<p>Circulating tumor cells (CTCs) have been
proved to possess great value and potential in detection, diagnosis, and <a>prognosis</a> of non-haematologic cancers. Their unique characteristics
in providing both phenotypic as well as genotypic information make them highly
valuable in liquid biopsy assays. A<a>t the same time, though
numerous studies and research have been done, identification and enumeration of
CTCs is still technically challenging due to their rarity and heterogeneity</a>.
The primary goal of the thesis is to develop a CTC detection and isolation system
with ultra-high
sensitivity and purity, while keeping it fast and scalable. We proposed a
microfluidic system that integrates
positive immunomagnetic capturing, high-throughput parallel flow and size
filtration. In this thesis, two generations of the system have been developed
to achieve the goal, and are approved to be able to effectively detect and
isolate CTCs from hundreds of breast cancer blood samples in real clinical
applications. </p>
<p>The first-generation system is based on a
sandwich-structured microfluidic chamber, which has a micro-aperture chip as
the core to detect and isolate immunomagnetically targeted CTCs. The system
achieves high detection yield (>95%) and purity (>99.9998% depletion of leukocytes) by streamlining the workflow and
using unprocessed whole blood (without centrifuging), as well as utilizing an
advanced surface coating approach to passivate the microchip surface. <a>We first demonstrate experiments for determining the optimal
detection parameters. Then we characterize the system by isolating deterministically
spiked 1, 10, and 100 single MCF-7 breast cancer cells into tubes of whole
blood, and show that >95% of cells were captured. A detection yield of 100%
from single cell spiking experiments (n = 6) demonstrates excellent detection
capability and repeatability of the system. We finally demonstrate the use of
the system for CTC detection in the context of a phase II clinical trial of
early-stage triple-negative breast cancer (TNBC) patients. As a part of the
trial, 182 blood samples were collected from 124 early-stage TNBC patients at
high-risk of relapse. We detected CTCs in 36.3% of patients who had already
completed chemotherapy and surgery for curative intent and were thus nominally
expected to have very few to zero CTCs. </a>Moreover, increasing CTC count from the
same patients shows good correlation with <a></a><a>their clinical course</a>. The ability to detect CTCs’ presence using this first-generation
system illustrates its important clinical utility.</p>
<p>The second-generation system applies a similar
detection strategy but employs an upgraded microchip and device, as well as a
further streamlined process flow to achieve an even higher detection efficiency,
especially for capturing the target cells with low surface marker expression
level. We first did modeling and simulation of the new system to find the optimal
magnet configuration and verify the detection sensitivity improvement on the
first-generation system. Then we characterized the new system by detecting
spiked JEG-3 and JAR cells in both cell culture medium and human blood. The
result demonstrates that the detection yield increased by ~20% using the
second-generation system under the same experiment condition. Next, we applied
the system to a phase I clinical trial for CTC detection from metastatic triple-negative breast cancer (mTNBC) patient blood samples. CTCs of mTNBC
are known to with in the low marker expression phenotype, which requires
ultra-high detection sensitivity. Our system captured CTCs from 48 out of 102
(47%) blood samples, the positivity rate agrees with the conclusions from other
studies and presents the reliability to the system. Finally, we explored a
novel 4-marker panel for CTC detection from mTNBC patient blood samples. We
conducted paired comparisons using the 4-marker panel versus a single marker
for detection. The 4-marker panel yielded more CTCs in 5/8 complete paired assessments,
and less CTCs in 1/8. The association missed the significance level only
slightly (p = 0.08), and the result strongly illustrates the potential for
using the panel to cover the mTNBC cells’ heterogeneity for enhanced CTC
detection. Furthermore, the presence of CTCs from blood samples correlates well
with the patient’s disease progression.</p>
<p>Finally, we demonstrated downstream analysis ability
of the CTCs detected by the second-generation system. Captured CTCs can be
readily released from our system without any loss or damage to a secondary
microchip device to be further isolated as single cells, and picked up
individually for downstream analysis like DNA/RNA sequencing or single-cell cultivation.
Directions for future work is also discussed. We envision this versatile and
efficient system to be highly beneficial in a broad range of clinical and
research applications regarding CTCs.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/14510979 |
| Date | 29 April 2021 |
| Creators | Yuan Zhong (10695393) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY-NC-SA 4.0 |
| Relation | https://figshare.com/articles/thesis/DETECTION_AND_ISOLATION_OF_CIRCULATING_TUMOR_CELLS_FROM_WHOLE_BLOOD_USING_A_HIGH-THROUGHPUT_MICROCHIP_SYSTEM/14510979 |
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