Hongshen Ma

Associate Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Mar 2019)
Deformability-based separation and single-cell sequencing of circulating tumor cells in prostate cancer (2018)

Circulating tumor cells (CTCs) are malignant cells shed from a primary tumor into the bloodstream, where they have the potential to seed metastases responsible for >90% of all cancer related deaths. CTCs are particularly interesting for prostate cancer because metastases occur predominantly bone tissue, which makes biopsies difficult and low yielding. Since CTCs are accessible from peripheral blood, these cells represent a potential source of highly relevant tumor materials, which could be used to reveal new biomarkers for monitoring disease progression and evaluating drug efficacy. A key challenge in CTC isolation and characterization has been their extreme rarity in blood and their cell-to-cell heterogeneity. Both of these issues suggest the need to develop robust methods to isolate and analyze individual CTCs. This dissertation presents a new workflow to isolate, extract, and sequence single CTCs from patients with metastatic prostate cancer. Initially, we investigated the morphology of CTCs from patients with prostate cancer, and observed that CTCs and leukocytes were similar in size, but distinct in nucleus-to-cytoplasm ratio, which suggests the potential to separate CTCs based on deformability. Based on this result, a microfluidic device that separates CTCs based on cell deformability, as well as an accompanying analytical pipeline to identify CTCs using immunofluorescence, were developed, optimized, and tested. This workflow was used to successfully enumerate CTCs from 20 patients with metastatic castrate resistant prostate cancer, as well as 25 patients with localized prostate cancer. For the former cohort, we compared our process against existing technology and demonstrated 25× greater yield. We then developed a process to isolate single CTCs using laser capture microdissection for genome sequencing. Using this process, we enriched and isolated 30 single CTCs from 3 patients with metastatic prostate cancer, and sequenced 5 of these single CTCs from a patient with matched cell-free DNA. The sequencing data confirmed the presence of major driver mutations, including PTEN and TP53, as well as heterogeneous characteristics of individual CTCs. These results demonstrate the potential of our single cell sequencing workflow to discover clinically relevant mutations from single CTCs that may aid in monitoring disease progression and guiding treatment.

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Deformability based sorting of red blood cells and white blood cells using microfluidic ratchets (2017)

There are many situations in medicine and biology where it is desirable to sort cells in a heterogeneous sample based on their mechanical deformability, which can potentially serve as a proxy for morphology or pathology. This biophysical characteristic is particularly relevant for cells in the circulatory system, such as red blood cells and white blood cells, because deformability determines the capacity for these cells to transit through the microvasculature. Since deformability is such a fundamental characteristics of blood cells, deviations in normal cell deformability can contribute to a range of pathological conditions, such as microvascular occlusion, tissue necrosis and organ failure, observed in diseases such as malaria caused by Plasmodium falciparum. A commonly employed approach for deformability-based cell sorting is microfiltration. However, this method suffers from cell clogging at the filter microstructures, leading to reduced selectivity and device malfunction. This dissertation presents an improved microfiltration strategy performed using the microfluidic ratchet mechanism, which relies on the deformation of individual cells through micrometer-scale tapered constrictions. Deforming single cells through such constrictions requires directionally asymmetrical forces, which enables oscillatory flow to create a ratcheting transport that depends on cell size and deformability. Simultaneously, oscillatory flow continuously agitates the cells to limit the contact time with the filter microstructure to prevent clogging and adsorption. This work demonstrates the utility of the ratchet mechanism for cell sorting by developing a microfluidic device to sort red blood cells based on deformability. The device is used to separate Plasmodium falciparum infected red blood cells from uninfected cells. The method was shown to dramatically improve the sensitivity of malaria diagnosis performed using both microscopy and rapid diagnostic tests by converting samples with difficult-to-detect parasitemia (0.1%). This work further demonstrates the utility of the microfluidic ratchet mechanism by developing a microfluidic device to isolate and sort leukocytes directly from whole blood. The method is capable of separating leukocytes from whole blood with 100% purity (i.e. no contaminant erythrocytes) and
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Master's Student Supervision (2010-2017)
Development of an epoxy-based microfluidic device for automated circulating tumour cell separation (2017)

Circulating tumour cells (CTCs) are cancer cells shed from a primary tumour site into the bloodstream, where they have the potential to invade other tissues in the body, and thus become the seed of metastases. CTCs have great potential to monitor disease progression and guide cancer treatment, but a key technical challenge for their isolation and characterization is their extreme rarity in blood. CTCs are commonly enriched using immunoaffinity, which while being highly selective, may fail to capture cells that have weak antigen expression. The biophysical properties of CTCs offer a compelling alternative to immunoenrichment. CTCs are much larger in size than erythrocytes, but are similar to leukocytes. Owing to their epithelial origin however, CTCs are likely to be more rigid than leukocytes which allows for deformability based methods to separate these cells. Previously, our group has demonstrated the continuous flow microfluidic ratchet device for deformability based separation of CTCs. Here, an improved version of the device has been developed to be compatible with pre-enrichment methods, allowing for a dramatic increase in throughput. While similar in principle to the previous version, this work specifically improves the design of the sample infusion area to increase the points of contact between the sorting matrix and sample inlet, in order to prevent the accumulation of cell debris. Using this new design, epoxy resin devices and supporting instrumentation were developed to provide a pathway towards scale-up production and automation. These combined improvements allow biology laboratory technicians to enrich CTCs without significant training. The improved device is capable of capturing > 80% CTCs from whole blood at a throughput of 1 mL/hr, which when combined with a red blood cell lysis pre-enrichment step, increases to 8 mL/hr. Finally, devices were used to enrich CTCs from patients with metastatic castration-resistant prostate cancer. CTCs were found in 3 out of 11 patients, with an average count of 78. The enriched cells were further processed to perform single cell genomic sequencing where CTCs were found to contain driver mutations including those commonly associated with prostate cancer.

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Spectral image cytometry for circulating tumor cell identification (2017)

Circulating tumor cells (CTCs) are exceedingly rare cancer cells shed from tumors into the bloodstream, where they have the potential to invade other tissues to seed metastases. CTCs are difficult to isolate but their critical role in tumor metastasis, as well as their proven prognostic value has attracted tremendous interest in recent years. While many methods have been developed to isolate CTCs, a major bottleneck to their clinical application has been the precise identification and characterization of these cells, owing to their tremendous phenotypic heterogeneity. To address these formidable challenges, a number of microscopy techniques have been applied to gather large amounts of information about captured cells. However, these studies are currently limited by two major concerns: First, due to the phenotypic plasticity of tumor cells, there may be significant variability in the properties of CTCs as observed using microscopy. Second, if the CTCs are subjected to multi-parameter analysis, the high-content data may be too expansive to analyze with a reasonable amount of time and effort. In this thesis, I developed an efficient and customizable spectral image cytometry platform to collect multi-spectral data from immunofluorescence micrographs of cell samples enriched for CTCs in order to quickly and easily analyze this information to facilitate CTC identification and characterization. This work includes the development of software tools to convert microscopy data for processing, to segment the images into single cell images, to rank potential CTCs, and to provide a user interface for rapid augmented review. The performance of this software platform has been evaluated by analyzing multi-spectral fluorescence imaging data previously collected by our group from ten patients with castrate resistant prostate cancer, and then comparing the result to unassisted manual reviews performed by blinded reviewers. The final CTC identification counts closely matched manual analysis with a slight increase in verified CTC counts, which is likely a result of the comprehensive nature of the automated screening process. The average computation time is 4.5 minutes per sample, which is faster than the time required to acquire the imaging data, and thus allows operators to quickly review results between acquisitions.

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Red blood cell trans-dispersion revealing biophysical signatures in malaria parasitism (2015)

Red blood cell (RBC) deformability plays an important role in the pathogenesis of Plasmodium falciparum malaria, and therefore could potentially enable simple, rapid, and reagent-free biophysical assays. A key challenge, however, is that pathological cells often only represent a small fraction of the sample, which requires testing a large number of individual cells to enable their detection. Additionally, it is often desirable to perform multiple assays simultaneously, which require technologies capable of parallelized analysis. Traditional technologies for analyzing RBC deformability are limited by their experimental difficulty, extensive instrumentation requirements, as well as their lack of throughput and parallelizability. Here, a new microfluidic mechanism called trans-dispersion is developed to address these issues, enabling a high-throughput and parallelized analysis of RBC deformability. The trans-dispersion mechanism transports single RBCs through a series of constrictions in a microfluidic channel, where their transit speed is a function of their deformability. This process is analogous to gel-electrophoresis, where the migration speed of molecules depends on their length. To ensure a sensitive and consistent measurement, the geometry of the constriction is sized such that the transiting cell forms a temporary seal with each constriction while supporting microchannels ensure consistent forces are applied to each deformation channel. After undergoing repeated deformations, the final position of each RBC, indicating its deformability, is determined using simple bright-field microscopy and automated image processing, and thereby resulting in a repeatable, high-throughput and parallelized process. The performance of this mechanism was evaluated by detecting changes in RBC deformability resulting from chemical degradation, malaria parasitism and exposure to anti-malarial drugs. This device can distinguish variation in RBC deformability following chemical degradation using small concentrations (0.0005%) of glutaraldehyde (GTA). P. falciparum-infected RBCs (iRBCs) show distinct deformability curves compared to the uninfected controls. The linear correlation between the parasitemia and the percentage of non-transiting cells could potentially be used to infer the parasitemia of clinical specimen. Furthermore, this device was able to simultaneously assess the efficacy of several antimalarial compounds; showing that rigidification of P. falciparum-iRBCs can potentially be used to evaluate antimalarial drug efficacy, as well as serve as a functional screen for new antimalarials.

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Separation of circulating tumor cells using resettable cell traps (2015)

Immunoenrichment of conventional circulating tumor cells (CTCs) may fail to capture cells with poor antigen expression. Micropore filtration is a compelling label-free alternative to separate CTCs based on their biophysical characteristics rather than biochemical characteristics. However, this strategy is prone to clogging of the filter microstructure, which dramatically reduces selectivity after processing large numbers of cells. Our group previously reported the resettable cell trap (RCT) mechanism to perform micropore filtration in a way that is resistant to clogging. We improved the selectivity of this label-free mechanism by filtering the samples multiple times on chip and dramatically improving the throughput by parallelization. The resettable cell trap device is a microfluidic mechanical constriction with adjustable apertures that can capture CTCs based on their distinct size and deformability. It can also be periodically cleared to release the trapped cells to prevent clogging. Three identical cell traps are aligned in series which improves selectivity by removing leukocytes that non-specifically adhere to the surface of microchannels.We validated this mechanism by doping UM-UC13 bladder cancer cells into diluted whole blood at a density of 1 UC13 to 1000 leukocytes. The first filtration step achieved 183-fold enrichment and 93.8% yield. The second and third traps together provided an additional enrichment of ~5 without significant change in yield. Furthermore, additional filtration steps provide even greater enrichment. In patients with metastatic castration-resistant prostate cancer (mCRPC, n=24) and localized prostate cancer (LPC, n=18), CTCs were successfully identified using the resettable cell trap device followed by single-cell spectral analysis. We additionally compared the RCT device to the CellSearch® System, the only FDA approved commercial CTC enumeration platform. The microfluidic RCT device identified 83.3% (20/24) patients with >=5 CTCs per 7.5 ml of blood with a mean of 329 counts. Within the same patient group, the CellSearch only measured >=5 CTCs in 37.5% (9/24) patients with a mean of 23 CTCs per 7.5 ml of blood. The RCT device identified significant more CTCs and positively identified more mCRPC patients than the CellSearch system.

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Microfluidic device for continuous deformability based separation of circulating tumor cells (2014)

Circulating tumor cells have been implicated as potential seeds of cancer metastasis and have strong prognostic and diagnostic value in cancer therapy. The primary challenge in CTC characterization is their extreme rarity in circulation relative to leukocytes. Conventional strategies employ CTC immunoenrichment that is highly selective but may fail to enrich for CTCs with poor antigen expression. However, CTCs exhibit unique morphological characteristics that distinguish them from leukocytes and deformability-based sorting mechanisms represent a compelling label-free CTC enrichment strategy. Our group previously reported the microfluidic ratchet mechanism capable of highly selective deformability based cell separation without clogging. Here, we developed a continuous version of this process that obviates the need for microvalves and operate with dramatically increased throughput. Implementation of the microfluidic ratchet consists of a matrix of funnel constrictions with microchannels for flow control. The openings of the funnel constrictions are gradually reduced from the bottom row to the top row. Cells enter at the bottom-left of the funnel matrix and are driven by a rightward flow simultaneously as a vertical oscillatory flow. Each cell traverses through the funnel matrix in a step-wise diagonal path until reaching a limiting funnel size. CTCs are the least deformable cells and reach their limiting funnel size relatively quickly. Leukocytes are more deformable and travel to a smaller funnel region. Finally, erythrocytes are extremely deformable and exit through the top row. We evaluated the selectivity of this mechanism using UM-UC13 bladder cancer cells doped into whole blood from healthy donors. UM-UC13 cells were enriched by ~10⁴ relative to leukocytes, with ~90% capture efficiency, and thus demonstrate significantly greater selectivity than separation based solely on size. We used the microfluidic ratchet device to enumerate CTCs from 58 samples with 52 patients with castrate resistant prostate cancer, in parallel with CellSearch, and 6 healthy control samples. The CTC capture rate is significantly higher for our device, which detected ≥5 CTCs in 67.3% of patients with an average count of 256, while the CellSearch system detected ≥5 CTCs in 40.4% of patients with an average count of 74.

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Multiplexed fluidic plunger mechanism for the measurement of red blood cell deformability (2014)

Red blood cell (RBC) deformability plays an important role in the pathology of various diseases, including malaria, hemoglobinopathies, and micronutrient deficiencies. Specifically, in malaria, the analysis of RBC deformability presents new approaches for detecting infections and for rapidly evaluating the response to drugs by patients. A key challenge, however, is that the infected RBCs represent only a small subpopulation of clinical blood specimens. Therefore effective detection of infection and analysis require methods that can measure a large number of individual RBCs. Traditional technologies for measuring RBC deformability either cannot evaluate single cells to identify diseased subpopulations or do not have sufficient measurement throughput to detect rare subpopulations. In additions, they require delicate experiments, expensive equipment, and skilled technicians. To address these issues, we developed a new microfluidic mechanism, known as the Multiplexed Fluidic Plunger (MFP), to measure RBC deformability using many microscale-tapered constrictions in parallel. The deformability of each RBC is determined by the threshold pressure required to squeeze the cell through a constriction. Our mechanism overcomes a key challenge where the pressure applied to each cell is dependent on the presence or absence of other cells and thereby produces in an inconsistent measurement result. We devised a mechanism to avoid this error and showed that a consistent measurement is obtained independent of constrictions occupancy. Furthermore, the sensitivity of the MFP device is comparable or superior to existing techniques since it can distinguish control and 0.0005% glutaraldehyde-treated RBCs (p
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Automated microfluidic device for the separation of cancer cells from blood (2013)

The ability to separate cells based on biomechanical properties such as size and deformability is emerging as a potential alternative to biochemical methods for cell separation, particularly in cases where biochemical markers are unknown or expressed at low levels. The separation of circulating tumor cells (CTCs) is an example problem where this type of technology is important because the cell surface markers currently used to capture these cells are known to be unreliable. The performance of existing biomechanical cell separation techniques is currently hindered by clogging, which reduces specificity of the separation process. We previously demonstrated a microfluidic ratchet mechanism that overcomes the reversible nature of low Reynolds number flow. In this thesis, we leverage this mechanism to prevent clogging while preserving high selectivity by periodically clearing the filter microstructure to create an automated microfluidic platform that demonstrates the size and deformability-based separation of cultured human bladder UC13 cancer cells from white blood cells (WBCs). This platform has two components: the first is a size-based hydrodynamic concentrator, which performs an initial sample preparation step to reduce the sample volume while removing a fraction of the contaminant WBCs. The second is an automated cell separation device where cells are transported through a 2D array of ratcheting funnel constrictions and sorted using an oscillatory flow. We evaluate the ability of this platform for separating rare cancer cells doped into WBCs at low concentration to assess the potential of this technology for biomechanical separation of CTCs. Specifically, using a sample where cancer cells are doped into WBCs at a ratio of 1:1000, the combined system achieved a cancer cell yield of 96.0±0.1%; the outlet had a purity of 75±3%; and the population of cancer cells in the mixture was enriched by a factor 3000 (+643, -278).

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Rare cell separation using resettable cell traps (2013)

Techniques for the separation of cells from heterogeneous samples that do not rely onbiological labels are important in applications where specific labels are unknown orunavailable. However, limitations of existing label-free separation techniques haveprevented their widespread adoption. Those techniques that separate based on cell sizetypically offer high throughput but lack specificity. Those that separate based on acombination of cell size and deformability have superior selectivity, but are slow and proneto clogging.This work reports a microfluidic device that employs novel resettable cell traps to separatecells based on size and deformability. The resettable cell trap is a microchannel withcontrollable cross-section, featuring recesses to temporarily store captured cells. Larger andless deformable cells flowing through a cell trap with constricted cross-section will beselectively captured due to size restriction, and can be released back into the flow forcollection by enlarging the channel cross-section. Smaller and more deformable cells willsimply pass through the constricted channel. The ability to enlarge the trap and purge it ofcaptured cells enables long term operation without clogging. The cell separation devicepresented is able to separate UM-UC13 cancer cells from human leukocytes with highenrichment (~100x), retention (~90%) and throughput (450,000 cells/hour). Serial separationusing this mechanism provides extremely high enrichment (~2500x) without sacrificingretention. The mechanism is also shown to resolve size differences of 1 µm betweenpolystyrene microspheres. The resettable cell trap is an improvement upon existingtechnology, providing greater enrichment than possible through size-based techniques whileimproving throughput and eliminating problems caused by clogging that are typical offiltration based techniques.

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Microfluidic device for measuring the deformability of single cells (2012)

The deformability of single cells can be used as a biomarker to evaluate the status of many diseases including cancer, malaria, and arthritis. Traditional techniques for measuring single cell deformability, such as micropipette aspiration, optical tweezers, and atomic force microscopy, involve delicate experiments performed by highly-skilled technicians using specialized equipment. This thesis presents a new mechanism for measuring the deformability of single cell using the pressure required to deform single cells through a micro-scale constriction. This technique is in principle similar to the micropipette aspiration, but involves considerably simpler operation, is less prone to errors, and requires less specialized equipment and technical skill. The ability of this mechanism to measure single cell deformability is initially verified by testing neutrophils, which demonstrated similar results and measurement precision as micropipette aspiration. Subsequently, this device was used to study the deformability of human red blood cells and specifically, the decrease in deformability of red blood cells parasitized by Plasmodium falciparum, the most common species of the parasite that causes malaria. Finally, this device was used to measure the directional asymmetry associated with the deformation of single cells along the direction of the funnel taper and against the direction of the funnel taper. This asymmetry was used to create a microfluidic ratchet to enable unidirectional transport of cells from a fluctuating fluid flow.

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Physical cell separation using microfluidic funnel ratchets (2012)

The separation of biological cells using non-chemical methods is important to many areas of medicine and biology. Filtration through microstructured constrictions is one such method where cells can be separated by a combination of size and deformability. This technique, however, is limited by unpredictable variations of the filter hydrodynamic resistance as cells accumulate in the microstructure. Applying a reverse flow to unclog the filter will undo the separation and reduce filter selectivity because of the reversibility of low-Reynolds number flow. This work introduces a microfluidic structural ratchet mechanism to separate cells using oscillatory flow through a 2-dimensional array of funnel-shaped structures. Devices are fabricated using multi-layer soft lithography of polydimethylsiloxane (PDMS) and flow is controlled using pressure sources and on-chip membrane valves. An iterative procedure of design and testing is used to produce a final device which is characterized by the sorting and separation of L1210 mouse lymphoma cells (MLCs), peripheral blood mononuclear cells (PBMCs) from healthy donors, as well as polystyrene microparticles. The ability of this mechanism to sort and separate cells/particles based on size and deformability is investigated and confirmed. Additionally, the spatial distribution of cells after sorting is demonstrated to be repeatable and the separation process is shown to be irreversible. This mechanism can be applied generally to separate cells that differ by size and/or deformability.

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