Cell-free DNA Methylation as a Predictive Biomarker of Response to Neoadjuvant Chemotherapy for Patients with Muscle-invasive Bladder Cancer in SWOG S1314.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard of care in muscle-invasive bladder cancer (MIBC). However, treatment is intense, and the overall benefit is small, necessitating effective biomarkers to identify patients who will benefit most. OBJECTIVE: To characterize cell-free DNA (cfDNA) methylation in patients receiving NAC in SWOG S1314, a prospective cooperative group trial, and to correlate the methylation signatures with pathologic response at radical cystectomy. DESIGN, SETTING, AND PARTICIPANTS: SWOG S1314 is a prospective cooperative group trial for patients with MIBC (cT2-T4aN0M0, ≥5 mm of viable tumor), with a primary objective of evaluating the coexpression extrapolation (COXEN) gene expression signature as a predictor of NAC response, defined as achieving pT0N0 or ≤pT1N0 at radical cystectomy. For the current exploratory analysis, blood samples were collected prospectively from 72 patients in S1314 before and during NAC, and plasma cfDNA methylation was measured using the Infinium MethylationEPIC BeadChip array. INTERVENTION: No additional interventions besides plasma collection. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Differential methylation between pathologic responders (≤pT1N0) and nonresponders was analyzed, and a classifier predictive of treatment response was generated using the Random Forest machine learning algorithm. RESULTS AND LIMITATIONS: Using prechemotherapy plasma cfDNA, we developed a methylation-based response score (mR-score) predictive of pathologic response. Plasma samples collected after the first cycle of NAC yielded mR-scores with similar predictive ability. Furthermore, we used cfDNA methylation data to calculate the circulating bladder DNA fraction, which had a modest but independent predictive ability for treatment response. In a model combining mR-score and circulating bladder DNA fraction, we correctly predicted pathologic response in 79% of patients based on their plasma collected at baseline and after one cycle of chemotherapy. Limitations of this study included a limited sample size and relatively low circulating bladder DNA levels. CONCLUSIONS: Our study provides the proof of concept that cfDNA methylation can be used to generate classifiers of NAC response in bladder cancer patients. PATIENT SUMMARY: In this exploratory analysis of S1314, we demonstrated that cell-free DNA methylation can be profiled to generate biomarker signatures associated with neoadjuvant chemotherapy response. With validation in additional cohorts, this minimally invasive approach may be used to predict chemotherapy response in locally advanced bladder cancer and perhaps also in metastatic disease.

Bladder cancer cells shift rapidly and spontaneously to cisplatin-resistant oxidative phosphorylation that is trackable in real time.

Genetic mutations have long been recognized as drivers of cancer drug resistance, but recent work has defined additional non-genetic mechanisms of plasticity, wherein cancer cells assume a drug resistant phenotype marked by altered epigenetic and transcriptional states. Currently, little is known about the real-time, dynamic nature of this phenotypic shift. Using a bladder cancer model of nongenetic plasticity, we discovered that rapid transition to drug resistance entails upregulation of mitochondrial gene expression and a corresponding metabolic shift towards the tricarboxylic acid cycle and oxidative phosphorylation. Based on this distinction, we were able to track cancer cell metabolic profiles in real time using fluorescence lifetime microscopy (FLIM). We observed single cells transitioning spontaneously to an oxidative phosphorylation state over hours to days, a trend that intensified with exposure to cisplatin chemotherapy. Conversely, pharmacological inhibition of oxidative phosphorylation significantly reversed the FLIM metabolic signature and reduced cisplatin resistance. These rapid, spontaneous metabolic shifts offer a new means of tracking nongenetic cancer plasticity and forestalling the emergence of drug resistance.

FOXC1 Binds Enhancers and Promotes Cisplatin Resistance in Bladder Cancer.

Chemotherapy resistance is traditionally attributed to DNA mutations that confer a survival advantage under drug selection pressure. However, in bladder cancer and other malignancies, we and others have previously reported that cancer cells can convert spontaneously to an aggressive drug-resistant phenotype without prior drug selection or mutational events. In the current work, we explored possible epigenetic mechanisms behind this phenotypic plasticity. Using Hoechst dye exclusion and flow cytometry, we isolated the aggressive drug-resistant cells and analyzed their chromatin accessibility at regulatory elements. Compared to the rest of the cancer cell population, the aggressive drug-resistant cells exhibited enhancer accessibility changes. In particular, we found that differentially accessible enhancers were enriched for the FOXC1 transcription factor motif, and that FOXC1 was the most significantly overexpressed gene in aggressive drug-resistant cells. ChIP-seq analysis revealed that differentially accessible enhancers in aggressive drug-resistant cells had a higher FOXC1 binding, which regulated the expression of adjacent cancer-relevant genes like ABCB1 and ID3. Accordingly, cisplatin treatment of bladder cancer cells led to an increased FOXC1 expression, which mediated cell survival and conversion to a drug-resistant phenotype. Collectively, these findings suggest that FOXC1 contributes to phenotypic plasticity by binding enhancers and promoting a mutation-independent shift towards cisplatin resistance in bladder cancer.

Non-Invasive Profiling of Advanced Prostate Cancer via Multi-Parametric Liquid Biopsy and Radiomic Analysis.

Integrating liquid biopsies of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) with other minimally invasive measures may yield more comprehensive disease profiles. We evaluated the feasibility of concurrent cellular and molecular analysis of CTCs and cfDNA combined with radiomic analysis of CT scans from patients with metastatic castration-resistant PC (mCRPC). CTCs from 22 patients were enumerated, stained for PC-relevant markers, and clustered based on morphometric and immunofluorescent features using machine learning. DNA from single CTCs, matched cfDNA, and buffy coats was sequenced using a targeted amplicon cancer hotspot panel. Radiomic analysis was performed on bone metastases identified on CT scans from the same patients. CTCs were detected in 77% of patients and clustered reproducibly. cfDNA sequencing had high sensitivity (98.8%) for germline variants compared to WBC. Shared and unique somatic variants in PC-related genes were detected in cfDNA in 45% of patients (MAF > 0.1%) and in CTCs in 92% of patients (MAF > 10%). Radiomic analysis identified a signature that strongly correlated with CTC count and plasma cfDNA level. Integration of cellular, molecular, and radiomic data in a multi-parametric approach is feasible, yielding complementary profiles that may enable more comprehensive non-invasive disease modeling and prediction.

Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype.

Highly tumorigenic, drug-resistant cancer stem-like cells drive cancer progression. These aggressive cells can arise repeatedly from bulk tumor cells independently of mutational events, suggesting an epigenetic mechanism. To test this possibility, we studied bladder cancer cells as they cyclically shifted to and from a cancer stem-like phenotype, and we discovered that these two states exhibit distinct DNA methylation and chromatin accessibility. Most differential chromatin accessibility was independent of methylation and affected the expression of driver genes such as E2F3, a cell cycle regulator associated with aggressive bladder cancer. Cancer stem-like cells exhibited increased E2F3 promoter accessibility and increased E2F3 expression that drove cell migration, invasiveness and drug resistance. Epigenetic interference using a DNA methylation inhibitor blocked the transition to a cancer stem-like state and reduced E2F3 expression. Our findings indicate that epigenetic plasticity plays a key role in the transition to and from an aggressive, drug-resistant phenotype.

Current status of liquid biopsies for the detection and management of prostate cancer.

In recent years, new therapeutic options have become available for prostate cancer (PC) patients, generating an urgent need for better biomarkers to guide the choice of therapy and monitor treatment response. Liquid biopsies, including circulating tumor cells (CTCs), circulating nucleic acids, and exosomes, have been developed as minimally invasive assays allowing oncologists to monitor PC patients with real-time cellular or molecular information. While CTC counts remain the most extensively validated prognostic biomarker to monitor treatment response, recent advances demonstrate that CTC morphology and androgen receptor characterization can provide additional information to guide the choice of treatment. Characterization of cell-free DNA (cfDNA) is another rapidly emerging field with novel technologies capable of monitoring the evolution of treatment relevant alterations such as those in DNA damage repair genes for poly (ADP-ribose) polymerase (PARP) inhibition. In addition, several new liquid biopsy fields are emerging, including the characterization of heterogeneity, CTC RNA sequencing, the culture and xenografting of CTCs, and the characterization of extracellular vesicles (EVs) and circulating microRNAs. This review describes the clinical utilization of liquid biopsies in the management of PC patients and emerging liquid biopsy technologies with the potential to advance personalized cancer therapy.

A Circulating Tumor Cell-RNA Assay for Assessment of Androgen Receptor Signaling Inhibitor Sensitivity in Metastatic Castration-Resistant Prostate Cancer.

Rationale: Our objective was to develop a circulating tumor cell (CTC)-RNA assay for characterizing clinically relevant RNA signatures for the assessment of androgen receptor signaling inhibitor (ARSI) sensitivity in metastatic castration-resistant prostate cancer (mCRPC) patients. Methods: We developed the NanoVelcro CTC-RNA assay by combining the Thermoresponsive (TR)-NanoVelcro CTC purification system with the NanoString nCounter platform for cellular purification and RNA analysis. Based on the well-validated, tissue-based Prostate Cancer Classification System (PCS), we focus on the most aggressive and ARSI-resistant PCS subtype, i.e., PCS1, for CTC analysis. We applied a rigorous bioinformatic process to develop the CTC-PCS1 panel that consists of prostate cancer (PCa) CTC-specific RNA signature with minimal expression in background white blood cells (WBCs). We validated the NanoVelcro CTC-RNA assay and the CTC-PCS1 panel with well-characterized PCa cell lines to demonstrate the sensitivity and dynamic range of the assay, as well as the specificity of the PCS1 Z score (the likelihood estimate of the PCS1 subtype) for identifying PCS1 subtype and ARSI resistance. We then selected 31 blood samples from 23 PCa patients receiving ARSIs to test in our assay. The PCS1 Z scores of each sample were computed and compared with ARSI treatment sensitivity. Results: The validation studies using PCa cell line samples showed that the NanoVelcro CTC-RNA assay can detect the RNA transcripts in the CTC-PCS1 panel with high sensitivity and linearity in the dynamic range of 5-100 cells. We also showed that the genes in CTC-PCS1 panel are highly expressed in PCa cell lines and lowly expressed in background WBCs. Using the artificial CTC samples simulating the blood sample conditions, we further demonstrated that the CTC-PCS1 panel is highly specific in identifying PCS1-like samples, and the high PCS1 Z score is associated with ARSI resistance samples. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). In the analysis of 8 patients who were initially sensitive to ARSI (ARSI-S) and later developed resistance (ARSI-R), we found that the PCS1 Z score increased from the time of ARSI-S to the time of ARSI-R (Pairwise T-test, P=0.016). Conclusions: Using our new methodology, we developed a first-in-class CTC-RNA assay and demonstrated the feasibility of transforming clinically-relevant tissue-based RNA profiling such as PCS into CTC tests. This approach allows for detecting RNA expression relevant to clinical drug resistance in a non-invasive fashion, which can facilitate patient-specific treatment selection and early detection of drug resistance, a goal in precision oncology.

Gelatinous bone marrow transformation and emergence of clonal Philadelphia-negative cytogenetic abnormalities with excess blasts in a patient with chronic myeloid leukemia treated with dasatinib.

Gelatinous bone marrow transformation (GBMT) is a rare pathologic entity of unclear etiology characterized by adipose cell atrophy, focal hematopoietic tissue hypoplasia, and a distinct eosinophilic substance that stains with Alcian blue at pH 2.5. It is traditionally described in the context of malnutrition and cachexia from generalized disease and is important to identify because of its potential reversibility. Several recent case reports have described GBMT in patients with chronic myeloid leukemia (CML) on the first-generation tyrosine-kinase inhibitor (TKI) imatinib. Here, we describe a case of gelatinous transformation in a patient with CML receiving the second-generation TKI dasatinib who subsequently developed clonal cytogenetic abnormalities in Philadelphia chromosome negative cells with excess peripheral blasts consistent with advanced secondary myelodysplastic syndrome. While the development of clonal cytogenetic abnormalities in Philadelphia-negative cells has been frequently described in the setting of TKI, most abnormalities are transient and generally do not effect disease progression and/or transformation like in this case. Remarkably, after TKI discontinuation, repeat bone marrow biopsies had markedly diminished amounts of gelatinous transformation - supporting reversible GBMT with TKI removal. We review the relevant pathophysiology underlying our patient's possible therapeutic-mediated complications during CML therapy in an attempt to better understand the role of TKIs in the pathogenesis of these conditions.

NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood samples because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1st-gen NanoVelcro Chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2nd-gen NanoVelcro Chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3rd-gen Thermoresponsive NanoVelcro Chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4th-gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC assays, and the clinical applications of each generation of devices.

Circulating tumor cells in prostate cancer: beyond enumeration.

Circulating tumor cells (CTCs) are a population of rare cancer cells that have detached from the primary tumor and/or metastatic lesions and entered the peripheral circulation. Enumeration of CTCs has demonstrated value as a prognostic biomarker, and newer studies have pointed to information beyond enumeration that is of critical importance in prostate cancer. Technologic advances that permit examination of the morphology, function, and molecular content of CTCs have made it possible to measure these factors as part of liquid biopsy. These advances provide a way to study tumor evolution and the development of resistance to therapy. Recent breakthroughs have created new applications for CTCs that will affect the care of patients with prostate cancer.

Keratin 13 expression reprograms bone and brain metastases of human prostate cancer cells.

Lethal progression of prostate cancer metastasis can be improved by developing animal models that recapitulate the clinical conditions. We report here that cytokeratin 13 (KRT13), an intermediate filament protein, plays a directive role in prostate cancer bone, brain, and soft tissue metastases. KRT13 expression was elevated in bone, brain, and soft tissue metastatic prostate cancer cell lines and in primary and metastatic clinical prostate, lung, and breast cancer specimens. When KRT13 expression was determined at a single cell level in primary tumor tissues of 44 prostate cancer cases, KRT13 level predicted bone metastasis and the overall survival of prostate cancer patients. Genetically enforced KRT13 expression in human prostate cancer cell lines drove metastases toward mouse bone, brain and soft tissues through a RANKL-independent mechanism, as KRT13 altered the expression of genes associated with EMT, stemness, neuroendocrine/neuromimicry, osteomimicry, development, and extracellular matrices, but not receptor activator NF-κB ligand (RANKL) signaling networks in prostate cancer cells. Our results suggest new inhibitors targeting RANKL-independent pathways should be developed for the treatment of prostate cancer bone and soft tissue metastases.

Clinical Applications of NanoVelcro Rare-Cell Assays for Detection and Characterization of Circulating Tumor Cells.

Liquid biopsy of tumor through isolation of circulating tumor cells (CTCs) allows non-invasive, repetitive, and systemic sampling of disease. Although detecting and enumerating CTCs is of prognostic significance in metastatic cancer, it is conceivable that performing molecular and functional characterization on CTCs will reveal unprecedented insight into the pathogenic mechanisms driving lethal disease. Nanomaterial-embedded cancer diagnostic platforms, i.e., NanoVelcro CTC Assays represent a unique rare-cell sorting method that enables detection isolation, and characterization of CTCs in peripheral blood, providing an opportunity to noninvasively monitor disease progression in individual cancer patients. Over the past decade, a series of NanoVelcro CTC Assays has been demonstrated for exploring the full potential of CTCs as a clinical biomarker, including CTC enumeration, phenotyping, genotyping and expression profiling. In this review article, the authors will briefly introduce the development of three generations of NanoVelcro CTC Assays, and highlight the clinical applications of each generation for various types of solid cancers, including prostate cancer, pancreatic cancer, lung cancer, and melanoma.

Applications of circulating tumor cells for prostate cancer.

One of the major challenges that clinicians face is in the difficulties of accurately monitoring disease progression. Prostate cancer is among these diseases and greatly affects the health of men globally. Circulating tumor cells (CTCs) are a rare population of cancer cells that have shed from the primary tumor and entered the peripheral circulation. Not until recently, clinical applications of CTCs have been limited to using enumeration as a prognostic tool in Oncology. However, advances in emerging CTC technologies point toward new applications that could revolutionize the field of prostate cancer. It is now possible to study CTCs as components of a liquid biopsy based on morphological phenotypes, biochemical analyses, and genomic profiling. These advances allow us to gain insight into the heterogeneity and dynamics of cancer biology and to further study the mechanisms behind the evolution of therapeutic resistance. These recent developments utilizing CTCs for clinical applications will greatly impact the future of prostate cancer research and pave the way towards personalized care for men.

A comparison of isolated circulating tumor cells and tissue biopsies using whole-genome sequencing in prostate cancer.

Previous studies have demonstrated focal but limited molecular similarities between circulating tumor cells (CTCs) and biopsies using isolated genetic assays. We hypothesized that molecular similarity between CTCs and tissue exists at the single cell level when characterized by whole genome sequencing (WGS). By combining the NanoVelcro CTC Chip with laser capture microdissection (LCM), we developed a platform for single-CTC WGS. We performed this procedure on CTCs and tissue samples from a patient with advanced prostate cancer who had serial biopsies over the course of his clinical history. We achieved 30X depth and ≥ 95% coverage. Twenty-nine percent of the somatic single nucleotide variations (SSNVs) identified were founder mutations that were also identified in CTCs. In addition, 86% of the clonal mutations identified in CTCs could be traced back to either the primary or metastatic tumors. In this patient, we identified structural variations (SVs) including an intrachromosomal rearrangement in chr3 and an interchromosomal rearrangement between chr13 and chr15. These rearrangements were shared between tumor tissues and CTCs. At the same time, highly heterogeneous short structural variants were discovered in PTEN, RB1, and BRCA2 in all tumor and CTC samples. Using high-quality WGS on single-CTCs, we identified the shared genomic alterations between CTCs and tumor tissues. This approach yielded insight into the heterogeneity of the mutational landscape of SSNVs and SVs. It may be possible to use this approach to study heterogeneity and characterize the biological evolution of a cancer during the course of its natural history.

Subclassification of prostate cancer circulating tumor cells by nuclear size reveals very small nuclear circulating tumor cells in patients with visceral metastases.

BACKGROUND: Although enumeration of circulating tumor cells (CTCs) has shown some clinical value, the pool of CTCs contains a mixture of cells that contains additional information that can be extracted. The authors subclassified CTCs by shape features focusing on nuclear size and related this with clinical information. METHODS: A total of 148 blood samples were obtained from 57 patients with prostate cancer across the spectrum of metastatic states: no metastasis, nonvisceral metastasis, and visceral metastasis. CTCs captured and enumerated on NanoVelcro Chips (CytoLumina, Los Angeles, Calif) were subjected to pathologic review including nuclear size. The distribution of nuclear size was analyzed using a Gaussian mixture model. Correlations were made between CTC subpopulations and metastatic status. RESULTS: Statistical modeling of nuclear size distribution revealed 3 distinct subpopulations: large nuclear CTCs, small nuclear CTCs, and very small nuclear CTCs (vsnCTCs). Small nuclear CTCs and vsnCTC identified those patients with metastatic disease. However, vsnCTC counts alone were found to be elevated in patients with visceral metastases when compared with those without (0.36 ± 0.69 vs 1.95 ± 3.77 cells/mL blood; P<.001). Serial enumeration studies suggested the emergence of vsnCTCs occurred before the detection of visceral metastases. CONCLUSIONS: There are morphologic subsets of CTCs that can be identified by fundamental pathologic approaches, such as nuclear size measurement. The results of this observational study strongly suggest that CTCs contain relevant information regarding disease status. In particular, the detection of vsnCTCs was found to be correlated with the presence of visceral metastases and should be formally explored as a putative blood-borne biomarker to identify patients at risk of developing this clinical evolution of prostate cancer.

miR-154* and miR-379 in the DLK1-DIO3 microRNA mega-cluster regulate epithelial to mesenchymal transition and bone metastasis of prostate cancer.

PURPOSE: MicroRNAs in the delta-like 1 homolog-deiodinase, iodothyronine 3 (DLK1-DIO3) cluster have been shown to be critical for embryonic development and epithelial to mesenchymal transition (EMT). DLK1-DIO3 cluster miRNAs are elevated in the serum of patients with metastatic cancer. However, the biologic functions of these miRNAs in the EMT and metastasis of cancer cells are poorly understood. We previously demonstrated the oncogenic and metastatic role of miR-409-3p/5p, a member of this cluster, in prostate cancer. In this study, we defined the role of miR-154* and miR-379, two key members of this cluster, in prostate cancer progression and bone metastasis in both cell line models and clinical specimens. EXPERIMENTAL DESIGN: Genetic manipulation of miR-154* and miR-379 was performed to determine their role in tumor growth, EMT, and bone metastasis in mouse models. We determined the expression of miR-154* in prostate cancer clinical samples and bone metastasis samples using in situ hybridization and quantum dot labeling. RESULTS: Elevated expression of miR-154* and miR-379 was observed in bone metastatic prostate cancer cell lines and tissues, and miR-379 expression correlated with progression-free survival of patients with prostate cancer. Intracardiac inoculation (to mimic systemic dissemination) of miR-154* inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival. CONCLUSION: miR-154* and miR-379 play important roles in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding has particular translational importance because miRNAs in the DLK1-DIO3 cluster can be attractive biomarkers and possible therapeutic targets to treat bone metastatic prostate cancer.

Nanostructure embedded microchips for detection, isolation, and characterization of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells that break away from either a primary tumor or a metastatic site and circulate in the peripheral blood as the cellular origin of metastasis. With their role as a "tumor liquid biopsy", CTCs provide convenient access to all disease sites, including that of the primary tumor and the site of fatal metastases. It is conceivable that detecting and analyzing CTCs will provide insightful information in assessing the disease status without the flaws and limitations encountered in performing conventional tumor biopsies. However, identifying CTCs in patient blood samples is technically challenging due to the extremely low abundance of CTCs among a large number of hematologic cells. To address this unmet need, there have been significant research endeavors, especially in the fields of chemistry, materials science, and bioengineering, devoted to developing CTC detection, isolation, and characterization technologies. Inspired by the nanoscale interactions observed in the tissue microenvironment, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with high efficiency. The working mechanism of NanoVelcro cell-affinity substrates mimics that of Velcro: when the two fabric strips of a Velcro fastener are pressed together, tangling between the hairy surfaces on two strips leads to strong binding. Through continuous evolution, three generations (gens) of NanoVelcro CTC chips have been established to achieve different clinical utilities. The first-gen NanoVelcro chip, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, was created for CTC enumeration. Side-by-side analytical validation studies using clinical blood samples suggested that the sensitivity of first-gen NanoVelcro chip outperforms that of FDA-approved CellSearch. In conjunction with the use of the laser microdissection (LMD) technique, second-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation. The individually isolated CTCs can be subjected to single-CTC genotyping (e.g., Sanger sequencing and next-generation sequencing, NGS) to verify the CTC's role as tumor liquid biopsy. Created by grafting of thermoresponsive polymer brushes onto SiNS, third-gen NanoVelcro chips (i.e., Thermoresponsive NanoVelcro) have demonstrated the capture and release of CTCs at 37 and 4 °C, respectively. The temperature-dependent conformational changes of polymer brushes can effectively alter the accessibility of the capture agent on SiNS, allowing for rapid CTC purification with desired viability and molecular integrity. This Account summarizes the continuous evolution of NanoVelcro CTC assays from the emergence of the original idea all the way to their applications in cancer research. We envision that NanoVelcro CTC assays will lead the way for powerful and cost-efficient diagnostic platforms for researchers to better understand underlying disease mechanisms and for physicians to monitor real-time disease progression.

miR-409-3p/-5p promotes tumorigenesis, epithelial-to-mesenchymal transition, and bone metastasis of human prostate cancer.

PURPOSE: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. EXPERIMENTAL DESIGN: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. RESULTS: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle-treated cells. CONCLUSION: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer.

NanoVelcro Chip for CTC enumeration in prostate cancer patients.

Circulating tumor cells (CTCs) are one of the most crucial topics in rare cell biology and have become the focus of a significant and emerging area of cancer research. While CTC enumeration is a valid biomarker in prostate cancer, the current FDA-approved CTC technology is unable to detect CTCs in a large portion of late stage prostate cancer patients. Here we introduce the NanoVelcro CTC Chip, a device composed of a patterned silicon nanowire substrate (SiNW) and an overlaid polydimethylsiloxane (PDMS) chaotic mixer. Validated by two institutions participating in the study, the NanoVelcro Chip assay exhibits very consistent efficiency in CTC-capture from patient samples. The utilized protocol can be easily replicated at different facilities. We demonstrate the clinical utility of the NanoVelcro Chip by performing serial enumerations of CTCs in prostate cancer patients after undergoing systemic therapy. Changes in CTC numbers after 4-10 weeks of therapy were compared with their clinical responses. We observed a statistically significant reduction in CTCs counts in the clinical responders. We performed long-term follow up with serial CTC collection and enumeration in one patient observing variations in counts correlating with treatment response. This study demonstrates the consistency of the NanoVelcro Chip assay over time for CTC enumeration and also shows that continuous monitoring of CTC numbers can be employed to follow responses to different treatments and monitor disease progression.

High-purity prostate circulating tumor cell isolation by a polymer nanofiber-embedded microchip for whole exome sequencing.

Handpick single cancer cells: a modified NanoVelcro Chip is coupled with ArcturusXT laser capture microdissection (LCM) technology to enable the detection and isolation of single circulating tumor cells (CTCs) from patients with prostate cancer (PC). This new approach paves the way for conducting next-generation sequencing (NGS) on single CTCs.