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Open Access Publications from the University of California

Scholarly Works (31 results)

  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2013)
A glowing new era in cancer surgery may be dawning. Using fluorescently labelled markers, surgical molecular navigation means that tumours and nerves can be displayed in real time intra-operatively in contrasting pseudocolours, which allows more complete tumour resection while preserving important structures. These advances can potentially cause a paradigm shift in cancer surgery, improving patient outcome and decreasing overall health-care costs.
    Cover page: Fluorescence-guided surgery with live molecular navigation — a new cutting edge
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2013)

    This thesis presents an optimized triple modality reporter combining genes for a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk). This schematic allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. This triple reporter improves on previous designs in three ways. 1) The shorter wavelength green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP1) has been replaced with E2-Crimson to increase penetration of fluorescence signal through mammalian tissues. 2) Firefly luciferase (fLuc), Renilla luciferase (hrl), or mutant thermally stable firefly luciferase (mtfl) has been replaced by Luc2, a codon optimized firefly luciferase for increased expression in mammalian cells and detection of single cells by bioluminescence. 3) Self-cleaving viral 2A sequences separate each component, ensuring equal stoichiometry of the reporter genes without requiring protein fusion or the use of internal ribosomal entry site (IRES) sequences. Cleavage between each protein gene product allows for proper protein folding, trafficking, and full activity of each modality. This optimized triple reporter construct was cloned into a second-generation lentiviral vector, and a lentivirus was produced. Two human cancer cell lines were virally transduced with the triple reporter construct, and expression of all three modalities was confirmed in both cell lines in vitro and in vivo. Finally, the therapeutic responses of the MDA231 human breast cancer cell line to the chemotherapeutic agents monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) were successfully quantified in vivo by optical imaging of the optimized triple modality reporter. This is the first reported use of both fluorescence and bioluminescence signals from a multi-reporter construct to measure drug efficacy in vivo.

    • 1 supplemental video
    Cover page: Viral Transduction of Human Cancer Cell Lines with an Optimized Triple Modality Reporter for Quantifiable Tumor Imaging and Therapy Evaluation In Vivo
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2011)

    Objectives/hypothesis

    By phage display, we have developed a novel peptide (NP41) that binds selectively to nerves following systemic administration. We evaluated the pattern of facial nerve labeling with fluorescently-labeled NP41 (F-NP41). We also tested whether F-NP41 highlights facial nerves well enough to identify nerve stumps accurately several weeks after nerve transection.

    Study design

    Forty-seven wild-type mice were studied prospectively. One surgeon performed the nerve transection, reanastomosis, and monitoring of functional recovery.

    Methods

    Fluorescent labeling: F-NP41 was administered intravenously (20 mice). Nerve labeling was studied with fluorescence microscopy. Transection and reanastomosis: the right facial nerve was transected (25 mice). Three weeks after transection, F-NP41 was administered intravenously and fluorescence microscopy was used to identify the nerve stumps and reanastomosis in one group. Nerve identification and reanastomosis was performed with white light in another group without F-NP41. The control group underwent sham surgery. Time to nerve identification was recorded. Functional recovery was monitored for at least 8 weeks.

    Results

    We found excellent labeling of intact and transected facial nerves following F-NP41 administration. Several weeks following nerve transection, F-NP41 provided accurate identification of the proximal and distal nerve stumps. Following reanastomosis, time to recovery and level of functional recovery was similar in the absence and presence of F-NP41.

    Conclusions

    We show improved visualization of facial nerves with a novel systemically applied fluorescently labeled probe. Use of F-NP41 resulted in accurate identification of facial nerve stumps several weeks following transection. Functional recovery was similar with and without the use of F-NP41.
      Cover page: Improved facial nerve identification with novel fluorescently labeled probe
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2015)

      Objective

      To compare outcomes in patients with idiopathic sudden sensorineural hearing loss (ISSNHL) treated with intratympanic (IT) dexamethasone (DEX) at either 10 mg/mL or 24 mg/mL.

      Study design

      Retrospective case series.

      Setting

      Tertiary referral center.

      Patients

      Thirty-seven adults with ISSNHL.

      Interventions

      In addition to concurrent prednisone taper, patients received a series of IT DEX injections for 2 weeks with either 10 mg/mL or 24 mg/mL.

      Main outcome measure

      Greater than 30-dB improvement in pure-tone average (PTA).

      Results

      Baseline characteristics were similar between groups. Mean follow-up was 10 weeks. Ten (53%) of 19 patients treated with 24 mg/mL had greater than 30-dB improvement in PTA compared with 3 (17%) of 18 treated with 10 mg/mL (p = 0.0382, Fisher's exact test). There was a trend toward improved word recognition score outcome with 24 mg/mL. The interval between onset and initiation of IT DEX significantly affected outcome, with earlier treatment resulting in greater improvement in PTA and word recognition score. Multivariate logistic regression confirmed that IT DEX dose and interval to starting treatment were both independent predictors of PTA outcome. Change in PTA was not significantly affected by age, sex, pretreatment hearing levels, or concurrent treatment with hyperbaric oxygen.

      Conclusion

      To our knowledge, this is the first demonstration of superiority of IT DEX at 24 mg/mL for the treatment of ISSNHL, with significantly better recovery of PTA. Our data suggest that treatment should be initiated as soon as possible. A prospective randomized trial to confirm the optimal dose is warranted.
        Cover page: Dose Effect of Intratympanic Dexamethasone for Idiopathic Sudden Sensorineural Hearing Loss
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2016)

        Background and objectives

        Molecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC.

        Methods

        Thirteen BRAFV600E mice with PTC were studied-seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy.

        Results

        The average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/- 0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals.

        Conclusions

        RACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone.
          Cover page: Molecular targeting of papillary thyroid carcinoma with fluorescently labeled ratiometric activatable cell penetrating peptides in a transgenic murine model
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2015)

          Purpose

          The ability to detect small malignant lesions with magnetic resonance imaging (MRI) is limited by inadequate accumulations of Gd with standard chelate agents. To date, no T1-targeted agents have proven superiority to Gd chelates in their ability to detect small tumors at clinically relevant field strengths. Activatable cell-penetrating peptides and their Gd-loaded dendrimeric form (ACPPD-Gd) have been shown to selectively accumulate in tumors. In this study we compared the performance of ACPPD-Gd vs. untargeted Gd chelates to detect small tumors in rodent models using a clinical 3T-MR system.

          Materials and methods

          This study was approved by the Institutional-Animal Care-and-Use Committee. 2 of 4 inguinal breast fat pads of 16 albino-C57BL/6 mice were inoculated with tumor Py8119 cells and the other 2 with saline at random. MRI at 3T was performed at 4, 9, and 14 days after inoculation on 8 mice 24-hours after injection of 0.036mmol Gd/kg (ACPPD-Gd), and before and 2-3 minutes after 0.1 mmol/kg gadobutrol on the other 8 mice. T1-weighted (T1w) tumor signal normalized to muscle, was compared among the non-contrast, gadobutrol, and ACPPD-Gd groups using ANOVA. Experienced and trainee readers blinded to experimental conditions assessed for the presence of tumor in each of the 4 breast regions. Receiver operator characteristic (ROC) curves and area-under-curve (AUC) values were constructed and analyzed.

          Results

          Tumors ≥1mm3 were iso-intense to muscle without contrast on T1w sequences. They enhanced diffusely and homogeneously by 57±20% (p<0.001) 24 hours after ACPPD-Gd and by 25±13% (p<0.001) immediately after gadobutrol. The nearly 2-fold difference was similar for small tumors (1-5mm3) (45±19% vs. 19±18%, p = 0.03). ACPPD-Gd tended to improve tumor detection by an experienced reader (AUC 0.98 vs 0.91) and significantly more for a trainee (0.93 vs. 0.82, p = 0.02) compared to gadobutrol. This improvement was more pronounced when obvious tumors (>5mm3) were removed from the ROC analysis for both the experienced observer (0.96 vs. 0.86) and more so for the trainee (0.86 vs. 0.69, p = 0.04).

          Conclusion

          ACPPD-Gd enhances MMP-expressing tumors of any size at 3T 24 hours after administration, improving their detection by blinded observers when compared to non-contrast and contrast groups given commercial Gd-chelates and imaged during the equilibrium phase.
            Cover page: Tumor Detection at 3 Tesla with an Activatable Cell Penetrating Peptide Dendrimer (ACPPD-Gd), a T1 Magnetic Resonance (MR) Molecular Imaging Agent
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2016)

            Background

            We evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable cell-penetrating peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model.

            Methods

            Surgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time, and tumor-free survival were compared.

            Results

            RACPP guidance aided tumor detection (positive histology in 90% [27/30] vs 48% [15/31] for WL; p < .001). An approximate 25% ratiometric signal increase as the threshold to distinguish between tumor and adjacent tissue, yielded >90% detection sensitivity and specificity. Operating time was reduced by 54% (p < .001), and tumor-free survival was increased with RACPP guidance (p = .025).

            Conclusion

            RACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications.
              Cover page: Surgical molecular navigation with ratiometric activatable cell penetrating peptide for intraoperative identification and resection of small salivary gland cancers.
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2014)
              We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.
                Cover page: An Optimized Triple Modality Reporter for Quantitative In Vivo Tumor Imaging and Therapy Evaluation
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2016)

                Background

                We evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable cell-penetrating peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model.

                Methods

                Surgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time, and tumor-free survival were compared.

                Results

                RACPP guidance aided tumor detection (positive histology in 90% [27/30] vs 48% [15/31] for WL; p < .001). An approximate 25% ratiometric signal increase as the threshold to distinguish between tumor and adjacent tissue, yielded >90% detection sensitivity and specificity. Operating time was reduced by 54% (p < .001), and tumor-free survival was increased with RACPP guidance (p = .025).

                Conclusion

                RACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications.
                  Cover page: Surgical molecular navigation with ratiometric activatable cell penetrating peptide for intraoperative identification and resection of small salivary gland cancers
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2016)

                  Background and objectives

                  Molecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC.

                  Methods

                  Thirteen BRAFV600E mice with PTC were studied-seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy.

                  Results

                  The average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/- 0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals.

                  Conclusions

                  RACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone.
                    Cover page: Molecular targeting of papillary thyroid carcinoma with fluorescently labeled ratiometric activatable cell penetrating peptides in a transgenic murine model.
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