Investigating the Clinical Utility of Circulating Tumor Cells Via Nanomaterial Based Microfluidic Platforms

Abstract

To realize personalized treatment for cancer patients, it is crucial to identify and monitor the molecular drivers of tumors. Currently, the molecular analysis of tumors is mostly performed on tissue biopsies. However, due to the invasiveness of the procedure, biopsies typically cannot be obtained repeatedly during the course of treatment and thus cannot reveal the dynamic evolution of tumors on both the genetic and epigenetic levels. There is a pressing need to monitor tumor evolution and to predict the treatment response to guide the clinical decision-making in the practice of personalized therapy. Circulating tumor cells (CTCs) shed from the primary tumor, travel through the blood, and have the potential to cause metastases. As CTCs can be frequently sampled from peripheral blood, CTC isolation and analysis hold great potential as a biomarker in real-time monitoring of tumor status. This work highlights the clinical utility of CTCs for providing prognostic and predictive information for specific treatments in cancer patients. First, dynamic changes of PD-L1(+) CTCs during radio(chemo)therapy were investigated in NSCLC. The real-time monitoring of PD-L1 expression in tumor microenvironment is crucial in guiding the therapeutic management of anti-PD-1/PD-L1 immunotherapy. CTCs were isolated using a nanomaterial based microfluidic device, the GO chip. PD-L1 (+) CTCs were detected in 25 out of 36 (69%) samples from 12 NSCLC patients undergoing radiation or radiochemotherapy. After the initiation of radiation, the proportion of PD-L1 (+) CTCs in total CTCs increased significantly (median 4% vs 24%, P=0.018). Furthermore, patients who were PD-L1 positive (5% of CTCs stained with PD-L1) at baseline had shorter PFS, suggesting the prognostic value of PD-L1 (+) CTCs (6.7 months vs 14.75 months, P = 0.017) Secondly, CTC number and the molecular features of CTCs were monitored at different time points during the course of treatment for locally advanced pancreatic patients. The reduction of CTC numbers after chemotherapy correlated with shorter progressed free survival (PFS), indicating that changes of CTC numbers may be an early indicator for treatment failure (6.5 months vs 13.5 months, P value= 0.002). Furthermore, in the mRNA profiling of CTCs, the expression levels of three genes that have been shown to play a role in drug resistance, BAX, CHK1 and EZH2, are associated with poor prognosis, which could act as makers to predict and monitor the treatment response. Thirdly, this work presents two technical advances of CTC technologies. A highly sensitive microfluidic device to capture and release circulating tumor cells from whole blood of cancer patients is developed. Graphene oxide is embeded into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. As the temperature decreases to around 5 °C, the polymer film dissolves and detaches from the device and captured cells are released. Over 90% capture efficiency and release efficiency have been achieved. Released CTCs were viable and structurally intact, enabling subsequent analysis such as standard clinical cytopathological and genetic testing. Finally, to develop a high throughput CTC isolation technology, a herringbone mixer is incorporated into the previously developed GO chip and optimized the structure of the herringbone mixer and the channel geometry to maximize the throughput while achieving high capture efficiency (> 80%) and cell viability (> 90%). The time required to process a 1-mL blood sample is reduced to 10 minutes, 6 times faster than in the previous design.