| Cancer is a heterogeneous disease,currently one of the leading causes of morbidity and mortality worldwide.Phenotypic and genotypic differences of tumor among patients and within the primary tumor and metastatic lesions indicate the dynamic nature of the disease.Dynamic nature of cancer demands frequent sampling of cancerous tissue in every step of cancer diagnosis,treatment,and follow-up.However,in most cancer types repeated tissue biopsies are clinically unfeasible and pose unfavorable risks on patients.On the other hand,the use of liquid biopsies,have been the current state of the art approach in sampling solid tumor that is readily available via blood draw.Circulating Tumor Cells(CTCs)are cells that detached from a primary tumor and transported via the circulatory systems to initiate colonization in distance area of the body.Being a candidate of liquid biopsies that can repeatedly sample,CTCs are currently a topic of investigation and clinical trials in diagnosis,treatment,and follow-up of cancer patients.Isolation,detection,and characterization of CTCs can serve as differential diagnosis and prognosis,risk determination,disease recurrence,and prediction of specific benefits from particular therapies for the management of cancer-related diseases.Further molecular and pharmacological analysis also provide a wealth of information about their genetic makeup,pathogenesis,and drug resistance pattern.Despite the scarcity of CTCs in blood,as few as 1-100 CTCs per 109 blood cells,a wide range of technologies have been developed for isolating and detecting CTCs.Among others,the use of microfluidic immunocapture methods that immobilize cells via antibody or aptamer chemistry has reported high capture efficiencies.However,these methods lack the required sensitivity to selectively isolate CTCs from a high background of blood cells especially in the early development of diseases.Other techniques are often limited by capture performance imbalance between high efficiency and high purity.Since the most cancer-related diseases are from the malignancy of a primary tumor,early detection of such event is a critical factor in determining the probability of survival for many cancers.In this respect,the need for a highly sensitive device for detecting CTCs during early tumor development and result with high purity capture is highly desirable.This dissertation is concerned with development,characterization and clinical evaluation of new hybrid size based and immunocapture microfluidic device for isolation and detection of CTCs from cancer patients’ blood sample with high sensitivity and purity.More specifically,it reviews existing knowledge in CTCs and methods for the isolation,detection,and characterization of CTCs in the first Chapter of this dissertation.The current work also employs a computation and simulated approach in design,optimization,and characterization of a new hybrid size dictated immunocapture microfluidic chip(SDI-Chip)which is presented in the second and third Chapters of the document.Followed by,fabrication,surface modification,pre-clinical and clinical evaluation of the device towards sensitive and efficient isolation,detection of CTCs from patients’blood sample and comparison with currently utilized technology in the fourth Chapter.The ease of applicability of our technology in different cell-line types,surface modification chemistry,and ligand,were also proved and discussed in the fifth Chapter of this dissertation.A microfluidic SDI-Chip composed of a channel with an array of triangular micropillars and three inlets and three outlets were designed.Micropillars were arranged according to the principle of DLD which enables us to provide size based differential interaction of CTCs with immunodecorated micropillars for immunocapture.Further optimization ensures extended duration of contact of CTCs with antibody-coated micropillars.Moreover,the hydrodynamic force gradient generated by optimized arrangement of micropillars provides optimum hydrodynamic forces for efficient CTC capture and enables spatial distribution of captured cells based on their antigen expression levels.CTCs,which are relatively larger than blood cells,interact frequently and intimately with immunodecorated micropillars and experience extended duration of contact in a region with a gradient of hydrodynamic forces to ensure more efficient capturing.On the other hand,blood cells only occasionally interact with the micropillars;instead,they flow between rows of micropillars where the hydrodynamic forces are hostile to nonspecific capture.Thereby we can enhance sensitivity,capture efficiency and purity of the conceptual design.Characterization of the capture performance of the SDI-Chip using antibodies and aptamers as a ligand to capture human colorectal cancer cells(SW480)and human acute lymphoblastic leukemia(CCRF-CEM)spiked into a buffer,and blood samples demonstrated a significant improvement in capture efficiency and purity compared with other related methods.Moreover,the current SDI-Chip allowed us to determine the level of antigen(EpCAM)expression by simply inferring the capture position around micropillars as a part of downstream molecular processing to provide additional relevant clinical information in a single run.Cross-sectional and longitudinal clinical studies with colorectal cancer patients also demonstrated the sensitivity of our chip to detect CTCs from 27 of 27(100%)non-metastasis colorectal cancer(CRC)patients’ blood samples with concentrations ranging from 8 to 161 CTCs per milliliter.Our SDI-chip also accurately demonstrated correlations between CTC count,clinical stage of tumor and disease progression in non-metastatic CRC patients.Thus,provide a new approach to hybrid size base and immunocapture device for improved performance of capture efficiency,purity and antigen-based profiling of CTCs for potential clinical application and inspire the future of integrated approach to solve the current challenge of sensitivity and tradeoff in capture performance of immunocapture devices. |
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