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Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis. Transcriptome analysis of circulating tumor cells (CTCs) provides insights into monitoring target therapeutics and underlying tumor metastasis. Here the authors present Hydro-Seq, a contamination-free high-throughput hydrodynamic scRNA-seq barcoding technique for rare CTCs.

Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses. Using this device, we isolated a small subpopulation of migratory cells with significantly greater tumor formation and metastasis in mouse models. Whole transcriptome sequencing of migratory versus non-migratory cells from two metastatic breast cancer cell lines revealed a unique set of genes as key regulators of tumor-initiating cells. We focused on phosphatidylserine decarboxylase (PISD), a gene downregulated by 8-fold in migratory cells. Breast cancer cells overexpressing PISD exhibited reduced tumor-initiating potential in a high-throughput microfluidic mammosphere device and mouse xenograft model. PISD regulated multiple aspects of mitochondria, highlighting mitochondrial functions as therapeutic targets against cancer stem cells. This research establishes not only a novel microfluidic technology for functional isolation of tumor-initiating cells regardless of cancer type, but also a new approach to identify essential regulators of these cells as targets for drug development.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC’s extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC’s hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM “primes” the IBC cells’ cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins −6, −8, and −10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.

Schistosomiasis is still one of the most significant neglected tropical diseases worldwide, and China is endemic for Schistosoma japonicum. With its great achievement in schistosomiasis control, the government of China has set the goal to eliminate the parasitic disease at the country level by 2030. However, one major challenge is the remaining huge areas of habitats for the intermediate host Oncomelania hupensis. This is further exacerbated by an increasing number of new emerging snail habitats reported each year. Therefore, population genetics on snails in such areas will be useful in evaluation of snail control effect and/or dispersal. We then sampled snails from new emerging habitats in Taicang of Jiangsu, China, a currently S. japonicum non-endemic area from 2014 to 2017, and performed population genetic analyses based on nine microsatellites. Results showed that all snail populations had low genetic diversity, and most genetic variations originated from within snail populations. The estimated effective population size for the 2015 population was infinitive. All snails could be separated into two clusters, and further DIYABC analysis revealed that both the 2016 and the 2017 populations may derive from the 2015, indicating that the 2017 population must have been missed in the field survey performed in 2016. These findings may have implications in development of more practical guidelines for snail monitoring and control.

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

Schistosomiasis is still one of the most significant neglected tropical diseases worldwide, and China is endemic for Schistosoma japonicum. With its great achievement in schistosomiasis control, the government of China has set the goal to eliminate the parasitic disease at the country level by 2030. However, one major challenge is the remaining huge areas of habitats for the intermediate host Oncomelania hupensis. This is further exacerbated by an increasing number of new emerging snail habitats reported each year. Therefore, population genetics on snails in such areas will be useful in evaluation of snail control effect and/or dispersal. We then sampled snails from new emerging habitats in Taicang of Jiangsu, China, a currently S. japonicum non-endemic area from 2014 to 2017, and performed population genetic analyses based on nine microsatellites. Results showed that all snail populations had low genetic diversity, and most genetic variations originated from within snail populations. The estimated effective population size for the 2015 population was infinitive. All snails could be separated into two clusters, and further DIYABC analysis revealed that both the 2016 and the 2017 populations may derive from the 2015, indicating that the 2017 population must have been missed in the field survey performed in 2016. These findings may have implications in development of more practical guidelines for snail monitoring and control.

In this research, a X-band dual-frequency reconfigurable reflectarray with independent control is realized. Employing a bowtie and dipoles as the unit-cell elements, different structures controlling phases at two frequencies independently. A linearly polarized horn antenna is used to feed to a reflectarray consists of 169 elements, a PIN diode is integrated in each element, beam scanning could be accomplished by 1-bit quantization method. To avoid blockage from the feed, scanning plane is orthogonal to the horn antenna. The measured results show that the gain of the reflectarray at 9 GHz and 11.5 GHz is 17.57dBi and 18.31dBi, 1-dB gain bandwidth is 26% and 30%, the scanning range below 3-dB gain variation is -40° to 40° and -50° to 50°.

Cancer is one of the leading causes of death worldwide, with recent research suggesting that a high degree of cancer cellular heterogeneity leads to different responses to therapies, making cancer a challenging disease to cure. As such, characterizing tumor cells at single-cell resolution promises greater insight into the mechanisms of cancer progression and facilitates the development of cancer treatments targeting different cell types. In recent years, microfluidics has emerged as a promising platform technology for single cell analysis. However, most existing single-cell analysis platforms cannot be applied to study certain rare, yet important, cancer cell populations. Cancer stem-like cells (CSCs), for instance, represent a small subpopulation (1-5%) of the tumor cells, but they are tumorigenic and cause tumor relapse and metastasis, which is the cause of over 90% cancer related death. In addition, samples harvested from microfluidic assays represent another critical rare-cell population for study. As microfluidics has become ubiquitous in labs for cellular assays, there is an emerging need to interface other microfluidics to further investigate the cells of interest. Finally, liquid biopsy of circulating tumor cells (CTCs) analysis has a high potential for cancer diagnostics and precision medicine. However, CTCs are very rare in blood with the concentration of only 1-20 cells/mL, which is a great challenge for cell capture and the downstream analysis. To achieve single-cell analysis of these rare cell populations, this thesis presents high-efficiency microfluidic technologies for single cell phenotypic and transcriptomic studies. First, to study the CSCs, we achieved the scaling and automation of high-throughput single-cell-derived tumor sphere assays, which are a strong indicator of disease outcome (e.g. tumor relapse and metastasis). With a highly parallel chamber capture structure, the array size of the chip can be scaled from 800 to 12,800 while maintaining a high single cell capture rate of ~76.5% to study the heterogeneity of CSCs. The assay elucidated a controversial hypothesis of the linkage between cell size and tumor-initiating potential. The cell capture scheme was also applied to cell-to-cell interaction and cell differentiation studies, highlighting its versatility in single cell analysis. Second, to interface the sample from other microfluidics, a proteolytic chip with a vacuum-driven single-cell capture scheme was developed to enable handling of small number of cells down to ~50 cells for a given sample volume of 4 μl. By a vacuum driven cell loading process, the entire input solution can be loaded into each cell capture chamber to minimize cell loss in the dead volume. After loading protease sensitive reagents through diffusion and performing air isolation between capture chambers, the proteolytic activity, an important process in metastasis, of each cell can be monitored at single cell resolution. Finally, Hydro-Seq, a scalable hydrodynamic bead-cell-pairing technique, was developed to analyze CTCs with high cell capture efficiency, high-throughput, and contamination removal capability. We successfully achieved whole transcriptome sequencing of 666 CTCs from 21 breast cancer patient samples, identifying critical cancer metastasis subpopulations of mesenchymal–epithelial transition (MET) and epithelial–mesenchymal transition (EMT) with a fraction of cells expressing epithelial and mesenchymal CSC markers such as CD44+/CD24- and ALDH. The presented technology offers the capability to analyze the phenotype and transcriptome of rare cancer cells, ultimately providing better diagnostics and treatment of cancer in the future.

Membrane filtration is widely applied to the treatment of sewage, wastewater from the biomedical industry, and flowback water from hydraulic fracturing, but fouling remains a key challenge in these applications. Membrane fouling increases mass transfer resistance and energy consumption. Severely fouled membranes require expensive cleaning or replacement, which increases operating costs and reduces filtration efficiency. Modelling membrane fouling can advance our understanding of filtration processes and improve our ability to predict the onset and severity of fouling. This study focuses on developing fouling models in constant flux crossflow operations, commonly used in industry, and on developing fouling-resistant coatings to mitigate membrane fouling in industrial filtration applications. Two fouling mechanisms from the accepted Hermia’s model, intermediate pore blocking and cake filtration, were modified and combined together to describe fouling in constant flux crossflow ultrafiltration (UF). The model gave a qualitatively good fit to experimental fouling results using rigid latex bead particles and deformable oil droplets. Observations of the model’s accuracy at different fluxes shed light on the physical meaning of the threshold flux: the flux below which cake buildup is negligible and above which cake filtration becomes the dominant fouling mechanism. Although the model that combines intermediate pore blocking (IPB) and cake filtration can qualitatively described fouling by latex beads and emulsified oil, the IPB model fails at high foulant concentrations or high permeate flux. To resolve this issue, the IPB model has been replaced with a complete coverage model (CCM). CCM was combined with the cake filtration model, and then compared to the previous combined IPB/cake filtration model. Constant flux crossflow fouling experiments were conducted using dilute latex bead suspensions and commercial poly(ether sulfone) flat sheet ultrafiltration membranes to investigate the influence of operating conditions (foulant concentration, permeate flux, etc.) on the evolution of transmembrane pressure profile. The CCM/cake filtration combined model provides better agreement with experimental data than does the IPB/cake filtration combined model. To mitigate fouling in oil-water separations, a new approach to preparing hydrophilic membrane coatings based on 1,4-benzoquinone and various commercially available polyetheramines was developed. These coatings, prepared specifically from 1,4-benzoquinone and Jeffamine® EDR 148, poly(benzoquinone-Jeffamine® EDR 148) (p(BQ-EDR 148)), were used to modify polysulfone (PS) ultrafiltration membranes. In fouling experiments using an oil/water emulsion, membranes exhibited comparable fouling resistance to that of a polydopamine (pDA) modified membrane, a commonly used surface-modified membrane. Based on contact angle measurements, p(BQ-EDR 148) and pDA modified membranes have similar levels of hydrophilicity, and both exhibited higher threshold flux values than did their unmodified analogs. Based on their similar threshold flux values, p(BQ-EDR 148) (76 LMH) and pDA modified membranes (74 LMH) should have similar fouling resistance. Moreover, the mean pore size of p(BQ-EDR 148) modified membranes can be tuned, while keeping the pure water permeance constant, by changing the deposition time and molar ratio of benzoquinone to EDR 148 in the modification solution.

Our study aims to determine the effects of race, insurance, and hospital characteristics on the management of threatened abortion and early pregnancy loss. In this retrospective cohort study using the National Hospital Ambulatory Medical Care Survey, patient record files from 2002-2010 with diagnoses of threatened abortion, hemorrhage in pregnancy, or incomplete, inevitable, or unspecified spontaneous abortion were examined using logistic regression. Primary outcomes were rates of admission and active management, defined as surgical termination or use of abortifacients misoprostol or Cytotec. Covariates included race/ethnicity, age, insurance, and hospital location, ownership, and metropolitan status. Of 5,882,623 ED visits for threatened abortion and early pregnancy loss, 15% were admitted and 1.3% were actively managed. Compared to white women, black women were 0.83 times as likely to be admitted (95% CI 0.83–0.84), but 4.37 times as likely be actively managed (95% CI 4.25–4.50). Admission was more likely for “Other” women (Asian, Native Hawaiian, Native Alaskan, Native American, mixed race; OR 2.14, 95% CI 2.11–2.17), Medicaid/SCHIP (OR 1.24, 95% CI 1.22–1.25) and Self-pay (OR 1.04, 95% 1.03–1.05) compared to reference groups of white and privately insured women. Historically-marginalized groups, including uninsured, black, and “Other” women, were more likely to be actively managed. Exceptions were Latina (OR 0.84, 95% CI 0.80–0.89) and Medicaid/SCHIP-insured women (OR 0.13, 95% CI 0.12–0.15). Nonwhite women were less likely to be treated for pain, especially Latinas (OR 0.29, 95% CI 0.28–0.29). The etiology of these disparities is complex, but providers may seek to better understand their own preconceptions of patient risk, and to strengthen social support, communication, and shared decision-making.