Zunfu Ke

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

Conspectus 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.

Programming thermoresponsiveness of NanoVelcro substrates enables effective purification of circulating tumor cells in lung cancer patients.

Unlike tumor biopsies that can be constrained by problems such as sampling bias, circulating tumor cells (CTCs) are regarded as the “liquid biopsy” of the tumor, providing convenient access to all disease sites, including primary tumor and fatal metastases. Although enumerating CTCs is of prognostic significance in solid tumors, it is conceivable that performing molecular and functional analyses on CTCs will reveal much significant insight into tumor biology to guide proper therapeutic intervention. We developed the Thermoresponsive NanoVelcro CTC purification system that can be digitally programmed to achieve an optimal performance for purifying CTCs from non-small cell lung cancer (NSCLC) patients. The performance of this unique CTC purification system was optimized by systematically modulating surface chemistry, flow rates, and heating/cooling cycles. By applying a physiologically endurable stimulation (i.e., temperature between 4 and 37 °C), the mild operational parameters allow minimum disruption to CTCs’ viability and molecular integrity. Subsequently, we were able to successfully demonstrate culture expansion and mutational analysis of the CTCs purified by this CTC purification system. Most excitingly, we adopted the combined use of the Thermoresponsive NanoVelcro system with downstream mutational analysis to monitor the disease evolution of an index NSCLC patient, highlighting its translational value in managing NSCLC.

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

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.

High expression of CTHRC1 promotes EMT of epithelial ovarian cancer (EOC) and is associated with poor prognosis

Collagen triple helix repeat-containing 1 (CTHRC1) is aberrantly overexpressed in multiple malignant tumors. However, the expression characteristics and function of CTHRC1 in epithelial ovarian cancer (EOC) remain unclear. We found that CTHRC1 expression was up-regulated in the paraffin-embedded EOC tissues compared to borderline or benign tumor tissues. CTHRC1 expression was positively correlated with tumor size (p = 0.008), menopause (p = 0.037), clinical stage (p = 0.002) and lymph node metastasis (p < 0.001) and was also an important prognostic factor for the overall survival of EOC patients, as revealed by Kaplan-Meier analysis. CTHRC1 increased the invasive capabilities of EOC cells in vitro by activating the Wnt/β-catenin signaling pathway. We showed that ectopic transfection of CTHRC1 in EOC cells up-regulated the expression of EMT markers such as N-cadherin and vimentin, and EMT-associated transcriptional factor Snail. Knockdown of CTHRC1 expression in EOC cells resulted in down-regulation of N-cadherin, vimentin, Snail and translocation of β-catenin. Collectively, CTHRC1 may promote EOC metastasis through the induction of EMT process and serve as a potential biomarker for prognosis as well as a target for therapy.

Detecting ALK-rearrangement of CTC enriched by nanovelcro chip in advanced NSCLC patients

Circulating tumor cells (CTC) shows great prospect to realize precision medicine in cancer patients. We developed the NanoVelcro Chip integrating three functional mechanisms. NanoVelcro CTC capture efficiency was tested in advanced NSCLC. Further, ALK-rearrangement status was examined through fluorescent in situ hybridization in CTCs enriched by NanoVelcro. NanoVelcro system showed higher CTC-capture efficiency than CellSearch in advanced NSCLC. CTC counts obtained by both methods were positively correlated (r=0.45, p<0.05). Further, CTC counts enriched by NanoVelcro correlated to the pTNM stage but CellSearch. All ALK-positive patients had 3 or more ALK-rearranged CTC per mL of blood. Less than 3 ALK-rearranged CTC was detected in ALK-negative patients. NanoVelcro can detect the ALK-rearranged status with consistent sensitivity and specificity compared to biopsy test. Furthermore, the ALK-rearranged CTC ratio correlated to the pTNM stage in ALK-positive patients. Following up with CTC enumeration by NanoVelcro and CellSearch observed their variations in reflecting clinical response of crizotinib treatment. NanoVelcro provides a sensitive method for CTC counts and characterization in advanced NSCLC. ALK-rearrangement can be detected in CTCs collected from advanced NSCLC patients by NanoVelcro, facilitating diagnostic test and prognosis analysis, most importantly offering one noninvasive method for real-time monitoring of treatment reaction.

AEG-1 activates Wnt/PCP signaling to promote metastasis in tongue squamous cell carcinoma

Despite advances in therapy, survival among patients with locally advanced squamous cell carcinoma of tongue (TSCC) and cervical lymph node metastasis remains dismal. Here, we estimated the functional effect of AEG-1 on TSCC metastasis and explored the molecular mechanism by which AEG-1 stimulates epithelial-mesenchymal transition (EMT). We initially found that AEG-1 mRNA levels were much higher in metastatic TSCC than in non-metastatic TSCC and that AEG-1 expression strongly correlates with EMT status. Receiver operating characteristic analysis showed that the combined AEG-1 and EMT statuses are predictive of the survival rate among TSCC patients. In addition, AEG-1 knockdown inhibited EMT in cultured TSCC cell lines and in a xenograft-mouse model. Recombinant AEG-1 activated Wnt/PCP-Rho signaling, and its stimulatory effects on TSCC cell invasiveness and EMT were reversed by an anti-Wnt5a neutralizing antibody or by inhibition of Rac1 or ROCK. These results highlight the critical stimulatory effect of AEG-1 on cancer cell invasiveness and EMT and indicate that AEG-1 may be a useful prognostic biomarker for TSCC patients.

Multidimensional Roles of Collagen Triple Helix Repeat Containing 1 (CTHRC1) in Malignant Cancers.

Tumor is one of the principal diseases that seriously threaten human health. Insight into sensitive cancer markers may open a new avenue for the early diagnosis and treatment of this disease. CTHRC1 has been identified as a cancer-related gene. It is a secretory glycoprotein that possesses multidimensional roles associated with wound repair, bone remodeling, hepatocytes fibrosis, adipose tissue formation, and so on. Our previous studies and numerous reports from other researchers have revealed that the ascended expression of CTHRC1 tends to go hand in hand with tumorigenesis, proliferation, invasion and metastasis in various human malignancies through a series of molecular mechanisms and signaling pathways. However, the detailed pathogenic mechanisms of CTHRC1 overexpression in human malignant cancers are not yet clear. Here, we shall focus our description on the functions, expression profile in several representative malignant tumors and a number of molecular mechanisms and signaling pathways involved with CTHRC1. This introductory discussion of CTHRC1 will serve as a reference for further research in understanding this intriguing cancer-related protein.

Auxiliary diagnostic value of p16 amplification combined with the detection of heterozygous and homozygous loss for urothelial carcinoma

The present study aimed to investigate the significance of detecting cyclin dependent kinase inhibitor 2A (p16) gene aberrations in the diagnosis of urothelial carcinoma (UC) using fluorescence in situ hybridization (FISH). A total of 77 voided urine specimens from 65 patients with UC and 12 patients with benign urinary disease were recruited into the current study. Under a fluorescence microscope, cells with large and irregular nuclei were assessed for chromosomal aberrations. The positive rate of p16 amplification in UC samples was 32.3% (21/65), which was significantly higher than that in benign urinary disease samples (16.7%, 2/12; P<0.05). Heterozygous and homozygous loss of p16 was identified in 12 (18.5%) and 23 (35.4%) patients with UC, respectively; p16 expression in the remainder of patients was normal. In addition, as tumor stage or grade advanced, the positive rate of p16 aberrations also increased significantly (P<0.05). In conclusion, p16 gene aberrations may serve important roles in the auxiliary diagnosis of UC by FISH and could be utilized to monitor UC progression.

CTHRC1 induces non-small cell lung cancer (NSCLC) invasion through upregulating MMP-7/MMP-9

BackgroundThe strong invasive and metastatic nature of non-small cell lung cancer (NSCLC) leads to poor prognosis. Collagen triple helix repeat containing 1 (CTHRC1) is involved in cell migration, motility and invasion. The object of this study is to investigate the involvement of CTHRC1 in NSCLC invasion and metastasis.MethodsA proteomic analysis was performed to identify the different expression proteins between NSCLC and normal tissues. Cell lines stably express CTHRC1, MMP7, MMP9 were established. Invasion and migration were determined by scratch and transwell assays respectively. Clinical correlations of CTHRC1 in a cohort of 230 NSCLC patients were analysed.ResultsCTHRC1 is overexpressed in NSCLC as measured by proteomic analysis. Additionally, CTHRC1 increases tumour cell migration and invasion in vitro. Furthermore, CTHRC1 expression is significantly correlated with matrix metalloproteinase (MMP)7 and MMP9 expression in sera and tumour tissues from NSCLC. The invasion ability mediated by CTHRC1 were mainly MMP7- and MMP9-dependent. MMP7 or MMP9 depletion significantly eradicated the pro-invasive effects mediated by CTHRC1 on NSCLC cells. Clinically, patients with high CTHRC1 expression had poor survival.ConclusionsCTHRC1 serves as a pro-metastatic gene that contributes to NSCLC invasion and metastasis, which are mediated by upregulated MMP7 and MMP9 expression. Targeting CTHRC1 may be beneficial for inhibiting NSCLC metastasis.

MP07-06 VERY-SMALL-NUCLEAR CIRCULATING TUMOR CELL (VSNCTC) AS A PUTATIVE BIOMARKER FOR VISCERAL METASTASIS IN METASTATIC CASTRATION-RESISTANT PROSTATE CANCER (MCRPC)

INTRODUCTION AND OBJECTIVES: The relation of serum androgens and the development of prostate cancer (PCa) is subject of debate. Lower total testosterone (TT) levels have been associated with increased PCa detection and worse pathological features after treatment. The aim of this study is to investigate the association of serum androgens levels and PCa detection in a prospective screening study of men at higher genetic risk due to BRCA mutation METHODS: Six hundred and fifty seven participants in the IMPACT study had sequential TT and sexual hormone binding globulin (SHBG) measurements in serum samples at annual visits, giving 2783 prospective androgen levels, from 234 BRCA1 carriers, 268 BRCA2 carriers and 155 mutation negative controls. Free and bioavailable testosterone were calculated from TT and SHBG using the Sordergaard equation. Age, BMI, PSA and hormonal concentrations were compared between men affected and unaffected with PCa within each genetic cohort using unpaired t-test. The Fisher exact test was used to correlate hypogonadic levels of testosterone and genetic status with PCa diagnosis within each genetic category RESULTS: There were no significant differences in the PSA levels and BMI at study entry. Mean ages were 53.52, 52.17, and 51.17 years, for the control, BRCA1 and BRCA2 groups, respectively (BRCA1 p1⁄40.24 and BRCA2 p1⁄40.01). In the BRCA2 cohort, serum TT levels were lower in carriers diagnosed with PCa, compared with unaffected carriers and controls (11.2, 13.78, 13.4 nmol/L, mean values per group, respectively, p1⁄40.02 and p1⁄40.009). In the BRCA1 cohort, no significant difference was observed between affected and unaffected carriers and controls (13.6, 13.5, 13.4, nmol/L, mean values per group respectively, p1⁄40.82 and p-0.69). SHBG levels were significantly higher in BRCA1 carriers with PCa than unaffected carriers and controls (47.62, 37.97, 40.34 nmol/L, respectively; p1⁄40.002 and p1⁄40.03). TT levels <8nmol/L were significantly more frequent among BRCA2 carriers with PCa then unaffected carriers and controls (OR 2.78, 95% CI: 1.3-5.8, p1⁄40.01). This finding was not observed in the BRCA1 cohort. There was no significant difference in the levels of free and bioavailable testosterone in both cohorts CONCLUSIONS: Our findings indicate that there is a difference between BRCA1 and BRCA2 carriers who develop PCa in relation to serumTT and SHBG levels. BRCA2 carriers affected with PCa had lower total testosterone levels, and affected BRCA1 carriers had higher levels of SHBG. These data are preliminary but may indicate that these values could be explored as biomarkers for BRCA-related prostate cancer