibodies to specifically capture cancer cells. This step is required to immobilize the cells
on a glass surface in a properly distance, for subsequent imaging.
In particular, live-cell PAINT imaging was used. PAINT is a super-resolution microscopy
technique that visualizes protein structures and their dynamics behaviour below the
diffraction limit of light, utilizing the transient interaction between a target marker and a
labelled probe. The method’s effectiveness to capture cancer cells was confirmed using
different chambers, although the results were hindered by inadequate blood collection
tubing.
The last step is to develop an identification method that addresses sample impurities and
optimizes technique yield. Cell models with varying markers expression were used to mirror
the Epithelial-to-Mesenchymal transition, adding heterogeneity in metastatic cells. Markers
such as Vimentin and EpCAM, along with Hoechst (cell nuclei marker) as a positive control,
successfully differentiated between different cell types. Differentiating phenotypes
combined with considerations about morphological characteristics proved effective for
distinguishing between cell types.
In conclusion, the microscopy-based method for analyzing circulating tumor cells holds
potential as a diagnostic technique for counting and identifying CTCs in patient blood
samples within a relatively short time. This method shows promise to diagnose and monitor
various types of solid tumors.