Gene expression profiling for metastatic risk in head and neck cutaneous squamous cell carcinoma.

OBJECTIVE: Over 50% of newly diagnosed cutaneous squamous cell carcinoma (cSCC) lesions occur in the head and neck (cSCC-HN), and metastasis to nodal basins in this region further complicates surgical and adjuvant treatment. The current study addressed whether the 40-gene expression profile (40-GEP) test can predict metastatic risk in cSCC-HN with improved accuracy and provide independent prognostic value to complement current risk assessment methods. STUDY DESIGN: Multicenter, retrospective cohort study. METHODS: Formalin-fixed paraffin-embedded primary tumor tissue and associated clinical data from patients with cSCC-HN (n = 278) were collected from 33 independent centers. Samples were analyzed via the 40-GEP test. Cases were staged per American Joint Committee on Cancer, Eighth Edition (AJCC8) and Brigham and Women's Hospital (BWH) criteria after comprehensive medical record and pathology report review. Metastasis-free survival (MFS) rates were determined, and risk factors were analyzed via Cox regression. RESULTS: The 40-GEP test classified the cohort into low (Class 1, n = 126; 45.3%), moderate (Class 2A, n = 134; 48.2%), and high (Class 2B, n = 18; 6.5%) metastatic risk at 3 years postdiagnosis. Regional/distant metastasis occurred in 54 patients (19.4%). MFS rates were 92.1% (Class 1), 76.1% (Class 2A), and 44.4% (Class 2B; p < .0001). Multivariate analysis of 40-GEP results with AJCC8 or BWH tumor stage, or clinicopathologic risk factors, demonstrated independent prognostic value of the 40-GEP test (p < .03). Accuracy of predicting metastatic risk was also improved using 40-GEP classification (p < .02). CONCLUSIONS: Improved metastatic risk stratification through the 40-GEP test could complement cSCC-HN risk assessment for better-informed decision-making for treatment and surveillance and ultimately improve patient outcomes. LEVEL OF EVIDENCE: 3.

Enhanced metastatic risk assessment in cutaneous squamous cell carcinoma with the 40-gene expression profile test.

Aim: To clinically validate the 40-gene expression profile (40-GEP) test for cutaneous squamous cell carcinoma patients and evaluate coupling the test with individual clinicopathologic risk factor-based assessment methods. Patients & methods: In a 33-site study, primary tumors with known patient outcomes were assessed under clinical testing conditions (n = 420). The 40-GEP results were integrated with clinicopathologic risk factors. Kaplan-Meier and Cox regression analyses were performed for metastasis. Results: The 40-GEP test demonstrated significant prognostic value. Risk classification was improved via integration of 40-GEP results with clinicopathologic risk factor-based assessment, with metastasis rates near the general cutaneous squamous cell carcinoma population for class 1 and ≥50% for class 2B. Conclusion: Combining molecular profiling with clinicopathologic risk factor assessment enhances stratification of cutaneous squamous cell carcinoma patients and may inform decision-making for risk-appropriate management strategies.

Plain language summary Cutaneous squamous cell carcinoma is a common skin cancer, with approximately 2 million cases diagnosed each year in the USA. Because substantial numbers of patients experience metastasis, which can result in death, accurate metastatic risk assessment is important. Clinicians use clinicopathologic factors to determine risk for disease progression. However, traditional methods miss pinpointing many patients who experience metastasis and sometimes categorize patients as at risk who do not develop metastasis, indicating that additional tools are needed. A molecular test, the 40-gene expression profile (40-GEP), was developed to predict metastatic risk based on the biology of the tumor. This study demonstrates that the 40-GEP, either as an independent tool or together with traditional methods, accurately identifies patients' risk of metastasis. Using the 40-GEP could improve patient management to improve patient outcomes.

Adjuvant therapy for high-risk cutaneous squamous cell carcinoma: 10-year review.

Standard of care for high-risk cutaneous squamous cell carcinoma (cSCC) is surgical excision of the primary lesion with clear margins when possible, and additional resection of positive margins when feasible. Even with negative margins, certain high-risk factors warrant consideration of adjuvant therapy. However, which patients might benefit from adjuvant therapy is unclear, and supporting evidence is conflicting and limited to mostly small retrospective cohorts. Here, we review literature from the last decade regarding adjuvant radiation therapy and systemic therapy in high-risk cSCC, including recent and current trials and the role of immune checkpoint inhibitors. We demonstrate evidence gaps in adjuvant therapy for high-risk cSCC and the need for prognostic tools, such as gene expression profiling, to guide patient selection. More large-cohort clinical studies are needed for collecting high-quality, evidence-based data for determining which patients with high-risk cSCC may benefit from adjuvant therapy and which therapy is most appropriate for patient management.

Integrating gene expression profiling into NCCN high-risk cutaneous squamous cell carcinoma management recommendations: impact on patient management.

Objective: To integrate gene expression profiling into the management of high-risk cutaneous squamous cell carcinoma (cSCC) within the National Comprehensive Cancer Network (NCCN) guidelines to improve risk-aligned management recommendations.Methods: A cohort of 300 NCCN-defined high-risk cSCC patients, along with the American Joint Committee on Cancer (AJCC) T stage, Brigham and Women's Hospital (BWH) T stage, and known patient outcomes were analyzed. Risk classifications using a validated 40-gene expression profile (40-GEP) test and T stage were applied to NCCN patient management guidelines. Risk-directed patient management recommendations within the NCCN guidelines framework were aligned based on risk for metastasis.Results: Of the 300 NCCN high-risk cSCC patients, 159 (53.0%) were 40-GEP Class 1 and AJCC T1-T2, and 173 (57.7%) were Class 1 and BWH T1-2a, indicating low risk for metastasis and, thereby, suggesting low management intensity. The 40-GEP integration suggested high intensity management for only 24 (8.0%) patients (all Class 2B), and moderate intensity management for the remainder of the cohort.Conclusions: The 40-GEP test can be integrated within existing NCCN guideline recommendations for managing cSCC patients to help refine risk-directed management decisions. Integration of the 40-GEP test would allow >50% of this NCCN-defined high-risk cohort to be managed with the lowest intensity recommendations within the broad NCCN guidelines. High intensity management was deemed risk-appropriate for a small subpopulation (8.0%). This study demonstrates that the 40-GEP test, in combination with T stage, has clinical utility to impact patient management decisions in NCCN high-risk cSCC for improving risk-aligned management within the NCCN guidelines framework.

CRISPR/Cas9-Based Dystrophin Restoration Reveals a Novel Role for Dystrophin in Bioenergetics and Stress Resistance of Muscle Progenitors.

Although the lack of dystrophin expression in muscle myofibers is the central cause of Duchenne muscular dystrophy (DMD), accumulating evidence suggests that DMD may also be a stem cell disease. Recent studies have revealed dystrophin expression in satellite cells and demonstrated that dystrophin deficiency is directly related to abnormalities in satellite cell polarity, asymmetric division, and epigenetic regulation, thus contributing to the manifestation of the DMD phenotype. Although metabolic and mitochondrial dysfunctions have also been associated with the DMD pathophysiology profile, interestingly, the role of dystrophin with respect to stem cells dysfunction has not been elucidated. In the past few years, editing of the gene that encodes dystrophin has emerged as a promising therapeutic approach for DMD, although the effects of dystrophin restoration in stem cells have not been addressed. Herein, we describe our use of a clustered regularly interspaced short palindromic repeats/Cas9-based system to correct the dystrophin mutation in dystrophic (mdx) muscle progenitor cells (MPCs) and show that the expression of dystrophin significantly improved cellular properties of the mdx MPCs in vitro. Our findings reveal that dystrophin-restored mdx MPCs demonstrated improvements in cell proliferation, differentiation, bioenergetics, and resistance to oxidative and endoplasmic reticulum stress. Furthermore, our in vivo studies demonstrated improved transplantation efficiency of the corrected MPCs in the muscles of mdx mice. Our results indicate that changes in cellular energetics and stress resistance via dystrophin restoration enhance muscle progenitor cell function, further validating that dystrophin plays a role in stem cell function and demonstrating the potential for new therapeutic approaches for DMD. Stem Cells 2019;37:1615-1628.

Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration.

BACKGROUND: Human muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study. METHODS: We isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells' age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA). RESULTS: We found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1. CONCLUSION: hMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.

Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

Detection of lymphangiogenesis by near-infrared fluorescence imaging and responses to VEGF-C during healing in a mouse full-dermis thickness wound model.

Noninvasive, longitudinal near-infrared fluorescence (NIRF) imaging was used to detect and quantify lymphangiogenesis following a full-dermis thickness incision in the presence and absence of locally administered vascular endothelial growth factor-C (VEGF-C), a well-known regulator of lymphangiogenesis. Peripheral cytokines/chemokines were also measured in treated and sham-injected animals. Lymphangiogenesis was detected via NIRF imaging by day 7-8 and confirmed by intravital microscopy, while angiogenesis was observed by day 2-3 postincision (PI). All lymph vessel parameters quantified were significantly greater on wounded vs. nonwounded sides of mice. Lymph vessel parameters appeared larger on wounded sides of VEGF-C- relative to NaCl-treated mice, although differences were not significant. Interleukin-1α and interleukin-22 were significantly elevated at day 7 PI relative to respective preincision levels in VEGF-C-treated mice, and decreased by day 21 PI to levels nearing those measured preincision. For the majority of cytokines/chemokines measured, mean responses were significantly greater in VEGF-C- vs. NaCl-treated animals. Local VEGF-C administration may stimulate lymphangiogenesis during tissue repair and regeneration via mediating systemic cytokine/chemokine levels. NIRF imaging can be utilized to detect lymphangiogenesis during wound healing, and offers a promising platform to complement current methods for monitoring wound status and studying the effects of growth factors on healing.

Tumor margin detection using quantitative NIRF molecular imaging targeting EpCAM validated by far red gene reporter iRFP.

PURPOSE: Wide-field surgical excision reduces the chance of residual disease, but can also lead to disfigurement and devastating morbidities when resection is close to critical structures. We hypothesize that near-infrared fluorescence (NIRF) imaging can enable accurate detection of tumor margins for image-guided resection. EXPERIMENTAL DESIGN: An orthotopic model of human prostate cancer (PCa) was used to assess primary tumor margins using a NIRF-labeled antibody against epithelial cell adhesion molecule (EpCAM). PCa cells stably expressing far red fluorescent gene reporter, iRFP, enabled colocalization with NIRF signals for direct assessment of tumor margins. RESULTS: Using receiver operating characteristic analysis, far red fluorescence was validated against standard pathology of primary and metastatic lesions with >96 % accuracy. Primary tumor margins were more accurately detected by quantitative NIRF imaging using the EpCAM-targeting antibody as compared to a NIRF-labeled isotype control antibody. CONCLUSIONS: NIRF molecular imaging may enable real-time and accurate assessment of tumor margins.

Non-invasive optical imaging of the lymphatic vasculature of a mouse.

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging using indocyanine green (ICG), a dye that has been used for 50 years in humans, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 µl) of ICG (645 µM) or cABD-IRDye800 (200 µM) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 µg/kg for ICG or 113-1,700 µg/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Multimodal chelation platform for near-infrared fluorescence/nuclear imaging.

Dual-labeled compounds containing nuclear and near-infrared fluorescence contrast have the potential to molecularly guide surgical resection of cancer by extending whole-body diagnostic imaging findings into the surgical suite. To simplify the dual labeling process for antibody-based agents, we designed a multimodality chelation (MMC) scaffold which combined a radiometal chelating agent and fluorescent dye into a single moiety. Three dye-derivatized MMC compounds were synthesized and radiolabeled. The IRDye 800CW conjugate, 4, had favorable optical properties and showed rapid clearance in vivo. Using 4, an epithelial cell adhesion molecule (EpCAM) targeting MMC-immunoconjugate was prepared and dual-labeled with (64)Cu. In vitro binding activity was confirmed after MMC conjugation. Multimodal imaging studies showed higher tumor accumulation of (64)Cu-7 compared to nontargeted (64)Cu-4 in a prostate cancer model. Further evaluation in different EpCAM-expressing cell lines is warranted as well as application of the MMC dual labeling approach with other monoclonal antibodies.

Comparison of mAbs targeting epithelial cell adhesion molecule for the detection of prostate cancer lymph node metastases with multimodal contrast agents: quantitative small-animal PET/CT and NIRF.

UNLABELLED: The proliferation of most carcinomas is associated with an overexpression of epithelial cell adhesion molecule (EpCAM), a 40-kDa type I transmembrane protein found on epithelial cells yet absent from other cell types. The absence of EpCAM in normal lymphatics makes it an attractive marker for studying lymph node (LN) metastases of carcinomas to improve LN staging accuracy. Herein, we developed and quantitatively compared dual-labeled monoclonal antibodies (mAbs) of varying affinities against EpCAM for both noninvasive and intraoperative detection of metastatic LNs in prostate cancer. METHODS: A panel of hybridoma-derived anti-EpCAM mAbs was generated and screened. Two high-affinity candidate mAbs with specificity for nonoverlapping epitopes on the EpCAM extracellular domain were chosen for further evaluation. After conjugation with DOTA for (64)Cu radiolabeling and IRDye 800CW as a fluorophore, dual-labeled specific or isotype control mAb was administered intravenously to male nu/nu mice at 10-12 wk after orthotopic implantation of DsRed-expressing PC3 cells. Within 18-24 h, noninvasive small-animal PET/CT and in vivo, in situ, and ex vivo DsRed reporter gene and near-infrared fluorescence (NIRF) imaging were performed to detect primary tumors and metastatic LNs. Using DsRed fluorescence as the true indicator of cancer-positive tissue, we performed receiver operating characteristic curve analyses of percentage injected dose per gram measured from quantitative small-animal PET/CT and fluorescence intensity measured from semiquantitative NIRF imaging for each LN examined to compare mAb sensitivity and specificity. RESULTS: mAbs 7 and 153 generated in-house were found to have higher affinity than commercial mAb 9601. Accuracy, as a function of sensitivity and specificity, for the detection of cancer-positive LNs during in vivo small-animal PET/CT was highest for mAbs 7 (87.0%) and 153 (78.0%) and significantly greater (P < 0.001) than random chance (50.0%). Rates for mAb 9601 (60.7%) and control mAb 69 (27.0%) were not significantly different from chance. Similarly, mAb 7 had significant detection accuracy by NIRF imaging (96.0%, P < 0.001). CONCLUSION: mAbs 7 and 153 are attractive, high-affinity candidates for further multimodal imaging agent optimization aimed at enhancing sensitivity and specificity for detection of metastatic LNs in prostate cancer. Fully quantitative NIRF imaging is needed for comprehensive analyses of NIRF-labeled agent accuracy for intraoperative guidance.

Quantifying multimodal contrast agent biological activity using near-infrared flow cytometry.

Prior to imaging agent use in preclinical studies and clinical diagnostics, biological activity must be validated. The Lindmo assay has been used conventionally to quantify radiolabeled antibody (Ab) immunoreactivity, although published findings suggest it does not provide consistently accurate results. We developed and tested a near-infrared (NIR) flow cytometry (FC) method for quantifying biological activity of a dual-labeled Ab for use as a multimodal contrast agent in small animal and human positron emission tomography and NIR fluorescence imaging. Antibody specific for epithelial cell adhesion molecule was conjugated to DOTA-NHS-ester, labeled with IRDye 800CW and further labeled with (64)Cu or nonradioactive Cu prior to reacting with human prostate cancer cells for testing by the Lindmo or FC method, respectively. Immunoreactivity of the dual-labeled agent was found to be 76.4 ± 15.7% by the Lindmo assay. When tested with and without Cu labeling using NIR FC, the biological activity was found to be 73.1 ± 7.7 and 79.4 ± 8.1%, respectively. No significant differences were found between these activity levels (p > 0.05), supporting NIR FC as an alternative method for measuring immunoreactivity and demonstrating that Cu labeling does not significantly affect the agent's ability to bind to its target. Biological activity was significantly reduced when the NIR dye-to-protein ratio was increased 3- to 4-fold in agent preparations when tested by FC and the Lindmo assay. In summary, NIR FC is an alternative with similar specificity and sensitivity, and greater reproducibility relative to the Lindmo assay for quantifying biological activity of NIR fluorophore-labeled, multimodal imaging agents.

Imaging prostate cancer lymph node metastases with a multimodality contrast agent.

BACKGROUND: Methods to detect lymph node (LN) metastases in prostate cancer (PCa) are limited. Pelvic LN dissection is commonly performed during prostatectomy, but often followed by morbid complications. More refined methods for detecting LN invasion are needed. METHODS: We developed a dual-labeled targeting agent having a near-infrared (NIR) fluorophore for intraoperative guidance, and a conventional radiotracer for detection of LN metastasis. Nu/Nu mice were orthotopically implanted with DsRed-expressing human PCa (PC3) cells. Antibody (Ab) specific for epithelial cell adhesion molecule was conjugated to DOTA, IRDye 800CW, and radiolabeled with (64) Cu. Dual-labeled Ab was administered intravenously at 10-12 weeks post-implantation, and positron emission tomography/computed tomography (PET/CT) and fluorescence imaging were performed within 18-24 hr. RESULTS: Metastasis to lumbar LNs was detected by DsRed fluorescence imaging, as well as pathology, in 75% of mice having pathology-confirmed primary prostate tumors. These metastases were also detected by NIR fluorescence imaging. In some cases, metastases to sciatic, medial, renal, and axillary nodes were also detected. For all LNs examined, no significant differences were found between the percentages of metastases detected by NIR imaging (63%) and µPET/CT (64%) (P = 0.93), or between those detected by DsRed imaging (25%) and pathological examination (19%) (P = 0.12). CONCLUSION: This study demonstrates that a multimodality contrast agent is useful for early detection of metastatic disease, and has applications for intraoperative PCa treatment. Further agent optimization is necessary to enhance specificity, and provide validation for prostate and other LN metastasizing epithelial cancers.

Detection of Cancer Metastases with a Dual-labeled Near-Infrared/Positron Emission Tomography Imaging Agent.

UNLABELLED: By dual labeling a targeting moiety with both nuclear and optical probes, the ability for noninvasive imaging and intraoperative guidance may be possible. Herein, the ability to detect metastasis in an immunocompetent animal model of human epidermal growth factor receptor 2 (HER-2)-positive cancer metastases using positron emission tomography (PET) and near-infrared (NIR) fluorescence imaging is demonstrated. METHODS: ((64)Cu-DOTA)(n)-trastuzumab-(IRDye800)(m) was synthesized, characterized, and administered to female Balb/c mice subcutaneously inoculated with highly metastatic 4T1.2neu/R breast cancer cells. ((64)Cu-DOTA)(n)-trastuzumab-(IRDye800)(m) (150 µg, 150 µCi, m = 2, n = 2) was administered through the tail vein at weeks 2 and 6 after implantation, and PET/computed tomography and NIR fluorescence imaging were performed 24 hours later. Results were compared with the detection capabilities of F-18 fluorodeoxyglucose ((18)FDG-PET). RESULTS: Primary tumors were visualized with (18)FDG and ((64)Cu-DOTA)(n)-trastuzumab-(IRDye800)(m), but resulting metastases were identified only with the dual-labeled imaging agent. (64)Cu-PET imaging detected lung metastases, whereas ex vivo NIR fluorescence showed uptake in regions of lung, skin, skeletal muscle, and lymph nodes, which corresponded with the presence of cancer cells as confirmed by histologic hematoxylin and eosin stains. In addition to detecting the agent in lymph nodes, the high signal-to-noise ratio from NIR fluorescence imaging enabled visualization of channels between the primary tumor and the axillary lymph nodes, suggesting a lymphatic route for trafficking cancer cells. Because antibody clearance occurs through the liver, we could not distinguish between nonspecific uptake and liver metastases. CONCLUSION: ((64)Cu-DOTA)(n)-trastuzumab-(IRDye800)(m) may be an effective diagnostic imaging agent for staging HER-2-positive breast cancer patients and intraoperative resection.

Hydrogel microsphere encapsulation of a cell-based gene therapy system increases cell survival of injected cells, transgene expression, and bone volume in a model of heterotopic ossification.

Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid endochondral bone formation, enhancing our preexisting cell-based gene therapy system by microencapsulating adenovirus-transduced cells in nondegradable poly(ethylene glycol) diacrylate (PEGDA) hydrogels before intramuscular delivery. This study evaluates the in vitro and in vivo viability, gene expression, and bone formation from transgenic fibroblasts encapsulated in PEGDA microspheres. Fluorescent viability and cytotoxicity assays demonstrated >95% viability in microencapsulated cells. ELISA and alkaline phosphatase assays established that BMP-2 secretion and specific activity from microencapsulated AdBMP2-transduced fibroblasts were not statistically different from monolayer. Longitudinal transgene expression studies of AdDsRed-transduced fibroblasts, followed through live animal optical fluorescent imaging, showed that microencapsulated cells expressed longer than unencapsulated cells. When comparable numbers of microencapsulated AdBMP2-transduced cells were intramuscularly injected into mice, microcomputed tomography evaluation demonstrated that the resultant heterotopic bone formation was approximately twice the volume of unencapsulated cells. The data suggest that microencapsulation protects cells and prolongs and spatially distributes transgene expression. Thus, incorporation of PEGDA hydrogels significantly advances current gene therapy bone repair approaches.

Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections.

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.

Humoral immune response in mice over-expressing or deficient in growth hormone.

Effects of growth hormone (GH) levels on the humoral immune response were investigated in metallothionein I (MT)-bovine (b) GH-transgenic (tg) and GH-deficient Ames dwarf (Prop1 df(-/-)) mice. Four-month-old mice were given primary and secondary injections of either normal saline or tetanus toxoid (TT) to induce specific antibody (Ab) production. MT-bGH-tg mice with high peripheral levels of bGH produced less TT-specific Ab than normal nontransgenic (Ntg) littermates, df, or nondwarf (Ndf) control mice. Titers reached maximum levels at 3-4 weeks post-primary immunization (PPI) and declined gradually through 24 weeks PPI in all groups of mice. Peripheral CD4(+) and CD8(+) T cell populations were significantly lower in tg than in Ntg, df, or Ndf mice. No significant differences were found in B cell numbers between tg, Ntg, or df mice. T helper 2 (Th2) cell populations were significantly greater in df mice compared to Ntg control mice. No significant differences were found in CD4(+):CD8(+) T cell ratios, interleukin (IL)-4 concentrations or interferon (IFN)-gamma levels between tg,Ntg, df, and Ndf mice. No patterns of significant sexual dimorphism were found for any of the immune parameters studied. Elevated levels of corticosterone were investigated as a possible immunosuppressant mechanism responsible for low Ab responses in the tg mice. Ab production was not enhanced by decreasing corticosterone in tg mice. Thus, high endogenous GH levels inhibit specific Ab production and peripheral T cell populations but not peripheral B cell numbers, Th2 cell populations, or IL-4 or IFN-gamma production. Elevated corticosterone levels do not appear to be responsible for suppressed humoral immune responses. Low levels of endogenous GH do not inhibit specific Ab production but may contribute to increased peripheral Th2 cell numbers.