Isolation and Analysis of Circulating Tumor Cells in Genitourinary Cancers

While accessible by a relatively noninvasive blood draw, circulating tumor cells (CTCs) remain difficult to study because of their rarity and their presence amongst the billions of surrounding normal blood cells. Of particular promise and utility to the in-depth study of CTCs are those technologies making use of microfluidics and nanomaterials, such as the graphene oxide (GO) Chip. The GO Chip has been applied to a 41-patient metastatic castrate resistant prostate cancer (mCRPC) cohort. CTCs were enumerated from whole blood for all patients (range: 3-166 CTCs/mL, median: 20 CTCs/mL). Clusters of CTCs, defined as two or more directly adjacent CTCs, were observed in 26/41 patients, and ranged in size from 2-8 CTCs/cluster. Within the CTC population, the percentage of CTCs present as clusters ranged from 0-54.8%. Additionally, a parallel device was run for 36 patients to ultimately obtain RNA to use in RT-qPCR to assess levels of 96 genes of interest. Enumeration and RNA expression data were compared with clinical outcomes including overall survival, radioclinical progression, and PSA progression. An eight-gene score was determined to be highly prognostic of overall survival (AUC: 0.88), with the genes comprising the score suggesting the importance of a dedifferentiated expression phenotype in poor prognosis. Follow-up work in prostate cancer investigated the role of HER2 and EGFR in prostate cancer metastasis. Analysis of tissue microarrays showed HER2 expression in prostate cancer and bone metastases. Primary and secondary prostate sphere formation was dependent on high EGFR expression as determined by FACS, but not on HER2 expression. EGFR was also implicated in survival in transit as shown by the presence of EGFR+ CTCs isolated by the GO Chip in 9/10 mCRPC patients assays, with an average of 35.5% of CTCs showing EGFR expression. Dual inhibition of HER2 and EGFR in mouse xenograft models prevented tumor growth. HER2 and EGFR as well as ADAM15 and CD31 were studied in bladder cancer CTCs as well. In a preliminary study primarily for optimization, antibodies were chosen for higher sensitivity capture as well as to stain bladder CTCs for the markers of interest. Ultimately, CTCs were isolated from five metastatic bladder cancer patients (range: 5-499 CTCs/mL), and a combination of staining antibodies that showed low background in the healthy control was chosen. EGFR+ and CD31+ CTCs were observed, while HER+ and ADAM15+ CTCs were not, and clusters of CTCs were isolated from some patients. To address drawbacks in the current technology, two strategies were attempted to enable cell release. A layer-by-layer (LbL) substrate enclosed in a microfluidic chamber featured different disadvantages based on film composition, but a thermosensitive polymer substrate enable release when cooled below its lower critical solution temperature of 12-13°C. The polymer-GO composite showed between 84.9 and 95.2% capture efficiency of EpCAM expressing cell lines and released over 91% of cells captured from whole blood. Using this device, CTCs were captured from 2/3 pancreatic cancer patients and 8/10 breast cancer patients. FISH for HER2 was performed on CTCs isolated from one breast cancer patient. With high performing technologies to separate them from the noise of other cells in the blood, CTCs can provide information about disease spread in genitourinary cancers. The future incorporation of CTC-related information into clinical decision making has the potential to better inform treatment selection and disease prognosis.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140934/1/mollykoz_1.pd

Detection of CTC Clusters and a Dedifferentiated RNAâ Expression Survival Signature in Prostate Cancer

Rates of progression and treatment response in advanced prostate cancer are highly variable, necessitating nonâ invasive methods to assess the molecular characteristics of these tumors in real time. The unique potential of circulating tumor cells (CTCs) to serve as a clinically useful liquid biomarker is due to their ability to inform via both enumeration and RNA expression. A microfluidic graphene oxideâ based device (GO Chip) is used to isolate CTCs and CTC clusters from the whole blood of 41 men with metastatic castrationâ resistant prostate cancer. Additionally, the expression of 96 genes of interest is determined by RTâ qPCR. Multivariate analyses are conducted to determine the genes most closely associated with overall survival, PSA progression, and radioclinical progression. A preliminary signature, comprising high expression of stemness genes and low expression of epithelial and mesenchymal genes, potentially implicates an undifferentiated CTC phenotype as a marker of poor prognosis in this setting.A microfluidic graphene oxideâ based device (GO Chip) is used to isolate circulating tumor cells (CTCs) and CTC clusters from the whole blood of 41 metastatic castrationâ resistant prostate cancer patients. A preliminary RNA signature, comprising high expression of stemness genes and low expression of epithelial and mesenchymal genes, potentially implicates an undifferentiated CTC phenotype as a marker of poor prognosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147791/1/advs887.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147791/2/advs887-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147791/3/advs887_am.pd

The promise of single-cell mechanophenotyping for clinical applications.

Cancer is the second leading cause of death worldwide. Despite the immense research focused in this area, one is still not able to predict disease trajectory. To overcome shortcomings in cancer disease study and monitoring, we describe an exciting research direction: cellular mechanophenotyping. Cancer cells must overcome many challenges involving external forces from neighboring cells, the extracellular matrix, and the vasculature to survive and thrive. Identifying and understanding their mechanical behavior in response to these forces would advance our understanding of cancer. Moreover, used alongside traditional methods of immunostaining and genetic analysis, mechanophenotyping could provide a comprehensive view of a heterogeneous tumor. In this perspective, we focus on new technologies that enable single-cell mechanophenotyping. Single-cell analysis is vitally important, as mechanical stimuli from the environment may obscure the inherent mechanical properties of a cell that can change over time. Moreover, bulk studies mask the heterogeneity in mechanical properties of single cells, especially those rare subpopulations that aggressively lead to cancer progression or therapeutic resistance. The technologies on which we focus include atomic force microscopy, suspended microchannel resonators, hydrodynamic and optical stretching, and mechano-node pore sensing. These technologies are poised to contribute to our understanding of disease progression as well as present clinical opportunities

DNA-Directed Patterning for Versatile Validation and Characterization of a Lipid-Based Nanoparticle Model of SARS-CoV-2

Lipid-based nanoparticles have risen to the forefront of the COVID-19 pandemic—from encapsulation of vaccine components to modeling the virus, itself. Their rapid development in the face of the volatile nature of the pandemic requires high-throughput, highly flexible methods for characterization. DNA-directed patterning is a versatile method to immobilize and segregate lipid-based nanoparticles for subsequent analysis. DNA-directed patterning selectively conjugates oligonucleotides onto a glass substrate and then hybridizes them to complementary oligonucleotides tagged to the liposomes, thereby patterning them with great control and precision. The power of this method is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of SARS-CoV-2 —S-liposomes— which present the SARS-CoV-2 spike (S) protein on their surfaces. Patterning of a mixture of S-liposomes and liposomes that display the tetraspanin CD63 into discrete regions of a substrate is used to show that ACE2 specifically binds to S-liposomes. Importantly, DNA-directed patterning of S-liposomes is used to verify the performance of a commercially available neutralizing antibody against the S protein. Ultimately, the introduction of S-liposomes to ACE2-expressing cells demonstrates the biological relevance of DNA-directed patterning. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle

Recapitulating complex biological signaling environments using a multiplexed, DNA-patterning approach.

Elucidating how the spatial organization of extrinsic signals modulates cell behavior and drives biological processes remains largely unexplored because of challenges in controlling spatial patterning of multiple microenvironmental cues in vitro. Here, we describe a high-throughput method that directs simultaneous assembly of multiple cell types and solid-phase ligands across length scales within minutes. Our method involves lithographically defining hierarchical patterns of unique DNA oligonucleotides to which complementary strands, attached to cells and ligands-of-interest, hybridize. Highlighting our method's power, we investigated how the spatial presentation of self-renewal ligand fibroblast growth factor-2 (FGF-2) and differentiation signal ephrin-B2 instruct single adult neural stem cell (NSC) fate. We found that NSCs have a strong spatial bias toward FGF-2 and identified an unexpected subpopulation exhibiting high neuronal differentiation despite spatially occupying patterned FGF-2 regions. Overall, our broadly applicable, DNA-directed approach enables mechanistic insight into how tissues encode regulatory information through the spatial presentation of heterogeneous signals

Tunable Thermal-Sensitive Polymer-Graphene Oxide Composite for Efficient Capture and Release of Viable Circulating Tumor Cells.

A highly sensitive microfluidic system to capture circulating tumor cells from whole blood of cancer patients is presented. The device incorporates graphene oxide into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. By decreasing the temperature, captured cells may be released for subsequent analysis

High Throughput Layer-by-Layer Films for Extracting Film Forming Parameters and Modulating Film Interactions with Cells

A high-throughput approach which automates the synthesis of polyelectrolyte-based layer-by-layer films (HT-LbL) to facilitate rapid film generation, systematic film characterization, and rational investigations into their interactions with cells is described. Key parameters, such as polyelectrolyte adsorption time and polyelectrolyte deposition pH, were used to modulate LbL film growth to create LbL films of distinct thicknesses using the widely utilized polyelectrolytes poly­(allylamine hydrochloride) (PAH) and poly­(acrylic acid) (PAA). We highlight how HT-LbL can be used to rapidly characterize film-forming parameters and robustly create linearly growing films of various molecular architectures. Film thickness and growth rates of HT-LbL films were shown to increase as a function of adsorption time. Subsequently, we investigated the role that polyelectrolyte solution pH (ranging from 2.5 to 9) has in forming molecularly distinct films of weak polyelectrolytes and report the effect this has on modulating cell attachment and spreading. Films synthesized at PAA-pH of 5.5 and PAH-pH 2.5–5.5 exhibited the highest cellular attachment. These results indicate that HT-LbL is a robust method that can shift the paradigm regarding the use of LbL in biomedical applications as it provides a rapid method to synthesize, characterize, and screen the interactions between molecularly distinct LbL films and cells

HER2 and EGFR Overexpression Support Metastatic Progression of Prostate Cancer to Bone.

Activation of the EGF receptors EGFR (ErbB1) and HER2 (ErbB2) drives the progression of multiple cancer types through complex mechanisms that are still not fully understood. In this study, we report that HER2 expression is elevated in bone metastases of prostate cancer independently of gene amplification. An examination of HER2 and NF-κB receptor (RANK) coexpression revealed increased levels of both proteins in aggressive prostate tumors and metastatic deposits. Inhibiting HER2 expression in bone tumor xenografts reduced proliferation and RANK expression while maintaining EGFR expression. In examining the role of EGFR in tumor-initiating cells (TIC), we found that EGFR expression was required for primary and secondary sphere formation of prostate cancer cells. EGFR expression was also observed in circulating tumor cells (CTC) during prostate cancer metastasis. Dual inhibition of HER2 and EGFR resulted in significant inhibition of tumor xenograft growth, further supporting the significance of these receptors in prostate cancer progression. Overall, our results indicate that EGFR promotes survival of prostate TIC and CTC that metastasize to bone, whereas HER2 supports the growth of prostate cancer cells once they are established at metastatic sites. Cancer Res; 77(1); 74-85. ©2016 AACR