Imaging Tumor Vascularity and Response to Anti-Angiogenic Therapy Using Gaussia Luciferase

We developed a novel approach to assess tumor vascularity using recombinant Gaussia luciferase (rGluc) protein and bioluminescence imaging. Upon intravenous injection of rGluc followed by its substrate coelenterazine, non-invasive visualization of tumor vascularity by bioluminescence imaging was possible. We applied this method for longitudinal monitoring of tumor vascularity in response to the anti-angiogenic drug tivozanib. This simple and sensitive method could be extended to image blood vessels/vasculature in many different fields

Directed Molecular Evolution Reveals Gaussia Luciferase Variants with Enhanced Light Output Stability

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics. Several variants with a 10–15 nm shift in their light emission peak were found. Further, a Gluc variant that catalyzes a glow-type bioluminescence reaction, suited for high-throughput applications, was also identified. These results indicate that molecular evolution could be used to modulate Gluc bioluminescence reaction characteristics

Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models

Guidelines of care for the management of primary cutaneous melanoma

The incidence of primary cutaneous melanoma continues to increase each year. Melanoma accounts for the majority of skin cancer–related deaths, but treatment is usually curative following early detection of disease. In this American Academy of Dermatology clinical practice guideline, updated treatment recommendations are provided for patients with primary cutaneous melanoma (American Joint Committee on Cancer stages 0-IIC and pathologic stage III by virtue of a positive sentinel lymph node biopsy). Biopsy techniques for a lesion that is clinically suggestive of melanoma are reviewed, as are recommendations for the histopathologic interpretation of cutaneous melanoma. The use of laboratory, molecular, and imaging tests is examined in the initial work-up of patients with newly diagnosed melanoma and for follow-up of asymptomatic patients. With regard to treatment of primary cutaneous melanoma, recommendations for surgical margins and the concepts of staged excision (including Mohs micrographic surgery) and nonsurgical treatments for melanoma in situ, lentigo maligna type (including topical imiquimod and radiation therapy), are updated. The role of sentinel lymph node biopsy as a staging technique for cutaneous melanoma is described, with recommendations for its use in clinical practice. Finally, current data regarding pregnancy and melanoma, genetic testing for familial melanoma, and management of dermatologic toxicities related to novel targeted agents and immunotherapies for patients with advanced disease are summarized

Effects of the selective MPS1 inhibitor MPS1-IN-3 on glioblastoma sensitivity to antimitotic drugs

Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest. We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3-7 mice/group). MPS1 expression levels were examined in relation to patient survival. Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity. Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drug

Selection criteria for genetic assessment of patients with familial melanoma

Approximately 5% to 10% of melanoma may be hereditary in nature, and about 2% of melanoma can be specifically attributed to pathogenic germline mutations in cyclin-dependent kinase inhibitor 2A (&lt;i&gt;CDKN2A&lt;/i&gt;). To appropriately identify the small proportion of patients who benefit most from referral to a genetics specialist for consideration of genetic testing for &lt;i&gt;CDKN2A&lt;/i&gt;, we have reviewed available published studies of &lt;i&gt;CDKN2A&lt;/i&gt; mutation analysis in cohorts with invasive, cutaneous melanoma and found variability in the rate of &lt;i&gt;CDKN2A&lt;/i&gt; mutations based on geography, ethnicity, and the type of study and eligibility criteria used. Except in regions of high melanoma incidence, such as Australia, we found higher rates of &lt;i&gt;CDKN2A&lt;/i&gt; positivity in individuals with 3 or more primary invasive melanomas and/or families with at least one invasive melanoma and two or more other diagnoses of invasive melanoma and/or pancreatic cancer among first- or second-degree relatives on the same side of the family. The work summarized in this review should help identify individuals who are appropriate candidates for referral for genetic consultation and possible testing