iCTC drug resistance (CDR) Testing ex vivo for evaluation of available therapies to treat patients with epithelial ovarian cancer.

GOALS: Management of epithelial ovarian cancer (EOC) could use serial measurements of invasive circulating tumor cells (iCTCs) for monitoring therapeutic response and early detection of disease progression/recurrence. Goals of this study are to develop an iCTC drug resistance (CDR) assay and to evaluate clinical significance of patient-derived, cultured iCTCs in selecting available therapies. METHODS: The CDR assay using Taxol-Carboplatin and eight other EOC drugs at the concentration used for patients was performed. Blood was donated by six patients before primary Taxol-Carboplatin chemotherapy, one recurrent patient, six patients during and after their course of chemotherapy, and two patients with benign disease for procedure control. CDR score above and below 100 indicates sensitivity and resistance, respectively, to that drug. RESULTS: Five of six pre-therapy samples had >20 iCTCs/>111 CDR for Taxol-Carboplatin sensitivity, and one had 40 iCTCs/23 CDR for resistance. The recurrent sample had 58 iCTCs/5 CDR for resistance. Four of six post-therapy patients had iCTCs decreased to 0/>153 CDR indicating sensitivity, while two patients had >45 iCTCs/<85 CDR indicating resistance. The patients' treatment history and follow-up confirmed that patients were in response or remission when iCTCs were sensitive to Taxol-Carboplatin, and patients were in relapse or recurrence when iCTCs were resistant to Taxol-Carboplatin. CONCLUSION: Further investigation on the CDR assay is warranted to examine its use in selecting drugs before treating a patient.

Transcriptional regulation of seprase in invasive melanoma cells by transforming growth factor-ß signaling.

The tumor invasive phenotype driven by seprase expression/activity has been widely examined in an array of malignant tumor cell types; however, very little is known about the transcriptional regulation of this critical protease. Seprase (also named fibroblast activation protein-α, antiplasmin-cleaving enzyme, and dipeptidyl prolyl peptidase 5) is expressed at high levels by stromal fibroblast, endothelial, and tumor cells in a variety of invasive tumors but is undetectable in the majority of normal adult tissues. To examine the transcriptional regulation of the gene, we cloned the human seprase promoter and demonstrated that endogenous seprase expression and exogenous seprase promoter activity are high in invasive melanoma cells but not in non-invasive melanoma cells/primary melanocytes. In addition, we identified a crucial TGF-ß-responsive cis-regulatory element in the proximal seprase promoter region that enabled robust transcriptional activation of the gene. Treatment of metastatic but not normal/non-invasive cells with TGF-ß1 caused a rapid and profound up-regulation of endogenous seprase mRNA, which coincided with an abolishment of the negative regulator c-Ski, and an increase in binding of Smad3/4 to the seprase promoter in vivo. Blocking TGF-ß signaling in invasive melanoma cells through overexpression of c-Ski, chemically using SB-431542, or with a neutralizing antibody against TGF-ß significantly reduced seprase mRNA levels. Strikingly, RNAi of seprase in invasive cells greatly diminished their invasive potential in vitro as did blocking TGF-ß signaling using SB-431542. Altogether, we found that seprase is transcriptionally up-regulated in invasive melanoma cells via the canonical TGF-ß signaling pathway, supporting the roles of both TGF-ß and seprase in tumor invasion and metastasis.

Treatment monitoring of patients with epithelial ovarian cancer using invasive circulating tumor cells (iCTCs).

GOALS: Contemporary management of epithelial ovarian cancer (EOC) uses biomarkers to monitor response to therapy. This study evaluates the role of invasive circulating tumor cells (iCTCs) in monitoring EOC treatment in comparison with serum cancer antigen 125 (CA125). METHODS: Molecular and microscopic analyses were used to identify seprase and CD44 as tumor progenitor (TP) markers. The iCTC flow cytometry assay was optimized using blood donated by 64 healthy donors, 49 patients with benign abdominal diseases and 123 EOC patients. Serial changes in iCTCs and CA125 were measured in 129 blood and 169 serum samples, respectively, from 31 EOC patients to assess their concordance during therapy and their relationship with risk of progressive disease (PD). RESULTS: The assay had 97% specificity and 83% sensitivity for detecting iCTCs in blood of EOC patients. iCTCs were detected in each monitoring patient (31/31, 100%) and in 110 of the 129 blood samples (85.3%). The concordance between changes in iCTCs/CA125 levels and changes in the intervals associated with no evidence of disease (NED) were markedly stronger (specificity: CA125 93.8%; iCTCs 90.6%), whereas increases in iCTCs (79.5%) were more sensitive than increases in CA125 (67.6%) to predict PD or relapse. Among the six patients who had greater than 6 measurements, iCTCs but not CA125 antedated changes in clinical status from PD to NED during and after chemotherapy and predated relapse. CONCLUSION: Serial measurements of iCTCs could predict therapeutic responsiveness in 31 EOC patients who underwent standard taxol/carboplatin therapy.

Detection and characterization of invasive circulating tumor cells derived from men with metastatic castration-resistant prostate cancer.

The Vitatex cell-adhesion matrix (CAM) platform allows for isolation of invasive circulating tumor cells (iCTCs). Here we sought to determine the utility of prostate-specific membrane antigen (PSMA) as a metastatic castration-resistant prostate cancer (mCRPC) iCTC biomarker, to identify solitary cells and clusters of iCTCs expressing either epithelial, mesenchymal, or stem cell markers, and to explore the feasibility of iCTC epigenomic analysis. CTCs were isolated and enumerated simultaneously using the Vitatex and CellSearch platforms in 23 men with mCRPC. CAM-avid iCTCs were identified as nucleated cells capable of CAM uptake, but without detectable expression of hematopoietic lineage (HL) markers including CD45. iCTCs were enumerated immunocytochemically (ICC) and by flow cytometry. Whole-genome methylation status was determined for iCTCs using the Illumina HumanMethylation27 BeadChip. Thirty-four samples were collected for iCTC analysis. A median of 27 (range 0-800) and 23 (range 2-390) iCTCs/mL were detected by ICC and flow, respectively. In a subset of 20 samples, a median of seven CTCs/mL (range 0-85) were detected by the CellSearch platform compared to 26 by the CAM platform. iCTC clusters were observed in 17% of samples. iCTCs expressing PSMA as well as markers of EMT and stemness were detectable. The iCTC methylation profile highly resembled mCRPC. More CTCs were recovered using the CAM platform than the CellSearch platform, and the CAM platform allowed for the detection of iCTC clusters, iCTCs expressing EMT and stem-cell markers, and characterization of the iCTC methylome. Correlation with clinical data in future studies may yield further insight into the functional significance of these findings.

Prognostic analysis of invasive circulating tumor cells (iCTCs) in epithelial ovarian cancer.

GOALS: Circulating tumor cells (CTCs) have been introduced as a biomarker in detecting advanced epithelial ovarian cancer (EOC). The goals are to examine the prevalence of the invasive subpopulation of CTCs (iCTCs) in patients at high risk of EOC and to compare this biomarker to serum CA125. METHODS: We used a unique cell adhesion matrix (CAM)-based, functional cell enrichment and identification platform to isolate iCTCs from 129 preoperative patients. We confirmed the identity of iCTCs using positive epithelial (Epi+) markers and negative hematopoietic lineage (HL-) markers. Sensitivity and specificity of the assays were examined and iCTCs/CA125 were correlated with overall survival (OS), progression-free survival (PFS) and clinical parameters. RESULTS: We found a 41.2% sensitivity, 95.1% specificity and 77.8% positive predictive value (PPV) of the iCTC assay in detecting patients with stage I and II EOC malignancy, and a 83% sensitivity and 97.3% PPV in detecting all stages of EOC malignancy. However, a positive CA125 test provided weak evidence to detect stage I and II malignancy (61.6% PPV) and all EOC (92.1% PPV), because of its 76.2% specificity. A significantly stronger concordance in OS and PFS of clinical factors (tumor stage, debulking and platinum sensitivity) was noted for elevated iCTCs than for serum CA125. CONCLUSION: The CAM-initiated CTC enrichment/identification method enabled the detection of early stage EOC. iCTCs were better correlated with worse OS and PFS, more specific and better PPV than CA125 in detecting EOC malignancy in patients at high risk of EOC.

Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients.

Recent research advances show that tumor cell intravasation (entry into the circulation) and metastasis occur very early in breast cancer progression. Clinical studies also illustrate the potential importance of detection of circulating tumor cells (CTCs) in outcomes of patients with metastatic breast cancer. Whether these cells exhibit the invasiveness and express tumor stem or progenitor markers, hallmark of the metastatic phenotype, is less well characterized. To detect CTCs with the invasive phenotype and to explore their molecular features, we applied a functional cell separation method, called collagen adhesion matrix (CAM) assay, as enrichment and identification steps. The CAM-coated device successfully recovered tumor cells spiked in 1 ml of blood with a 54% +/- 9% (n = 18) recovery rate and 0.5-35% purity, and detected invasive tumor cells in 10/10 blood samples (100% yield) from patients with metastatic breast cancer with a range of 18-256 CTCs/ml and average of 126 +/- 25 (mean +/- SD) CTCs/ml. CTCs were detected in blood samples of 28/54 (52%) Stage I-III breast cancer patients with a mean count of 61 CTCs/ml. Furthermore, the relative frequency of these cells correlated to the staging, lymph node-status and survival of patients with early stage breast cancer. CAM-captured cells were capable of propagation in culture. Gene expression and multiplex flow cytometric analyses on CAM-captured cells demonstrated the existence of distinct populations of CTCs including these of epithelial lineage and stem or progenitor cells. Thus, CAM-initiated CTC detection provides advantages for examining invasiveness and tumor progenitor phenotypes.

Elevation of seprase expression and promotion of an invasive phenotype by collagenous matrices in ovarian tumor cells.

Tumor cells do not constitutively exhibit invasive activity, but rather, can be transiently induced to adhere and form lesions. We report here that the expression of seprase, a dominant EDTA-resistant gelatinase in malignant tumors, is dependent on tumor cell exposure to type I collagen gel (TICg). The induced seprase expression of ovarian tumor cells influences their collagen contraction and invasion capability. Importantly, tumor cells with reduced seprase expression, due to manipulation by RNA interference, showed a reduction of TICg contraction in the gel contractility assay, inhibition of tumor cell invasion through TICg as shown by a transwell migration assay and inhibition of peritoneal membrane tumor lesion in a mouse model. In addition, mAb C27, an antibody against beta1 integrin, which blocks cellular avidity to TICg, can induce seprase RNA expression and promote the invasive phenotype and metastatic potential of ovarian tumor cells. Thus, collagenous matrices in the tumor cell niche induce the expression of seprase and initiate tumor invasion and metastatic cascades.

Clinical significance of circulating tumor cells detected by an invasion assay in peripheral blood of patients with ovarian cancer.

OBJECTIVES: The invasive growth of circulating tumor cells (CTCs) propagates cancer metastasis. The aims of this study were to evaluate the association of invasive CTCs, detected by a novel cell invasion assay, with disease stage, CA-125 level and patient survival. METHODS: Peripheral blood samples from 71 patients undergoing evaluation for ovarian malignancy were assessed for the presence of invasive CTCs using a cell invasion assay that enriches and identifies tumor cells with a cell adhesion matrix (CAM). Invasive CTCs were identified as cells exhibiting CAM invasion (CAM+) and expressing standard epithelial markers (Epi+). RESULTS: 43 (60.6%) patients had detectable CTCs: 0/5 benign patients, 1/10 (10%) early stage, 39/52 (73.1%) late stage and 3/4 (75%) unstaged patients (p-value <0.001). CTC counts ranged from 0-149 CTCs/ml with stage III/IV patients exhibiting significantly higher mean counts (41.3 CTCs/ml) than stage I/II patients (6.0 CTCs/ml) and benign patients (0 CTCs/ml, p-value=0.001). A positive correlation between CTC count and CA-125 level was observed (Spearman correlation coefficient r=0.309, p-value=0.035). Kaplan-Meier curves revealed a significant decrease in disease-free survival in patients with detectable CTCs (median survival 15.0 months vs. 35.0 months, log-rank p-value=0.042). Tumor grade and tumor histology did not influence CTC detection. CONCLUSIONS: Invasive CTCs can be detected in a majority of epithelial ovarian cancer patients and may predict shorter disease-free survival. Furthermore, higher CTC counts may reflect later stage disease and higher CA-125 levels.

The propensity of invasive circulating tumor cells (iCTCs) in metastatic progression and therapeutic responsiveness.

Circulating tumor cells (CTCs) are important clinical indicators of metastatic progression and treatment efficacy. However, because of their low number and heterogeneity, reliable patient-derived CTC models are not readily available. We report here the isolation and characterization of the invasive population of CTCs, iCTCs, from blood of 10 patients with epithelial ovarian cancer (EOC) and one pancreatic cancer patient based on the avidity of tumor cells toward an artificial collagen-based adhesion matrix (CAM), in comparison with tumor progenitor (TP) cells isolated from tumor cell lines, tumors and ascites from EOC patients. CAM-avid cells identified to be iCTCs were indistinguishable with TP cells using either functional CAM uptake or surface markers (seprase and CD44). In addition, iCTCs were characterized using peritoneal and spontaneous metastasis models in vivo to evaluate their metastatic propensity and therapeutic response. TP cells and iCTCs had a doubling time of about 34-42 hours. TP cells were rare (<3.5%) in most patient-derived specimens, however, iCTCs emigrated into blood, at a high frequency, 64.2% (n = 49). Approximately 500 patient-derived iCTCs recapitulated formation of iCTCs in mouse blood and formed micrometastases in the liver and/or lung, a degree of metastatic spread equivalent to the inoculation of 5 × 105 bulk tumor cells isolated from ascites and tumors. iCTCs were shown to be novel therapeutic targets for blocking metastasis using the reduced formation of iCTCs and micrometastases by RNAi, peptides, and monoclonal antibodies against seprase.

Seprase, dipeptidyl peptidase IV and urokinase-type plasminogen activator expression in dysplasia and invasive squamous cell carcinoma of the esophagus. A study of 229 cases from Anyang Tumor Hospital, Henan Province, China.

OBJECTIVE: Seprase, dipeptidyl peptidase IV (DPPIV) and urokinase-type plasminogen activator (uPA) play a crucial role in the degradation of the extracellular matrix and in the progression of various human tumors. However, their pathophysiologic significance in esophageal carcinoma has not yet been fully elucidated. METHODS: The expression of seprase, DPPIV and uPA in esophageal dysplasia, squamous cell carcinoma (SCC) and normal epithelium was examined by immunohistochemistry. RESULTS: Seprase, DPPIV and uPA immunoreactivity was found in dysplastic and cancer cells as well as in stromal cells adjacent to dysplasia and cancer sites, but not in normal epithelium. We found a significant association between uPA expression and sex, tumor size and histological classification in carcinomas. High expression of DPPIV in cancer cells correlated with longer survival of the patients. No significant associations between seprase and clinicopathological features either in dysplasia or in carcinomas were found. Finally, we demonstrated higher levels of seprase, DPPIV and uPA in SCC cell lines than in normal esophageal epithelial cell lines. CONCLUSIONS: Our results showed that seprase, DPPIV and uPA are expressed in both premalignant and malignant forms of SCC, but are lacking in normal esophageal epithelia, suggesting that they are involved in SCC neoplastic progression.

Clinical implications of fibroblast activation protein in patients with colon cancer.

PURPOSE: Human fibroblast activation protein (FAP)/seprase is a 97-kDa surface glycoprotein expressed on tumor associated fibroblasts in the majority of epithelial cancers including colon adenocarcinomas. FAP overexpression in human tumor cells has been shown to promote tumor growth in animal models, and clinical trials targeting FAP enzymatic activity have been initiated. The primary objective of this study was to evaluate the clinical significance of stromal FAP in human colon cancers by immunohistochemisty. EXPERIMENTAL DESIGN: Sections of paraffin-embedded resected primary human colon cancer specimens from 1996 through 2001 within the Fox Chase Cancer Center tumor bank were stained with D8 antibody directed against FAP/seprase. Xenotransplanted human colorectal tumors in mice were examined similarly for stromal FAP in tumors of different sizes. Overall percentage of stromal FAP staining of the primary tumor was assessed semiquantitatively (0, 1+, 2+, 3+) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models. RESULTS: One hundred thirty-eight patients with resected specimens were available for study (mean follow-up, 1,050 days) with 6 (4%) stage I, 52 (38%) stage II, 43 (31%) stage III, and 37 (27%) stage IV patients. FAP was detected in >93% of specimens. Semiquantitative staining was scored as 1+ in 28 (20%), 2+ in 52 (38%), and 3+ in 49 (35%). FAP staining intensity was graded as weak in 45 (33%), intermediate in 48 (35%), and dark in 36 (26%). Stromal FAP was found to correlate inversely with tumor stage (semiquantitative, P = 0.01; intensity, P = 0.009) and with tumor size of the tumor xenograft model (correlation coefficient, -0.61; P = 0.047), suggesting that stromal FAP may have a greater role in the early development of tumors. Furthermore, greater stromal FAP for patients with known metastatic disease was associated with a decreased survival. CONCLUSION: Our data indicate that patients whose colon tumors have high levels of stromal FAP are more likely to have aggressive disease progression and potential development of metastases or recurrence. This study affirms the rationale for ongoing clinical investigations using FAP as a therapeutic target to disrupt FAP-driven tumor progression in patients with metastatic disease. It also suggests that the effects of FAP inhibition should be investigated in earlier-stage tumors, given its high levels and potential effect earlier in the course of the disease.

The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices.

Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein alpha are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH(2) terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices.

Activation of EDTA-resistant gelatinases in malignant human tumors.

Among the many proteases associated with human cancer, seprase or fibroblast activation protein alpha, a type II transmembrane glycoprotein, has two types of EDTA-resistant protease activities: dipeptidyl peptidase and a 170-kDa gelatinase activity. To test if activation of gelatinases associated with seprase could be involved in malignant tumors, we used a mammalian expression system to generate a soluble recombinant seprase (r-seprase). In the presence of putative EDTA-sensitive activators, r-seprase was converted into 70- to 50-kDa shortened forms of seprase (s-seprase), which exhibited a 7-fold increase in gelatinase activity, whereas levels of dipeptidyl peptidase activity remained unchanged. In malignant human tumors, seprase is expressed predominantly in tumor cells as shown by in situ hybridization and immunohistochemistry. Proteins purified from experimental xenografts and malignant tumors using antibody- or lectin-affinity columns in the presence of 5 mmol/L EDTA were assayed for seprase activation in vivo. Seprase expression and activation occur most prevalently in ovarian carcinoma but were also detected in four other malignant tumor types, including adenocarcinoma of the colon and stomach, invasive ductal carcinoma of the breast, and malignant melanoma. Together, these data show that, in malignant tumors, seprase is proteolytically activated to confer its substrate specificity in collagen proteolysis and tumor invasion.

Assessing Clinical Outcomes in Colorectal Cancer with Assays for Invasive Circulating Tumor Cells.

Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality. The goals of this study are to evaluate the association between levels of invasive circulating tumor cells (iCTCs) with CRC outcomes and to explore the molecular characteristics of iCTCs. Peripheral blood from 93 patients with Stage I⁻IV CRC was obtained and assessed for the detection and characterization of iCTCs using a functional collagen-based adhesion matrix (CAM) invasion assay. Patients were followed and assessed for overall survival. Tumor cells isolated by CAM were characterized using cell culture and microarray analyses. Of 93 patients, 88 (95%) had detectable iCTCs, ranging over 0⁻470 iCTCs/mL. Patients with Stage I⁻IV disease exhibited median counts of 0.0 iCTCs/mL (n = 6), 13.0 iCTCs/mL (n = 12), 41.0 iCTCs/mL (n = 12), and 133.0 iCTCs/mL (n = 58), respectively (p < 0.001). Kaplan⁻Meier curve analysis demonstrated a significant survival benefit in patients with low iCTC counts compared with in patients with high iCTC counts (log-rank p < 0.001). Multivariable Cox model analysis revealed that iCTC count was an independent prognostic factor of overall survival (p = 0.009). Disease stage (p = 0.01, hazard ratio 1.66; 95% confidence interval: 1.12⁻2.47) and surgical intervention (p = 0.03, HR 0.37; 95% CI: 0.15⁻0.92) were also independent prognostic factors. Gene expression analysis demonstrated the expression of both endothelial and tumor progenitor cell biomarkers in iCTCs. CAM-based invasion assay shows a high detection sensitivity of iCTCs that inversely correlated with overall survival in CRC patients. Functional and gene expression analyses showed the phenotypic mosaics of iCTCs, mimicking the survival capability of circulating endothelial cells in the blood stream.

Expression of seprase in effusions from patients with epithelial ovarian carcinoma.

BACKGROUND: Seprase plays an important role in malignant cell invasion and metastasis by degrading the extracellular matrix. However, its clinical significance remains largely unknown. The objective of the current study was to evaluate the expression of seprase in effusions from patients with epithelial ovarian carcinoma and its clinical values. METHODS: Immunohistochemistry was used to examine the expression of seprase protein in a series of 74 malignant peritoneal (n = 64) and pleural (n = 10) effusions from Norwegian patients with epithelial ovarian carcinoma. Additionally, 34 effusions were evaluated using the Western blotting. Nine reactive effusions, obtained from patients with benign lesions, served as a control group. Statistical analyses were carried out by Chi-square test and Kaplan-Meier method. RESULTS: In the 74 malignant effusion specimens, 57 (77.02%) were positive for seprase, while only 2 (22.22%) of the control group were positively stained (P = 0.001). In the malignant effusions, 17 (22.97%), 22 (29.73%), 22 (29.73%), 13 (17.57%) had negative, weak, moderate and strong seprase protein expression, respectively. The expression of seprase protein was predominant in cytoplasm of carcinoma cells. Increased seprase protein was negatively associated with the overall survival rate of the patients (P = 0.03). However, there was no significant correlation between protein expression and FIGO stage, age, histology, and histological grade. By Western blotting, 27 of the 34 effusions showed the presence of both 170-kD dimeric form and 97-KD monomeric form of seprase while only 1 of the 34 had 170-KD dimeric form, which was consistent with the results of immunohistochemistry (P = 0.05). CONCLUSIONS: Seprase may be involved in the development of ovarian cancer, and is a potential predictive marker for the disease.

Vita-Assay™ Method of Enrichment and Identification of Circulating Cancer Cells/Circulating Tumor Cells (CTCs).

The ability to capture, enrich, and propagate circulating cancer cells/circulating tumor cells (CTCs) for downstream analyses such as ex vivo drug-sensitivity testing of short-term cultures of CTCs, single cell sorting of CTCs by fluorescence activated cell sorting (FACS), animal injection tumor and/or metastasis formation studies, next generation sequencing (NGS), gene expression profiling, gene copy number determination, and epigenomic analyses is of high priority and of immense importance to both the basic research and translational/clinical research communities. Vitatex Inc.'s functional cell separation technology, constructed as Vita-Assay™ (AG6W, AN6W, AR6W) culture plates, is based on the preferential adhesion of invasive rare blood cells of tissue origin to a tissue or tumor microenvironment mimic-the so-called cell adhesion matrix (CAM), which has a demonstrated ability to enrich viable CTCs from blood up to one-million fold.The CAM-scaffold allows for the functional capture and identification of invasive CTCs (iCTCs) including invasive tumor progenitor (TP) cells from cancer-patients' blood. CAM-captured CTCs are capable of ingesting the CAM (CAM+) itself. Green and red fluorescent versions of Vita-Assay™ (AG6W and AR6W) allow for direct visualization of CAM-uptake by cancer cells. Vita-Assay™ CAM-enrichment has allowed for sensitive multiplex flow cytometric and microscopic detection of iCTCs from patients with cancers of the breast, ovary, prostate, pancreas, colorectum, and lung; it has also been successfully utilized for ex vivo drug-sensitivity testing of ovarian-cancer patient CTCs. The CAM enrichment method is equally suitable for the separation of iCTCs and TP cells in ascites and pleural fluid.

'Increased expression of seprase, a membrane-type serine protease, is associated with lymph node metastasis in human colorectal cancer'.

Seprase is a membrane-bound serine proteinase with gelatinase activity, which may be involved in cancer invasion and metastasis. We examined seprase expression in colorectal cancer specimens obtained from 109 patients. Seprase immunoreactivity was found in cancer cells and adjacent stromal cells. Immunoblotting showed higher levels of seprase protein in colorectal cancer tissue than in normal colorectal tissue. A semiquantitative assessment of the immunohistochemistry results revealed a significant correlation between seprase expression and lymph node metastasis. These results suggested that an abundant expression of seprase in colorectal cancer tissue is associated with lymph node metastasis.

Plasma seprase and DPP4 levels as markers of disease and prognosis in cancer.

Seprase (fibroblast activation protein α) has been examined as an invasion biomarker for various types of solid tumors. We studied whether plasma levels of seprase and homologous protease, DPP4 in cancer might serve as tumor biomarkers. We developed sensitive and specific Enzyme-Linked Immunosorbent Assays (ELISAs) to measure these proteases. In 747 plasma samples (from 139 healthy volunteers and 561 cancer patients), mean seprase and DPP4 levels were 0.51 ± 0.30 and 4.65 ± 6.37 µg/mL, respectively, and they were correlated with each other (R(2) = 0.382). Plasma DPP4 and seprase levels were significantly lower in cancer patients compared with healthy subjects (4.38 versus 5.65 µg/mL, p< 0.001 for DPP4; 0.46 versus 0.66 µg/mL, p< 0.001 for seprase). Higher DPP4 was associated with better survival in all cancers combined (n=346) as well as in head and neck malignancies (n=38). Higher seprase was associated with better survival in all non-metastatic cancers combined (n=151) as well as head and neck malignancies, but worse survival in colorectal cancers (n=47). This study demonstrates that in contrast to the high expression in solid tumors, plasma concentrations of seprase and DPP4 are reduced and correlate inversely with survival in most types of cancer, suggesting that these circulating proteases represent useful tumor markers.