Siddarth Rawal

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Fourier ptychographic microscopy for filtration-based circulating tumor cell enumeration and analysis

Abstract. Circulating tumor cells (CTCs) are recognized as a candidate biomarker with strong prognostic and predictive potential in metastatic disease. Filtration-based enrichment technologies have been used for CTC characterization, and our group has previously developed a membrane microfilter device that demonstrates efficacy in model systems and clinical blood samples. However, uneven filtration surfaces make the use of standard microscopic techniques a difficult task, limiting the performance of automated imaging using commercially available technologies. Here, we report the use of Fourier ptychographic microscopy (FPM) to tackle this challenge. Employing this method, we were able to obtain high-resolution color images, including amplitude and phase, of the microfilter samples over large areas. FPM’s ability to perform digital refocusing on complex images is particularly useful in this setting as, in contrast to other imaging platforms, we can focus samples on multiple focal planes within the same frame despite surface unevenness. In model systems, FPM demonstrates high image quality, efficiency, and consistency in detection of tumor cells when comparing corresponding microfilter samples to standard microscopy with high correlation (R2=0.99932). Based on these results, we believe that FPM will have important implications for improved, high throughput, filtration-based CTC analysis, and, more generally, image analysis of uneven surfaces.

Identification of cancer associated fibroblasts in circulating blood from patients with metastatic breast cancer

Metastasis is facilitated by cancer-associated fibroblasts (CAF) in the tumor microenvironment through mechanisms yet to be elucidated. In this study, we used a size-based microfilter technology developed by our group to examine whether circulating CAF identified by FAP and α-SMA co-expression (cCAF) could be distinguished in the peripheral blood of patients with metastatic breast cancer. In a pilot study of patients with breast cancer, we detected the presence of cCAFs in 30/34 (88%) patients with metastatic disease (MET group) and in 3/13 (23%) patients with localized breast cancer (LOC group) with long-term disease-free survival. No cCAFs as defined were detected in healthy donors. Further, both cCAF and circulating tumor cells (CTC) were significantly greater in the MET group compared with the LOC group. Thus, the presence of cCAF was associated with clinical metastasis, suggesting that cCAF may complement CTC as a clinically relevant biomarker in metastatic breast cancer.

Experimental study of PDMS bonding to various substrates for monolithic microfluidic applications

This paper presents a comprehensive experimental study and characterization of material and bonding of PDMS based structures to various substrates. A previously published method [1] of bonding is further improved with the inclusion of more substrate material and additional characteristics. Uncured PDMS is used as an adhesive to bond PDMS devices reversibly to various substrates including a number of commonly used substrate materials that are not supported by the widely used plasma treatment method. We have optimized parameters such as PDMS base to curing agent ratio, curing temperature, and PDMS device age to obtain better bond strengths and quality. Bond strengths are presented for semiconductor substrates (silicon, zinc oxide, and silicon dioxide), metals (gold, aluminum), photoresists (SU-8, AZxx) and glass. Silicon based substrates experienced minor amounts of surface residue, but the method is fully reversible for other tested substrates. Bond strengths were measured as maximum endurable pressure between PDMS and substrates. Maximum average bond strengths of more than 0.4 MPa were achieved for substrates with Si-O groups. Other substrates exhibited maximum average bond strengths in the range 0.2–0.3 MPa. Also presented is a method that avoids alignment step for PDMS microfluidic device bonding, named the non-aligned method. This method provides bond strengths of more than 0.1 MPa. Presented methods do not need special equipment or processes such as plasma generators or temperature increases. Biocompatibility tests are performed for materials used in fabrications to ensure applicability in bio-sensing related devices.

Molecular Histopathology Using Gold Nanorods and Optical Coherence Tomography

PURPOSE To examine the novel application of a commercially available optical coherence tomography (OCT) system toward molecular histopathology using gold nanorod (GNR) linked antibodies as a functionalized contrast agent to evaluate ocular surface squamous neoplasia (OSSN). METHODS GNRs were synthesized and covalently attached to anti-glucose transporter-1 (GLUT-1) antibodies via carbodiimide chemistry. Three specimens from each of three distinct categories of human conjunctival tissue were selected for analysis, including conjunctiva without epithelial atypia (controls); conjunctival intraepithelial neoplasia, carcinoma in situ (CIS); and conjunctival squamous cell carcinoma (SCC). Tissue sections were incubated initially with GNR tagged anti-GLUT-1 antibodies and then with a fluorescent-tagged secondary antibody. Immunofluorescence and OCT imaging of the tissue was performed and the results were correlated to the light microscopic findings on traditional hemotoxyin and eosin stained sections. RESULTS No binding of the functionalized GNRs was observed within the epithelium of three normal conjunctiva controls. While immunofluorescence disclosed variable binding of the functionalized GNRs to atypical epithelial cells in all six cases of OSSN, the enhancement of the OCT signal in three cases of CIS was insufficient to distinguish these specimens from normal controls. In two of three cases of SCC, binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to atypical epithelial cells which stained intensely on immunofluorescence imaging. Binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to regions of erythrocytes and hemorrhage which stained intensely on immunofluorescence imaging within all nine tested samples. CONCLUSIONS We have demonstrated the use of OCT for molecular histopathology using functionalized gold nanorods in the setting of OSSN. Our results suggest a threshold concentration of functionalized GNRs within tissue is required to achieve a detectable enhancement in scattering of the OCT signal.

A surface acoustic wave biosensor for interrogation of single tumour cells in microcavities

In this study, biological cells are sensed and characterized with surface acoustic wave (SAW) devices utilising microcavities. After tumour cells in media are transported to and trapped in microcavities, the proposed platform uses SAW interaction between the substrate and the cells to extract their mechanical stiffness based on the ultrasound velocity. Finite element method (FEM) analysis and experimental results show that output phase information is an indicator of the stiffness modulus of the trapped cells. Small populations of various types of cells such as MCF7, MDA-MB-231, SKBR3, and JJ012 were characterized and characteristic moduli were estimated for each cell population. Results show that high frequency stiffness modulus is a possible biomarker for aggressiveness of the tumour and that microcavity coupled SAW devices are a good candidate for non-invasive interrogation of single cells.

Thermoresponsive release of viable microfiltrated Circulating Tumor Cells (CTCs) for precision medicine applications

Stimulus responsive release of Circulating Tumor Cells (CTCs), with high recovery rates from their capture platform, is highly desirable for off-chip analyses. Here, we present a temperature responsive polymer coating method to achieve both release as well as culture of viable CTCs captured from patient blood samples.

Identification and Quantitation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are shed from the primary tumor into the circulatory system and act as seeds that initiate cancer metastasis to distant sites. CTC enumeration has been shown to have a significant prognostic value as a surrogate marker in various cancers. The widespread clinical utility of CTC tests, however, is still limited due to the inherent rarity and heterogeneity of CTCs, which necessitate robust techniques for their efficient enrichment and detection. Significant recent advances have resulted in technologies with the ability to improve yield and purity of CTC enrichment as well as detection sensitivity. Current efforts are largely focused on the translation and standardization of assays to fully realize the clinical utility of CTCs. In this review, we aim to provide a comprehensive overview of CTC enrichment and detection techniques with an emphasis on novel approaches for rapid quantification of CTCs.

Clinical translation of a novel microfilter technology Capture, characterization and culture of circulating tumor cells

The most important determinant of prognosis and management of cancer is the presence or absence of metastasis [1]. The road to metastasis involves tumor cells to become detached from the primary tumor and travel in the blood to distant sites, causing secondary tumors. These tumor cells traveling in blood are termed Circulating tumor cells (CTC). Capture of CTC from whole blood has been a challenging feat. The fact that these CTC are few in number, to effectively and efficiently isolate them from whole blood can be thought of as looking for a needle in a haystack. Our microfilter technology exploits the use of size based capture of the larger CTC from the smaller white blood cells and components of whole blood. The effective capture potential of the microfilter platform has driven the area of CTC analysis into a new age of research in the field of cancer. The ability to finally analyze CTC at a molecular level, leads to a deeper understanding of metastatic process, while providing an opportunity to evaluate, monitor and manage treatment options as well as the adherent possibility of having an “on-chip” drug sensitivity assay for focused treatment options. We have demonstrated through clinical trials the ability to effectively identify, enumerate and characterize CTC based on immunfluorescence and FISH assays and provide a companion endpoint for monitoring and evaluating treatment management. Our work on viable CTC capture has resulted in successfully capturing and culturing CTC from blood in mouse models that have been inoculated with breast cancer cell lines to form primary and secondary metastatic cancer sites. The future potential within the microfilter technology to capture viable CTC for culture, will catapult therapeutic interventions to a new level of personalized medicine in cancer management.

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 °C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 °C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.