Poster Presentations


P-29: Recovery and Analysis of Circulating Tumour Cells from the Peripheral Blood of Stages III and IV Colorectal and Breast Cancer Patients

Mr. Aaron Geekie-Sousa

Deep Patel, MPharm*, 
Mala Bahl, MD MSc FRCP, 
Jonathan Blay, PhD FRSM FRSB 

University of Waterloo
School of Pharmacy

Topic: Clinical Cancer Research

Objective
Optimising future approaches for the personalized treatment of patients with metastatic cancers will benefit from our ability to sample cells emigrating from the tumour and detectable in peripheral blood. These could then be analyzed for functional markers that will predict their ability to form extensive metastases. Existing methods to identify circulating tumour cells (CTCs) do not recover all tumour cells. Most current methods take advantage of the cells’ physical properties and marker based-biological properties for capture. We are using a different approach that takes advantage of the binding properties of proteins from the tissue extracellular matrix (ECM) to capture CTCs more optimally based on their function. We are then able to further examine the individual CTCs for features of aggressive behavior.

Methods
A group of 40 patients with Stage III or IV colorectal or breast cancers provide peripheral blood samples (10-12mL) at the time of their initial clinic visit (clinical research protocols ORE #21303 and THREB 2016-0586). Patients also provide samples at 6- and 12-months follow-up visits. The blood is transported on ice to our laboratory within 1 h of collection. The cells are then fractionated by density centrifugation on Ficoll-Paque PLUS®. CTCs are recovered from the interface together with leukocytes, washed and placed on substrata composed of selected ECM proteins including collagen, fibronectin, laminin and their combinations. After 18h of incubation at 37C, non-adherent cells (the majority of the leukocytes) are washed away and the remaining cells are lightly fixed with 3.7% paraformaldehyde, followed by immunofluorescence staining for marker proteins. Parallel experiments in which green fluorescent-dyed HCT116 colorectal cancer cells are added to normal human blood samples (protocol ORE #22816) are carried out for optimization and quantification of recovery.  

Results
Data show differences in recovery depending upon the ECM proteins used as the capture substratum, with CTCs staining positive for epithelial markers EpCAM and pan-cytokeratin and negative for leukocytic markers. Intact cells are also confirmed using the nuclear stain DAPI. At this point, a minimum of ~50% recovery has consistently been achieved through the calibration experiments, with individual recoveries reaching as high as ~70%. Current efforts are directed toward identifying markers of disease progression such as chemokines.

Conclusions
Capture of CTCs using an ECM-based substratum offers a different approach to tracking and characterising cancer cells from the peripheral blood, in that it focuses on a functional ability that is related to metastasis. Tracking differences in the abundance and disease markers of CTCs isolated using this approach in different patients, as well as identifying shifts in these parameters over the 6-12 months following the commencement of therapy, may allow us to distinguish different patterns of aggression in the disease progress.