Jen-chia Wu

Circulating Tumor Cell Count Correlates with Colorectal Neoplasm Progression and Is a Prognostic Marker for Distant Metastasis in Non-Metastatic Patients

Enumeration of circulating tumor cells (CTCs) has been proven as a prognostic marker for metastatic colorectal cancer (m-CRC) patients. However, the currently available techniques for capturing and enumerating CTCs lack of required sensitivity to be applicable as a prognostic marker for non-metastatic patients as CTCs are even more rare. We have developed a microfluidic device utilizing antibody-conjugated non-fouling coating to eliminate nonspecific binding and to promote the multivalent binding of target cells. We then established the correlation of CTC counts and neoplasm progression through applying this platform to capture and enumerate CTCs in 2 mL of peripheral blood from healthy (n = 27), benign (n = 21), non-metastatic (n = 95), and m-CRC (n = 15) patients. The results showed that the CTC counts progressed from 0, 1, 5, to 36. Importantly, after 2-year follow-up on the non-metastatic CRC patients, we found that those who had ≥5 CTCs were 8 times more likely to develop distant metastasis within one year after curable surgery than those who had <5. In conclusion, by employing a sensitive device, CTC counts show good correlation with colorectal neoplasm, thus CTC may be as a simple, independent prognostic marker for the non-metastatic CRC patients who are at high risk of early recurrence.

Antibody conjugated supported lipid bilayer for capturing and purification of viable tumor cells in blood for subsequent cell culture

Interest in the identification and isolation of circulating tumor cells (CTCs) has been growing since the introduction of CTCs as an alternative to the tumor tissue biopsy, which can potentially be important indices for prognosis and cancer treatment. However, the contamination of non-specific binding of normal hematologic cells makes high purity CTCs detection problematic. Furthermore, preserving the viability of CTCs remains a challenge. In this study, we proposed to construct an anti-EpCAM functionalized supported lipid bilayer (SLB), a biomimetic and non-fouling membrane coating, for CTCs capturing, purification and maintaining the viability. Healthy human blood spiked with pre-stained colorectal cancer cell lines, HCT116 and colo205, were used to investigate interaction of cells with the anti-EpCAM functionalized SLB surfaces. Over 97% of HCT116, and 72% of colo205 were captured and adhered by the surface anti-EpCAM; conversely, the majority of blood cells were easily removed by gentle buffer exchange, with the overall purity of cancer cells exceeding 95%. The bound cancer cells were subsequently detached for cell culture. Both HCT116 and colo205 continued to proliferate over 2-week observation period, indicating that the anti-EpCAM functionalized SLB platform providing a simple strategy for capturing, purifying, and releasing viable targeted rare cells.

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.

Efficient elusion of viable adhesive cells from a microfluidic system by air foam

We developed a new method for releasing viable cells from affinity-based microfluidic devices. The lumen of a microchannel with a U-shape and user-designed microstructures was coated with supported lipid bilayers functionalized by epithelial cell adhesion molecule antibodies to capture circulating epithelial cells of influx solution. After the capturing process, air foam was introduced into channels for releasing target cells and then carrying them to a small area of membrane. The results show that when the air foam is driven at linear velocity of 4.2 mm/s for more than 20 min or at linear velocity of 8.4 mm/s for more than 10 min, the cell releasing efficiency approaches 100%. This flow-induced shear stress is much less than the physiological level (15 dyn/cm(2)), which is necessary to maintain the intactness of released cells. Combining the design of microstructures of the microfluidic system, the cell recovery on the membrane exceeds 90%. Importantly, we demonstrate that the cells released by air foam are viable and could be cultured in vitro. This novel method for releasing cells could power the microfluidic platform for isolating and identifying circulating tumor cells.