Matrilin-1 is an inhibitor of neovascularization.

In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2 m NaCl, 0.01 m HEPES buffer at 4 °C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography, and fractions were then screened for their ability to inhibit capillary endothelial cell (EC) proliferation in vitro. Fractions containing EC inhibitory activity were pooled and further purified by cation exchange chromatography. The resulting fractions from this step were then screened to isolate the antiangiogenic activity in vitro. This activity was identified by tandem mass spectrometry as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay by the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1(-/-) mice compared with the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin.

The anti-angiogenic peptide, loop 6, binds insulin-like growth factor-1 receptor.

Tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, have been shown to possess biological functions that are independent of their ability to inhibit matrix metalloproteinases. We have previously shown that the C-terminal domain of TIMP-2 and, in particular, Loop 6 inhibit capillary endothelial cell proliferation and angiogenesis both in vitro and in vivo. To elucidate the mechanism by which Loop 6 inhibits angiogenesis, we sought to determine whether its biological effects were the result of a known TIMP-2 protein-protein interaction or of a receptor-mediated event. In this study, we identify insulin-like growth factor-1 receptor as a binding partner of Loop 6/TIMP-2 and characterize this interaction on the endothelial cell surface and the consequences of this interaction on downstream receptor signaling.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

SMOOT libraries and phage-induced directed evolution of Cas9 to engineer reduced off-target activity.

RNA-guided endonucleases such as Cas9 provide efficient on-target genome editing in cells but may also cleave at off-target loci throughout the genome. Engineered variants of Streptococcus pyogenes Cas9 (SpCas9) have been developed to globally reduce off-target activity, but individual off-targets may remain, or on-target activity may be compromised. In order to evolve against activity at specific off-targets while maintaining strong on-target editing, we developed a novel M13 bacteriophage-mediated selection method. Using this method, sequential rounds of positive and negative selection are used to identify mutations to Cas9 that enhance or diminish editing activity at particular genomic sequences. We also introduce scanning mutagenesis of oligo-directed targets (SMOOT), a comprehensive mutagenesis method to create highly diverse libraries of Cas9 variants that can be challenged with phage-based selection. Our platform identifies novel SpCas9 mutants which mitigate cleavage against off-targets both in biochemical assays and in T-cells while maintaining higher on-target activity than previously described variants. We describe an evolved variant, S. pyogenes Adapted to Reduce Target Ambiguity Cas9 (SpartaCas), composed of the most enriched mutations, each of unknown function. This evolved Cas9 mutant reduces off-target cleavage while preserving efficient editing at multiple therapeutically relevant targets. Directed evolution of Cas9 using our system demonstrates an improved structure-independent methodology to effectively engineer nuclease activity.

Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells.

Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.

A novel approach to using matrix metalloproteinases for bladder cancer.

PURPOSE: Given the steadily growing cancer survivor population, increasing pressure has been placed on more effective clinical approaches and biomarker assays to manage care. For bladder cancer despite the high probability of recurrence the number of patients with recurrent disease is significantly lower than the number that remains cancer free at any monitoring interval. We developed a noninvasive urine assay using a novel approach to identify patients without recurrent cancer with extremely high confidence. MATERIALS AND METHODS: Previous studies show that matrix metalloproteinases are increased in the urine of patients with cancer compared to that in disease-free individuals. To determine the clinical usefulness of these markers as monitors for bladder cancer recurrence we measured and compared metalloproteinase-2, metalloproteinase-9 and metalloproteinase-9/neutrophil gelatinase-associated lipocalin by enzyme-linked immunosorbent assay and zymography in a set of 530 samples, including 84 samples from patients with bladder cancer. RESULTS: Initial studies using urine metalloproteinase to discriminate disease-free patients from those with bladder cancer resulted in 80% sensitivity (67 of 84) and 71% specificity (318 of 446) for metalloproteinase-9. By applying our novel Clinical Intervention Determining Diagnostic() clinical approach to metalloproteinase-9 we correctly identified 42% of cases that were cystoscopy negative with 98% negative predictive value. CONCLUSIONS: A noninvasive urine diagnostic assay that uses metalloproteinases with the Clinical Intervention Determining Diagnostic could lead to more efficient treatment in bladder cancer survivors by decreasing the number of negative cystoscopies (42%), allowing physicians to more selectively monitor those at high risk.

Establishment and biochemical characterization of primary cells of the upper aerodigestive tract.

The upper aerodigestive tract, composed of the oral cavity, the pharynx and the esophagus, is a complex system whose components function in both organ-specific ways as well in serving as a protective barrier against the enzymes which initiate digestion as well as against the mechanical functions which serve to ensure movement of food through the upper aerodigestive tract. Given these diverse functional requirements, the study of the anatomy and physiology of this region are uniquely complex and significantly understudied. The goal of the current study was to develop a simple and reproducible method for the isolation, growth, and maintenance of primary epithelial cells from the oral cavity, the pharynx and the esophagus. In addition, given the increased interest in diseases characterized by a loss of mucosal integrity in these areas which is often accompanied by a diminished wound healing capability, these cells were biochemically characterized with a focus on the components of the extracellular matrix remodeling axis including the activity and inhibition of the matrix metalloproteinases (MMPs).

The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.

PURPOSE: Having previously shown that the binding of neutrophil gelatinase-associated lipocalin (NGAL) to matrix metalloproteinase-9 (MMP-9) protects this extracellular matrix remodeling enzyme from autodegradation, we hypothesized that the addition of NGAL to breast cancer cells, which do not express this protein but do express MMP-9, might result in a more aggressive phenotype in vivo. Based on our previous reports that MMPs can be detected in the urine of cancer patients, we also asked whether MMP-9/NGAL could be detected in the urine of breast cancer patients and whether it might be predictive of disease status. EXPERIMENTAL DESIGN: Clones of MCF-7 human breast cancer cells differentially expressing NGAL were generated by stable transfection with human NGAL expression constructs. The established clones were then implanted s.c. in immunodeficient mice and tumor growth was monitored. In addition, we analyzed the urine of individuals with breast cancer and age-matched, sex-matched controls using gelatin zymography for the presence of MMP-9/NGAL. RESULTS: Increased NGAL expression resulted in significant stimulation of tumor growth. Immunohistochemical analysis of MCF-7 tumors revealed that the NGAL-overexpressing ones exhibited increased growth rates that were accompanied by increased levels of MMP-9, increased angiogenesis, and an increase in the tumor cell proliferative fraction. In addition, MMP-9/NGAL complex was detected in 86.36% of the urine samples from breast cancer patients but not in those from healthy age and sex-matched controls. CONCLUSIONS: These findings suggest, for the first time, that NGAL may play an important role in breast cancer in vivo by protecting MMP-9 from degradation thereby enhancing its enzymatic activity and facilitating angiogenesis and tumor growth. Clinically, these data suggest that the urinary detection of MMP-9/NGAL may be useful in noninvasively predicting disease status of breast cancer patients.

Noninvasive multianalyte diagnostic assay for monitoring bladder cancer recurrence.

BACKGROUND: The purpose of this study was to establish the clinical performance of a urine-based assay, called a multianalyte diagnostic readout, in monitoring for bladder cancer recurrence. METHODS: This was a prospective, multicenter, single assessment observational study. The multianalyte diagnostic readout uses a combination of one protein and three DNA biomarkers. Urine samples from 733 patients undergoing monitoring for bladder cancer recurrence were analyzed for matrix metalloproteinase-2 levels, the presence of mutant FGFR3 DNA, and hypermethylation of the NID2 and VIM genes. The probability of a patient having (positive predictive value) or not having (negative predictive value) recurrent bladder cancer was determined by FGFR3 alone or all four biomarkers combined, respectively. RESULTS: Cystoscopy/biopsy diagnosed 63 patients with bladder cancer recurrence at the time of study assessment. The four-biomarker assay identified 237 patients as having a low probability of disease recurrence, 231 of whom were determined by cystoscopy as not having recurrent cancer, resulting in a negative predictive value of 97.5% at 90.5% sensitivity. The FGFR3 assay identified 49 patients with FGFR3 mutations, 19 of whom were confirmed by biopsy as having cancer, resulting in a positive predictive value of 38.8%, with 95.5% specificity. CONCLUSION: The urine-based multianalyte diagnostic readout assay was able to delineate the patient population into those highly likely to have bladder cancer recurrence, those unlikely to have recurrent disease, and those with an average risk for bladder cancer recurrence.

A noninvasive multianalyte urine-based diagnostic assay for urothelial cancer of the bladder in the evaluation of hematuria.

OBJECTIVE: To test whether a noninvasive urine-based multianalyte diagnostic readout assay that uses protein and DNA biomarkers can risk stratify patients with hematuria into those who are or are not likely to have bladder cancer and those who should receive standard care. PATIENTS AND METHODS: This prospective, observational, multicenter, single-assessment study was conducted between June 12, 2009, and April 15, 2011. Eligible patients presented with hematuria and as part of their evaluation underwent cystoscopy. Urine samples were analyzed for the presence of mutant FGFR3 and quantified matrix metalloproteinase 2 and the hypermethylation of TWIST1 and NID2. A patient's chance of having (positive predictive value [PPV]) or not having (negative predictive value [NPV]) cancer was determined by FGFR3 alone or by all 4 biomarkers, respectively. RESULTS: Cystoscopy/biopsy diagnosed 690 of 748 patients as negative and 58 as positive for bladder cancer. Of 21 patients identified by FGFR3 as highly likely to have cancer, 20 were also positive by cystoscopy/biopsy, resulting in a PPV of 95.2% (20 of 21), with specificity of 99.9% (689 of 690). The 4-marker combination identified 395 patients as having a low likelihood of cancer. Of these, 56.2% (388 of 690) also had negative biopsy/cystoscopy findings, resulting in an NPV of 98.2% (388 of 395). In total, 416 of the 748 patients with hematuria (55.6%) were identified with extremely high NPV and PPV to have or not have bladder cancer. CONCLUSION: This multianalyte assay accurately stratified patients with high confidence into those who likely do or do not have bladder cancer. This test was developed to enhance and not to eliminate referrals for urologic evaluation.

Detection of low frequency FGFR3 mutations in the urine of bladder cancer patients using next-generation deep sequencing.

Biological fluid-based noninvasive biomarker assays for monitoring and diagnosing disease are clinically powerful. A major technical hurdle for developing these assays is the requirement of high analytical sensitivity so that biomarkers present at very low levels can be consistently detected. In the case of biological fluid-based cancer diagnostic assays, sensitivities similar to those of tissue-based assays are difficult to achieve with DNA markers due to the high abundance of normal DNA background present in the sample. Here we describe a new urine-based assay that uses ultradeep sequencing technology to detect single mutant molecules of fibroblast growth factor receptor 3 (FGFR3) DNA that are indicative of bladder cancer. Detection of FGFR3 mutations in urine would provide clinicians with a noninvasive means of diagnosing early-stage bladder cancer. The single-molecule assay detects FGFR3 mutant DNA when present at as low as 0.02% of total urine DNA and results in 91% concordance with the frequency that FGFR3 mutations are detected in bladder cancer tumors, significantly improving diagnostic performance. To our knowledge, this is the first practical application of next-generation sequencing technology for noninvasive cancer diagnostics.

Molecular grading of tumors of the upper urothelial tract using FGFR3 mutation status identifies patients with favorable prognosis.

BACKGROUND: Mutations in FGFR3 have been shown to occur in tumors of the upper urothelial tract and may be indicative of a good prognosis. In bladder tumors, the combination of FGFR3 mutation status and Ki-67 level has been used to define a tumor's molecular grade and predict survival. Pathological evaluation of upper urothelial tumors is currently the best predictor of prognosis, but suffers from variability in pathological assessments. This study investigated the association with prognosis of FGFR3 mutations alone and in combination with Ki-67 in this patient population. METHODS: Genomic DNA was isolated from tumor samples of 80 patients with upper urothelial cancer. The presence of mutation in FGFR3 was evaluated using real-time polymerase chain reaction. Ki-67 protein expression was determined by immunohistochemistry. Kaplan-Meier survival analysis evaluated the relationship of FGFR3 mutations and Ki-67 levels with survival. RESULTS: FGFR3 mutations were identified in 40% of tumors and were predominantly associated with noninvasive tumors. Overall survival was higher in patients with FGFR3 mutant tumors (P = 0.02) and in molecular grade 1 tumors as determined by FGFR3 and Ki-67 (P = 0.02). CONCLUSION: In this study, we confirm the occurrence of FGFR3 mutations in tumors of the upper urothelial tract and its association with a good prognosis. Both FGFR3 and molecular grading are predictors of overall survival. Molecular grading can help to assess the prognosis of patients with upper urinary tract cancer and may represent a new tool for managing this population of patients.

A noninvasive multi-analyte diagnostic assay: combining protein and DNA markers to stratify bladder cancer patients.

PURPOSE: The authors recently reported the development of a noninvasive diagnostic assay using urinary matrix metalloproteinases (MMPs) as monitors of disease-free status and bladder cancer in high-risk populations. Using an approach called clinical intervention determining diagnostic (CIDD), they identified with high confidence those patients who could be excluded from additional intervention. To maximize performance, MMPs were combined with DNA-based markers and CIDD was applied to a population of patients undergoing monitoring for recurrence. PATIENTS AND METHODS: Urine samples were obtained from 323 patients, 48 of whom had a recurrence and 275 of whom did not have cancer upon cytoscopic evaluation. Twist1 and Nid2 methylation status was determined using methylation-specific polymerase chain reaction, FGFR3 mutational status by quantitative PCR, and MMP levels by enzyme-linked immunosorbent assay. RESULTS: Using a combination of these DNA and protein markers, the authors identified with high confidence (97% negative predicted value) those patients who do not have cancer. Cutoffs were adjusted such that at 92% sensitivity, 51% of disease-free patients might be triaged from receiving further tests. CONCLUSION: The multi-analyte diagnostic readout assay described here is the first to combine protein and DNA biomarkers into one assay for optimal clinical performance. Using this approach, the detection of FGFR3 mutations and Twist1 and Nid2 methylation in the urine of patients undergoing bladder cancer recurrence screening increase the sensitivity and negative predictive value at an established MMP protein cutoff. This noninvasive urinary diagnostic assay could lead to the more efficient triage of patients undergoing recurrence monitoring.

Bone morphogenetic protein 1 processes prolactin to a 17-kDa antiangiogenic factor.

In addition to classical expression patterns in pituitary and placenta and functions in growth and reproduction, members of the small family of hormones that includes prolactin (PRL), growth hormone (GH), and placental lactogen are expressed by endothelia and have angiogenic effects. In contrast, 16- to 17-kDa proteolytic fragments of these hormones have antiangiogenic effects. Here we show that PRL and GH are bound and processed by members of the bone morphogenetic protein 1 (BMP1) subgroup of extracellular metalloproteinases, previously shown to play key roles in forming extracellular matrix and in activating certain TGFbeta superfamily members. BMP1 has previously been suggested to play roles in angiogenesis, as high throughput screens have found its mRNA to be one of those induced to highest levels in tumor-associated endothelia compared with resting endothelia. PRL and GH cleavage is shown to occur in each hormone at a single site typical of sites previously characterized in known substrates of BMP1-like proteinases, and the approximately 17-kDa PRL N-terminal fragment so produced is demonstrated to have potent antiangiogenic activity. Mouse embryo fibroblasts are shown to produce both PRL and GH and to process them to approximately 17-kDa forms, whereas GH and PRL processing activity is lost in mouse embryo fibroblasts doubly null for two genes encoding BMP1-like proteinases.

Modulation of angiogenesis by tissue inhibitor of metalloproteinase-4.

Despite the importance of MMP activity in the regulation of angiogenesis, relatively little is known about the role of TIMP-4, the most recently discovered endogenous MMP inhibitor, in modulating neovascularization. It has largely been assumed that all TIMPs are capable of inhibiting angiogenesis in vivo. However, it is now widely appreciated that TIMPs-1, -2, and -3 differ significantly in their ability to modulate angiogenic processes in vitro and angiogenesis in vivo. In order to study the effect of TIMP-4 in controlling angiogenesis, we have cloned and expressed TIMP-4 in a Pichia pastoris expression system, purified it to homogeneity, and tested its ability to regulate angiogenesis in vivo and in vitro. Our studies demonstrate that TIMP-4 is an inhibitor of capillary endothelial cell migration, but not of proliferation or of angiogenesis in vivo.

A role for antiangiogenic therapy in breast cancer.

The success of the angiogenesis inhibitor bevacizumab, the vascular endothelial growth factor antagonist that was recently shown to significantly improve the survival of patients with metastatic colon cancer when administered in combination with conventional chemotherapy, has provided proof of principle in clinical trials that antiangiogenesis can be an important strategy in the treatment of cancer. This report reviews the contemporary therapeutic approaches for breast cancer, the essential role that angiogenesis plays in the initiation and progression of this disease, and the strategies that should be considered to make antiangiogenic therapy a successful component of breast cancer treatment.

Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor.

Tissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression, and angiogenesis in a variety of experimental cancer models and in human malignancies. Results from numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, a series of structure-function studies were conducted to identify and to characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation as well as angiogenesis in vivo. We demonstrate that the COOH-terminal domain of TIMP-2 (T2C) inhibits the proliferation of capillary EC at molar concentrations comparable with those previously reported for intact TIMP-2, while the NH2-terminal domain (T2N), which inhibits MMP activity, has no significant anti-proliferative effect. Interestingly, although both T2N and T2C inhibited embryonic angiogenesis, only T2C resulted in the potent inhibition of angiogenesis driven by the exogenous addition of angiogenic mitogen, suggesting that MMP inhibition alone may not be sufficient to inhibit the aggressive neovascularization characteristic of aberrant angiogenesis. We further mapped the anti-proliferative activity of T2C to a 24-amino acid peptide corresponding to Loop 6 of TIMP-2 and show that Loop 6 is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other, the first represented by its MMP-dependent inhibitory activity which can inhibit only embryonic neovascularization and the second represented by an MMP-independent activity which inhibits both normal angiogenesis and mitogen-driven angiogenesis in vivo. In addition, we report, for the first time, the discovery of Loop 6 as a novel and potent inhibitor of angiogenesis.