An introduction to the Marburg virus vaccine consortium, MARVAC.

The emergence of Marburg virus (MARV) in Guinea and Ghana triggered the assembly of the MARV vaccine "MARVAC" consortium representing leaders in the field of vaccine research and development aiming to facilitate a rapid response to this infectious disease threat. Here, we discuss current progress, challenges, and future directions for MARV vaccines.

Pre- and Postlicensure Animal Efficacy Studies Comparing Anthrax Antitoxins.

BACKGROUND: The deliberate use of Bacillus anthracis spores is believed by the US government to be a high bioweapons threat. The first line of defense following potential exposure to B. anthracis spores would be postexposure prophylaxis with antimicrobials that have activity against B. anthracis. Additional therapies to address the effects of toxins may be needed in systemically ill individuals. Over the last 2 decades, the United States government (USG) collaborated with the private sector to develop, test, and stockpile 3 antitoxins: anthrax immunoglobulin intravenous (AIGIV), raxibacumab, and obiltoxaximab. All 3 products target protective antigen, a protein factor common to the 2 exotoxins released by B. anthracis, and hamper or block the toxins' effects and prevent or reduce pathogenesis. These antitoxins were approved for licensure by the United States Food and Drug Administration based on animal efficacy studies compared to placebo. METHODS: We describe USG-sponsored pre- and postlicensure studies that compared efficacy of 3 antitoxins in a New Zealand White rabbit model of inhalation anthrax; survival following a lethal aerosolized dose of B. anthracis spores was the key measure of effectiveness. To model therapeutic intervention, intravenous treatments were started following onset of antigenemia. RESULTS: In pre- and postlicensure studies, all 3 antitoxins were superior to placebo; in the postlicensure study, raxibacumab and obiltoxaximab were superior to AIGIV, but neither was superior to the other. CONCLUSIONS: These data illustrate the relative therapeutic benefit of the 3 antitoxins and provide a rationale to prioritize their deployment.

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation.

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a ß(-) emitter such as (90)Y with an optical imager.

The influence of vibrational excitation on the photoisomerization of trans-stilbene in solution.

Preparing electronically excited trans-stilbene molecules in deuterated chloroform using both one-photon excitation and excitation through an intermediate vibrational state explores the influence of vibrational energy on excited-state isomerization in solution. After infrared excitation of either two quanta of C-H stretch vibration |2ν(CH)> at 5990 cm(-1) or the C-H stretch-bend combination |ν(CH) + ν(bend)> at 4650 cm(-1) in the ground electronic state, an ultraviolet photon intercepts the vibrationally excited molecules during the course of vibrational energy flow and promotes them to the electronically excited state. The energy of the infrared and ultraviolet photons together is the same as that added in the one-photon excitation. Transient broadband-continuum absorption monitors the lifetime of electronically excited molecules. The lifetime of excited-state trans-stilbene after one-photon electronic excitation with 33,300 cm(-1) of energy is (51 +/- 6) ps. The excited-state lifetimes of (55 +/- 9) ps and (56 +/- 7) ps for the cases of excitation through |2ν(CH)> and |ν(CH) + ν(bend)>, respectively, are indistinguishable from that for the one-photon excitation. Vibrational relaxation in the electronically excited state prepared by the two-photon excitation scheme is most likely faster than the barrier crossing, making the isomerization insensitive to the method of initial state preparation.

Spectroscopic identification of higher-order rare gas-dihalogen complexes with different geometries: He(2,3)...Br(2) and He(2,3)...ICl.

Rovibronic transitions of multiple conformers of the He(2)...(79)Br(2)(X, v'' = 0), He(3)...(79)Br(2)(X, v'' = 0), He(2)...I(35)Cl(X, v'' = 0), and He(3)...I(35)Cl(X, v'' = 0) complexes stabilized in a pulsed, supersonic expansion are observed in action spectra recorded in the B-X region of the dihalogens. In addition to features associated with He(2)...(79)Br(2) and He(2)...I(35)Cl complexes with the rare gas atoms localized in the toroidal potential well lying in a plane perpendicular to the dihalogen bond, those associated with a ground-state conformer that has one He atom localized in the toroidal potential and the other He atom localized in the linear well at the end of the dihalogen moiety are also identified. Transitions of at least three conformers of the He(3)...Br(2) complex and two conformers of the He(3)...ICl complex are also observed. The relative populations of the different conformers are found to depend on where along the supersonic expansion the spectra are recorded, and thus on the local temperature regime sampled. The He(2)...(79)Br(2) and He(2)...I(35)Cl conformers with one He atom in each well are found to be the more stable conformers.

ImmunoPET Imaging of αvß6 Expression Using an Engineered Anti-αvß6 Cys-diabody Site-Specifically Radiolabeled with Cu-64: Considerations for Optimal Imaging with Antibody Fragments.

PURPOSE: Increased expression of the αvß6 integrin correlates with advanced tumor grade and poor clinical outcome, identifying αvß6 as a prognostic indicator and an attractive target for molecular imaging. This work investigated the ability of a disulfide-stabilized [64Cu]NOTA-αvß6 cys-diabody to image αvß6 expression in vivo using a nu/nu mouse model bearing human melanoma xenografts and positron-emission tomography. PROCEDURES: Small-animal positron emission tomography (PET) imaging, quantitative ROI analysis, and ex vivo biodistribution were conducted to ascertain tumor uptake and organ distribution of the [64Cu]NOTA-αvß6 cys-diabody. Immunohistochemical staining of tumors and mouse organs and immunoreactivity assays were utilized to correlate in vivo and ex vivo observations. RESULTS: PET imaging of the [64Cu]NOTA-αvß6 cys-diabody revealed low tumor uptake at 24 h p.i. in DX3Puroß6 tumors (2.69 ± 0.45 %ID/g) with comparable results found in the DX3Puro tumors (2.24 ± 0.15 %ID/g). Quantitative biodistribution confirmed that DX3Puroß6 tumor uptake was highest at 24 h p.i. (4.63 ± 0.18 %ID/g); however, uptake was also observed in the stomach (4.84 ± 2.99 %ID/g), small intestines (4.50 ± 1.69 %ID/g), large intestines (4.73 ± 0.97 %ID/g), gallbladder (6.04 ± 1.88 %ID/g), and lungs (3.89 ± 0.69 %ID/g). CONCLUSIONS: Small-animal PET imaging was successful in visualizing αvß6-positive tumor uptake of the [64Cu]NOTA-αvß6 cys-diabody. Cys-diabody cross-reactivity was observed between human and murine αvß6 and immunohistochemical staining confirmed the presence of an endogenous αvß6 antigen sink, which led to suboptimal tumor contrast in this mouse model. Future investigations will focus on dose escalation studies to overcome the endogenous antigen sink while increasing DX3Puroß6 tumor uptake.

A Functional Approach to Solubility Parameter Computations.

The determination of solubility parameters for solutes represents a challenging mathematical problem of locating the central tendency of solvent affinity based on a limited set of data taken from experimental observations. At present, the most commonly used methods for computing solubility parameters of a solute require a binary classification of solvent affinity for the solute and employ a spherical/ellipsoidal compatibility region in the three-dimensional Hansen solubility parameter space. Utilizing a binary classification requires an arbitrary solubility threshold, and an ellipsoidal fitting model imposes a symmetry on the intermolecular forces that is rarely reflected by the experimental data. To overcome these issues, an approach that makes use of accurate solubility data to describe a three-dimensional solubility function, f, is introduced. The principles of the approach are discussed in detail and the procedures for constructing the solubility function and computing solubility parameters are described. An example using PCBM solubility data available in the literature demonstrates the new method. Lastly, a method that employs f as a predictor of solubility in arbitrary solvents with a proposed measure of reliability is presented.

Characterization of a novel androgen receptor mutation in a relapsed CWR22 prostate cancer xenograft and cell line.

CWR22 has been a valuable xenograft model for the study of prostate cancer progression from an androgen-dependent tumor to one that grows in castrated animals. Herein, we report the identification and characterization of a novel androgen receptor (AR) mutation occurring in a relapsed tumor (CWR22R-2152) and in the CWR22Rv1 cell line established from it. The mutation was not detected in the original, hormone-dependent CWR22 xenograft, indicating that this change occurred during the progression to androgen independence. It is characterized by an in-frame tandem duplication of exon 3 that encodes the second zinc finger of the AR DNA-binding domain. Accordingly, immunoblot analyses demonstrated the expression of an AR species having an approximately 5-kDa increase in size relative to the LNCaP AR. This was accompanied by a COOH-terminally truncated AR species migrating with a relative mass of 75-80 kDa, referred to as ARDeltaLBD because it lacks the ligand-binding domain. By recreating the exon 3 duplication mutation in a wild-type AR expression construct, the generation of ARDeltaLBD could be recapitulated. Whereas ARDeltaLBD exhibited constitutive nuclear localization and DNA binding, these functions in the full-length AR remained androgen dependent. The CWR22Rv1 AR repertoire displayed dose-dependent, androgen-responsive transcriptional transactivation in reporter assays, albeit to a lesser extent in comparison with LNCaP. This cell line also expressed low levels of prostate-specific antigen mRNA and did not express or secrete detectable levels of prostate-specific antigen protein in androgen-depleted medium or in response to physiological androgenic stimulation. In summary, the CWR22Rv1 cell line displays both androgen-responsive and androgen-insensitive features due, at least in part, to a novel insertional mutation of the AR.

Solubility Characteristics of PCBM and C60.

Empirical data indicate that several good solvents for C60 and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) have substantial polar and hydrogen-bonding components, which are not intrinsic to the structure of the C60 and PCBM molecules themselves. Functional solubility parameter (FSP) and convex solubility parameter (CSP) computations are performed on C60 and PCBM using solubility data available in the literature. The CSP and FSP results are compared to previously reported Hansen solubility parameters (HSPs) and to the parameters calculated using additive functional group contribution methods. The CSP and FSP methods confirm the anomalously large polar and hydrogen-bonding parameters, δP and δH, obtained experimentally for C60 and PCBM. This behavior, which is quite irregular given the structure of the molecules, is due to the fact that several good solvents have high δP and δH values. Thus, these irregularities are highlighted by the CSP and FSP calculations. Additional contradictory solubility characteristics are disclosed by comparing the experimental solubility parameters to a linear solvation energy relationship (LSER) model, additive functional group calculations, and COSMO-RS computations. The FSP solubility function strongly suggests that the solubility parameters do not accurately represent the cohesive energy density properties of C60 and PCBM, as intended, but rather they manifest the properties of the solvents, e.g., high δP and δH values, that are necessary to accommodate these molecules in the liquid phase.

A Prospective Evaluation of Circulating Tumor Cells and Cell-Free DNA in EGFR-Mutant Non-Small Cell Lung Cancer Patients Treated with Erlotinib on a Phase II Trial.

PURPOSE: Genotype-directed therapy is the standard of care for advanced non-small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression. EXPERIMENTAL DESIGN: EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression. RESULTS: Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival. CONCLUSIONS: cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010-20. ©2016 AACR.

Development and characterization of an αvß6-specific diabody and a disulfide-stabilized αvß6-specific cys-diabody.

INTRODUCTION: This work describes the development and characterization of two antibody fragments that specifically target the α(v)ß(6) integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound α(v)ß(6). Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([(64)Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [(18)F]-FB-α(v)ß(6) diabody, [(18)F]-FB-α(v)ß(6) cys-diabody, and the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. METHODS: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-α(v)ß(6) intact antibody. The anti-α(v(ß(6) cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to α(v)ß(6). The diabodies were radiolabeled with [(18)F]-SFB and in addition, the anti-α(v)ß(6) cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [(64)Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroß(6) (α(v)ß(6)+) and DX3Puro (α(v)ß(6)-)cell lines. RESULTS: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvß6 was confirmed by ELISA (diabody IC(50)=0.8 nM, cys-diabody IC(50)=0.6 nM) and flow cytometry revealed high specificity only to the DX3Puroß(6) cell line for both diabodies. RCYs were 22.6%±3.6% for the [(18)F]-FB-α(v)ß(6) diabody, 8.3%±1.7% for the [(18)F]-FB-α(v)ß(6) cys-diabody and 43.5%±5.5% for the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([(18)F]-FB-α(v)ß(6) diabody=58.7%±6.7%, [(18)F]-FB-α(v)ß(6) cys-diabody=80.4%±4.4%, [(64)Cu]-NOTA-α(v)ß(6) cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. CONCLUSIONS: Two novel diabodies with excellent binding affinity and specificity for the α(v)ß(6) integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([(18)F]) and [(64)Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.

Comparison of blood gas and electrolyte test results from the Gem-Premier and the ABL-70 versus a conventional laboratory analyzer.

Blood gas analyzers serve a critical role in providing information that reflects patient homeostasis. This study was undertaken to evaluate the accuracy, reliability, consistency, and bias of the Radiometer ABL-70 point of care blood gas analyzer. Thirty samples were gathered for analysis of pH, pCO2, pO2, sodium, potassium, sodium bicarbonate, and base excess. Twenty-nine samples were gathered for hematocrit, 31 for ionized calcium, and 33 for venous pO2 and saturations. The data were compared with the Gem-Premier point of care analyzer and the hospital blood gas machine and electrolyte analyzer. There was statistical significance between the pH, pCO2, sodium, potassium, calcium, hematocrit, and venous pO2 and saturations when comparing the ABL-70 with the Gem-Premier. When comparing the ABL-70 with the Corning 278/270 blood gas machine/Co-Ox, the AVL-9180, and the Dimension XL, there was statistical significance seen between the pH, pCO2, pO2, sodium, calcium, hematocrit, and base excess. Although this statistical significance was observed between the ABL-70 and the other analyzers, the significance was not of clinical importance. The ABL-70 demonstrated acceptable accuracy, reliability, consistency, and bias.

Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer.

INTRODUCTION: Identification of some somatic molecular alterations in non-small-cell lung cancer (NSCLC) has become evidence-based practice. The success and failure rate of using commercially available tumor genotyping techniques in routine day-to-day NSCLC pathology samples is not well described. We sought to evaluate the success and failure rate of EGFR mutation, KRAS mutation, and ALK FISH in a cohort of lung cancers subjected to routine clinical tumor genotype. METHODS: Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 381 patient-tumor samples. RESULTS: From these 381 patients with lung cancer, the mean age was 65 years, 61.2% were women, 75.9% were white, 27.8% were never smokers, 73.8% had advanced NSCLC and 86.1% had adenocarcinoma histology. The tumor tissue was obtained from surgical specimens in 48.8%, core needle biopsies in 17.9%, and as cell blocks from aspirates or fluid in 33.3% of cases. Anatomic sites for tissue collection included lung (49.3%), lymph nodes (22.3%), pleura (11.8%), bone (6.0%), brain (6.0%), among others. The overall success rate for EGFR mutation analysis was 94.2%, for KRAS mutation 91.6% and for ALK FISH 91.6%. The highest failure rates were observed when the tissue was obtained from image-guided percutaneous transthoracic core-needle biopsies (31.8%, 27.3%, and 35.3% for EGFR, KRAS, and ALK tests, respectively) and bone specimens (23.1%, 15.4%, and 23.1%, respectively). In specimens obtained from bone, the failure rates were significantly higher for biopsies than resection specimens (40% vs. 0%, p=0.024 for EGFR) and for decalcified compared to non-decalcified samples (60% vs. 5.5%, p=0.021 for EGFR). CONCLUSIONS: Tumor genotype techniques are feasible in most samples, outside small image-guided percutaneous transthoracic core-needle biopsies and bone samples from core biopsies with decalcification, and therefore expansion of routine tumor genotype into the care of patients with NSCLC may not require special tissue acquisition or manipulation.

Adequacy of lymph node transbronchial needle aspirates using convex probe endobronchial ultrasound for multiple tumor genotyping techniques in non-small-cell lung cancer.

INTRODUCTION: Adequate tumor acquisition is essential to identify somatic molecular alterations in non-small-cell lung cancer (NSCLC), such as epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations. The success and failure rates for tumor genotyping of tissue obtained from fine-needle aspirates of nodal tissue using a convex probe endobronchial ultrasound (CP-EBUS) and other diagnostic modalities in routine NSCLC care have not been described. METHODS: Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 207 patient-tumor samples sent for routine tumor genotype in clinical practice, including 42 patient-tumor samples obtained from hilar or mediastinal lymph nodes using CP-EBUS. RESULTS: The median age of the patients was 65 years, 62.3% were women, 77.8% were white, 26.6% were never smokers, 73.9% had advanced NSCLC, and 84.1% had adenocarcinoma histology. Tumor tissue was obtained from CP-EBUS-derived hilar or mediastinal nodes in 42 cases (20.2% of total). In this latter cohort, the overall success rate for EGFR mutation analysis was 95.2%, for Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation 90.5%, and for ALK fluorescence in situ hybridization 90.5%. In the complete 207 tumors, the success rate for EGFR was 92.3%, for KRAS 91.8%, and for ALK 89.9%. The failure rates were not significantly different when comparing CP-EBUS-derived nodal tissue versus all other samples or versus surgical biopsies of mediastinal nodes, but were significantly lower than image-guided percutaneous transthoracic core-needle biopsies. CONCLUSIONS: The success rate of multiple tumor genomic analyses techniques for EGFR, KRAS, and ALK gene abnormalities using routine lung cancer tissue samples obtained from hilar or mediastinal lymph nodes by means of CP-EBUS exceeds 90%, and this method of tissue acquisition is not inferior to other specimen types. Tumor genotype techniques are feasible in most CP-EBUS-derived samples and therefore further expansion of routine tumor genotype for the care of patients with NSCLC may be possible using targeted sample acquisition through CP-EBUS.

Smoking status and self-reported race affect the frequency of clinically relevant oncogenic alterations in non-small-cell lung cancers at a United States-based academic medical practice.

INTRODUCTION: The identification of somatic genomic aberrations in non-small-cell lung cancer (NSCLC) is part of evidence-based practice guidelines for care of patients with NSCLC. We sought to establish the frequency and correlates with these changes in routine patient-tumor sample pairs. METHODS: Clinicopathologic data and tumor genotype were retrospectively compiled and analyzed from an overall cohort of 381 patient-tumor samples. RESULTS: Of these patients, 75.9% self-reported White race, 13.1% Asian, 6.5% Black, 27.8% were never-smokers, 54.9% former-smokers and 17.3% current-smokers. The frequency of EGFR mutations was 23.9% (86/359), KRAS mutations 34.2% (71/207) and ALK FISH positivity 9.1% (23/252) in tumor samples, and almost all had mutually exclusive results for these oncogenes. In tumors from White, Black and Asian patients, the frequencies of EGFR mutations were 18.4%, 18.2% and 62%, respectively; of ALK FISH positivity 7.81%, 0% and 14.8%, respectively; and of KRAS mutations 41.6%, 20% and 0%. These patterns changed significant with increasing pack-year history of smoking. In White patients, the frequencies of EGFR mutations and ALK FISH positivity decreased with increasing pack-year cohorts; while the frequencies of KRAS mutations increased. Interestingly, in Asian patients the frequencies of EGFR mutations were similar in never smokers and in the cohorts with less than 45pack-year histories of smoking and only decreased in the 45pack-year plus cohort. CONCLUSIONS: The frequencies of somatic EGFR, KRAS, and ALK gene abnormalities using routine lung cancer tissue samples from our United States-based academic medical practice reflect the diverse ethnicity (with a higher frequency of EGFR mutations in Asian patients) and smoking patterns (with an inverse correlation between EGFR mutation and ALK rearrangement) of our tested population. These results may help other medical practices appreciate the expected results from introduction of routine tumor genotyping techniques into their day-to-day care of NSCLC.

Modeling truncated AR expression in a natural androgen responsive environment and identification of RHOB as a direct transcriptional target.

Recent studies identifying putative truncated androgen receptor isoforms with ligand-independent activity have shed new light on the acquisition of androgen depletion independent (ADI) growth of prostate cancer. In this study, we present a model system in which a C-terminally truncated variant of androgen receptor (TC-AR) is inducibly expressed in LNCaP, an androgen-dependent cell line, which expresses little truncated receptor. We observed that when TC-AR is overexpressed, the endogenous full length receptor (FL-AR) is transcriptionally downmodulated. This in essence allows us to "replace" FL-AR with TC-AR and compare their individual properties in exactly the same genetic and cellular background, which has not been performed before. We show that the TC-AR translocates to the nucleus, activates transcription of AR target genes in the absence of DHT and is sufficient to confer ADI growth to the normally androgen dependent LNCaP line. We also show that while there is significant overlap in the genes regulated by FL- and TC-AR there are also differences in the respective suites of target genes with each AR form regulating genes that the other does not. Among the genes uniquely activated by TC-AR is RHOB which is shown to be involved in the increased migration and morphological changes observed in LN/TC-AR, suggesting a role of RHOB in the regulation of androgen-independent behavior of prostate cancer cells.

Establishment of clonal MIN-O transplant lines for molecular imaging via lentiviral transduction & in vitro culture.

As the field of molecular imaging evolves and increasingly is asked to fill the discovery and validation space between basic science and clinical applications, careful consideration should be given to the models in which studies are conducted. The MIN-O mouse model series is an established in vivo model of human mammary precancer ductal carcinoma in situ with progression to invasive carcinoma. This series of transplant lines is propagated in vivo and experiments utilizing this model can be completed in non-engineered immune intact FVB/n wild type mice thereby modeling the tumor microenvironment with biological relevance superior to traditional tumor cell xenografts. Unfortunately, the same qualities that make this and many other transplant lines more biologically relevant than standard cell lines for molecular imaging studies present a significant obstacle as somatic genetic re-engineering modifications common to many imaging applications can be technically challenging. Here, we describe a protocol for the efficient lentiviral transduction of cell slurries derived from precancerous MIN-O lesions, in vitro culture of "MIN-O-spheres" derived from single cell clones, and the subsequent transplantation of these spheres to produce transduced sublines suitable for optical imaging applications. These lines retain the physiologic and pathologic properties, including multilineage differentiation, and complex microanatomic interaction with the host stroma characteristic of the MIN-O model. We also present the in vivo imaging and immunohistochemical analysis of serial transplantation of one such subline and detail the progressive multifocal loss of the transgene in successive generations.