Open Access
Add to BibSonomyAbstract
Each year almost 300,000 individuals worldwide are diagnosed with oral cancer, more than 90% of these being oral carcinoma [N. Engl. J. Med. 328, 184 1993]. Surgical resection is the standard of care, but accurate delineation of the tumor boundaries is challenging, resulting in either under-resection with risk of local recurrence or over-resection with increased functional loss and negative impact on quality of life. This study evaluates, in two pre-clinical in vivo tumor models, the potential of fluorescence-guided resection using molecular beacons activated by metalloproteinases, which are frequently upregulated in human oral cancer. In both models there was rapid (<15 min) beacon activation upon local application, allowing clear fluoresecence imaging in vivo and confirmed by ex vivo fluorescence microscopy and HPLC, with minimal activation in normal oral tissues. Although the tissue penetration was limited using topical application, these findings support further development of this approach towards translation to first-in-human trials.
© 2016 Optical Society of America
1. Introduction
Multiple optical spectroscopic and imaging techniques, including the use of endogenous tissue (auto)fluorescence, have been widely investigated for the identification of a variety of malignant diseases, including in the lung, skin, bladder, cervix, brain and gastrointestinal tract [1–3]. Autofluoresecnce imaging (AFI) has also recently been evaluated for detection of oral carcinoma [4–7]. The 5-year survival rate for patients with oral carcinoma is as low as 30% [8] and it has one of the highest local recurrence rates of all cancers, mainly attributed to the inability to accurately identify tumor boundaries at time of resection. An ongoing multicenter clinical trial, the COOLS study, uses AFI of oral carcinoma during surgical resection, based on an earlier clinical study that showed marked improvement in the localization of tumor boundaries compared with standard clinical assessment [9]. Preliminary results from the COOLS trial show decreased recurrence rates when AFI guidance is used as an adjuvant technique in oral cancer resection [10]. However, autofluorescence cannot reliably discern malignant lesions from benign lesions, potentially leading to high rates of false positives and unnecessarily large surgical margins that can be disfiguring or lead to functional impairment. The high false positive rates with AFI has been a general finding in a number of different malignancies, especially in more advanced disease or where there is background tissue inflammation [11–13]. On the other hand, AFI has the significant advantage that no exogenous contrast is required, thereby reducing costs and regulatory hurdles.
As an alternative to AFI, there have been numerous studies in different tumors of fluorescence imaging or point spectroscopy using exogenous fluorescent contrast agents [14–17]. In terms of surgical guidance, for example, indocyanine green, a vascular agent, is used widely in multiple surgical specialties including oncologic for imaging tissue perfusion. For tumor-specific imaging, the most clinically advanced exogenous agent is aminolevulinic acid (ALA) that results in preferential synthesis in tumor cells of the fluorophore protoporphyin IX (PpIX), and this is now an established technique, particularly in Europe, where it is approved in neurosurgery [18]. ALA-PpIX fluorescence has been reported for detection of oral cancer and premalignant lesions (leukoplakia, dysplasia) [19–21] and status, while treatment using red light-activated ALA-PpIX (photodynamic therapy) has also shown positive results [22–24]. There has also been significant preclinical work in developing and evaluating molecularly-targeted fluorescent contrast agents, typically based on tumor-specific antibodies or peptides conjugated to fluorescent dyes or nanoparticles [25,26]. The advantages of exogenous fluorophores over AFI are that the signal is usually much stronger and the spectral characteristics are known, both of which facilitate the optimum design of the imaging system. In addition, there is the potential for high specificity through the targeting of tumor-specific biomarkers [27,28] where these are known and suitable for targeting (typically expressed on the cell surface). As an alternative to biomarker targeting, several groups have investigated activatable fluorescent molecular beacons (MBs) [29,30], which consists of a fluorophore with a short linker to a second molecule or nanoparticle (quencher). Upon the fluorophore absorbing light, the energy is transferred directly to the quencher, so that there is no fluorescence emitted. However, when the fluorophore and quencher are physically separated upon interaction with the intended target, the fluorescence is recovered. There are several classes of MB, depending on the form and mode of activation of the linker [31–33]. Here we use beacons with an enzyme–cleavable peptide sequence as the linker, shown in Fig. 1, so that the beacon serves as a reporter for the presence of the specific enzyme.
Fig. 1 Molecular beacons comprising a fluorophore (F) conjugated to a quencher (Q) by a short disease-specific linker. In normal tissue the linker remains intact and no fluorescence is observed, while in diseased tissue the linker is specifically cleaved, activating the beacon and enabling fluorescence detection.
We have previously reported peptide-linked MBs with high quenching and that are efficiently cleaved by metalloproteinases (MMPs) over-expressed in malignant tissue compared with normal host tissue. We have demonstrated these in preclinical models of KB tumors in vitro and in vivo for both tumor-specific fluorescence imaging and photodynamic therapy [34]. These beacons have also been validated pre-clinically in other malignancies, most recently in vertebral metastases of breast cancer [35], but they have not been investigated in oral cancer. MMPs are proteolytic enzymes that have long been associated with various stages of tumor progression, including oral carcinoma [36,37]. Specifically, studies have demonstrated that MMP-1, −2, −3, −7, −9, −10, −11 and −13 are expressed in oral carcinoma and are critical to oral cancer progression [36,38–40]. This is particularly timely with the recent publication by Whitley et al., demonstrating first-in-human data using a protease-activated fluorescent probe for imaging cancer [41].
We have demonstrated that our MMP beacon, consisting of pyropheophorbide (pyro) as the fluorophore linked to black hole quencher 3 (BHQ3) by an MMP-cleavable peptide sequence, can be specifically and efficiently activated by MMP-3, −7, −9, −10, −12 and −13 [42]. Therefore, we hypothesize that the MMP-activatable beacons will allow visualization of oral carcinoma. Further, we hypothesize that the topically applied beacon will be activated in a time window sufficiently short to ensure its clinical applicability for oral cancer treatment. We report here in vivo wide-field fluorescence imaging and ex vivo fluorescence microscopy of this beacon’s behaviour in two different oral cancer models.
2. Materials and methods
2.1 PPMMPB synthesis
The beacon, referred to as PPMMPB, consists of pyro conjugated to BHQ3 via a MMP-cleavable peptide sequence, GPLGLARK, where italics indicate the cleavage site. PPMMPB was synthesized as described previously [34]. A positive control, PPMMP, was synthesized by the same protocol, consisting of pyro conjugated to GPLGLARK but without the addition of BHQ3, the quencher.
2.2 Cell line
The human oral carcinoma cell line UM-SCC-1 [43] was grown and maintained in Dulbecco’s Modified Eagles’ Medium (D-MEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM non-essential amino acids and 2 mM L-glutamine, at 37°C in a humidified incubator with 5% CO2.
2.3 In vivo xenograft model and imaging
All animal studies were carried out with institutional approval (University Health Network, Toronto, Canada). Adult male athymic nude mice (Charles River, Wilmington, USA) were inoculated with 1x105 tumor cells in 20 μL of media at the tip of the tongue and maintained in pathogen-free conditions in autoclaved microisolator cages. Two weeks after injection, the tumors reached 2-3mm in diameter. A 50 nmol dose of either PPMMPB or control PPMMP was formulated in 20 μL of aqueous solution with 5% dimethyl sulfoxide (DMSO, Sigma Aldrich) and 1.5% Tween-80. Two negative controls were also used. The first was a non-beacon control consisting of 20 μL of aqueous solution with 5% DMSO and 1.5% Tween-80 injected into the tongue of tumor-bearing animals. The other consisted of healthy tongues that received 50 nmol of PPMMPB formulated in 20 μL of aqueous solution with 5% DMSO and 1.5% Tween-80 to assess the role of surgical resection. Under general inhalation anaesthesia (isofluorane in oxygen), the tongues were imaged using an in-house fluorescence endoscopy system (650 ± 20 nm excitation, 700 ± 25 nm detection, various integration times, set manually), similar to studies described previously in different tumor models [44]. The PPMMPB, PPMMP, or aqueous solution negative control was then injected directly into the tumor and surrounding tongue tissue using a 31G needle, at one injection site. Injection was used rather than topical application because topical application was not logistically possible. At 15 min post-injection the tongues were imaged again. The mice were then immediately euthanized by CO2, and the tongues were harvested and snap-frozen and stored in the dark at −80°C. Frozen sections (6 μm thick) were cut, immersed in phosphate buffered saline (PBS) for 5 min and dried. 5 μL of mounting solution with DAPI (4’,6-diamidino-2-phenylindole, Vector Laboratories Inc.) was added as a nuclear stain. The sections were covered with a coverslip and imaged on a confocal fluorescence microscope (Olympus BX50, DAPI excitation 387 nm, emission 440 ± 20 nm emission, pyro 460 nm excitation, 690 ± 40 nm emission). A total of N = 12 mice were used for the above studies.
2.4 In vivo hamster model: imaging and resection
Male 6-8 week old hamsters (Syrian, Harlan, Indianapolis, USA) with chemically-induced tumors were used as a model to mimic human oral carcinoma. Under general inhalation anaesthesia, 0.5% DMBA (7,12-dimethylbenz(a)anthracene) in DMSO was applied to both cheeks three times every week over 16-20 weeks, as per published protocols [45]. Hamster buccal cheek model is ideal as tumors develop similarly to human oral carcinoma, in location and preceded by dysplasia [46]. Additionally, chemical agents are the main etiological factors responsible for the development for oral carcinoma [47], making DMBA application a relevant modality. Gauze was packed into the cheek pouch prior to DMBA application to minimize spillage down the throat and a 5 mm non-absorbent swab was used to apply the DMBA for 6 s at a time. The gauze was then removed. After 16-20 weeks of application, the tumors reached 5-10 mm in size. For each animal, a 50 nmol dose of PPMMPB or PPMMP was formulated in 100 μL of aqueous solution with 5% DMSO and 1.5% Tween-80. Other animals received a non-beacon negative control, consisting of 100 μL of aqueous solution with 5% DMSO and 1.5% Tween-80. Under general anaesthesia (80 mg/kg ketamine and 5 mg/kg xylazine), a 90-95% resection of the tumor of interest was performed as follows (Fig. 2) in 15 tumor-bearing animals, with tumors on each cheek pouch: 5 animals received topical PPMMPB, 5 animals served as positive controls using PPMMP, and 5 animals served as negative controls, treated with and aqueous solution with 5% DMSO and 1.5% Tween-80 but no fluorophore. Additionally, 5 animals without tumors underwent resection of healthy cheek pouch and received topical PPMMPB to serve as an additional negative control.
Fig. 2 Representative example from the mouse xenograft model using interstitial administration of PPMMPB or (control) PPMMP, demonstrating tumor-associated fluorescence and whole tongue fluorescence, respectively. A) tongue injected with PPMMPB: i) color white light image pre-injection, ii) monochrome white light image pre-injection, iii) monochrome white light image15 min post injection, iv) fluorescence image pre-injection, v) fluorescence image 15 min post injection showing fluorescence localized to the tumor at the tip of the tongue. B) corresponding images using PPMMP, with the 15 min post injection image (v) showing fluorescence throughout tongue in both healthy and tumor tissue. C) representative H&E stained (i) and confocal fluorescence (ii) of tongue at 15 min post injection of PPMMPB (red: beacon, blue: DAPI). D) corresponding images with PPMMP. E) corresponding images for tongue injected with negative control (DMSO and Tween-80 solution). N = 3 for each group.
All resections were performed by a single surgeon (NW) to ensure consistent technique. The cheek pouches were exposed and held under tension. Tumors were selected for resection if they were clearly identifiable by visual inspection and surrounded by healthy mucosa. A #15 blade was used to incise the oral mucosa circumferentially around the tumor down to the subcutaneous tissue. This plane was then followed to ensure total removal of the tumor save for the remaining 10-15%. In patients, oral squamous cell carcinoma is typically resected with a 1 cm margin but this could not be accommodated in this model, so that a 2 mm margin of visibly-healthy mucosa was resected en-bloc with the tumor. Care was taken to avoid the venous tributaries to the rostral and caudal facial vessels to minimize bleeding. Any bleeding was controlled locally with pressure or with monopolar cautery. After this initial resection, the cheek was imaged in situ (prescan). The animals were placed on their sides and cheek pouch positioned such that solution could be applied to the tumor bed. Then the 50 nmol solution of either PPMMPB or PPMMP was applied topically to the tumors and surrounding healthy cheek tissue for 15 min. After 15 min the MB solution was removed and the cheek was washed thoroughly with sterile saline solution and re-imaged. The other cheek was then used. Once both cheeks were studied, the animals were euthanized by pentobarbital overdose (Euthanyl, Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada). The whole cheeks were harvested immediately following euthanasia and prepared for fluorescence microscopy as per the xenograft model above. A total of N = 20 hamsters were used for the above studies.
In addition, in a separate cohort of 6 animals, high pressure liquid chromatography (HPLC) and mass spectrometry was used to confirm the specificity of beacon activation. Whole tumors were topically incubated in PPMMPB solution for 15 min, either in vivo prior to being immediately imaged in situ and resected (N = 3) or ex vivo immediately following resection (N = 3). The tumors were then submerged in 200 μL of DMSO for 2 h to extract all PPMMPB and the extract was run through HPLC-MS. Additionally, the applied solution was removed and run through the HPLC-MS. The cleavage rate of PPMMPB was calculated by the integration area of the cleaved peak divided by the total integration area of the intact peak plus the cleaved peak at pyro specific absorption of 665 nm.
3. Results
3.1 MMP beacon for fluorescence imaging of xenograft tumor model
Following injection of 50 nmol of PPMMPB into the tongue tumors and surrounding healthy tongue, strong fluorescence was observed in the tumor within 15 min, while minimal fluorescence was observed in surrounding healthy tissue, indicating a fast tumor-specific activation of PPMMPB in mouse tongue tumor, Fig. 2(A). To further validate this activation specificity, the same procedure using the positive control, PPMMP, showed fluorescence in both tumor and surrounding healthy tissue, Fig. 2(B). When the tumors were treated with a non-beacon control, the same aqueous solution without beacon, consisting of the aqueous solution without PPMMPB, no increase in fluorescence was observed over the 15 min, indicating that the fluorescence imaging system did not detect significant tissue autofluorescence background, so that the positive images were due to the exogenous fluorophore.
This tumor-specific beacon activation was then validated by correlation of confocal fluorescence microscopy and histology, which confirmed that PPMMPB was activated, displaying remarkable fluorescence, only within cancerous tissue, but not in healthy tissue, Fig. 2(C). However, the positive control PPMMP exhibited fluorescence in both tumor and healthy tissues alike, Fig. 2(D).
3.2 MMP beacon for fluorescence imaging of a hamster oral carcinoma model
Having demonstrated fast and specific activation of PPMMPB in the mouse tumor xenograft model, the effectiveness of the MMP beacon for visualizing oral carcinoma in a more clinically-relevant carcinogenesis animal model under surgical conditions was examined. Disease progression in the DMBA-induced hamster cheek pouch model closely mimics that in humans [48]. Clinically, it is envisioned that surgeons would resect tumor under standard white-light visualization before topically applying the beacon to identify and locate any residual disease. Some background autofluorescence was observed on the periphery of some tumors prior to PPMMPB application. It is presumed that the background autofluorescence was associated with inflammation, as it was mainly observed in large inflamed tumors. After topical application of the beacon following incomplete resection, there was significant tumor-associated fluorescence seen in vivo, Fig. 3(Biii), whereas the surrounding healthy tissue showed no fluorescent signal. Using fluorescence microscopy ex vivo it is also clear that fluorescence activation of PPMMPB is confined to tumor components, Fig. 3(C). However, it appears that the topically-applied PPMMPB has limited depth penetration into the tissue, with fluorescence seen only at the outermost layers of the tumor, Fig. 3(C).
Fig. 3 Representative example of a hamster cheek treated with topical PPMMPB. A) images following 90-95% resection before PPMMPB application: i) color white light, ii) monochrome white light, iii) fluorescence image showing some background fluorescence that is too weak to accurately identify lesions. B) following 15 min topical application of PPMMPB: i) color white light, ii) monochrome white light, iii) fluorescence image showing that the resected area and remnant tumor can be easily visualized. C) representative microscopy images: i) en-face image of H&E stained tissue, and ii) corresponding confocal fluorescence image showing activated PPMMPB at the outer layer of tumor tissue but absent in healthy tissue (red: beacon, blue: DAPI). N = 5 for each group. Note that, for spatial reference, hamster bottom teeth are 1.5cm in length.
To further validate the tumor specific activation of PPMMPB beacon, tumor-bearing hamsters, after incomplete tumor resection, were treated with either the positive control PPMMP (no quencher) or the non-beacon negative control. In all positive control animals, significant fluorescence was seen in both normal or tumor tissue, Fig. 4(A), while the non-beacon negative control showed no fluorescence, Fig. 4(B) in either tissue. Additionally, healthy hamster cheeks that underwent similar treatment (surgery + PPMMPB) displayed negligible fluorescence in the surgery area, eliminating any concern that surgical intervention might cause beacon activation, Fig. 4(C).
Fig. 4 Representative images of different control hamster cheeks. A) PPMMP-treated, B) negative control-treated (DMSO and Tween-80 solution), C) healthy cheek following surgery and treated with PPMMPB: i) pre-beacon color white light, ii) pre-beacon monochrome white light, iii) pre-beacon fluorescence, iv) post-beacon color white light, v) post-beacon monochrome white light, vi) post-beacon fluorescence. Both healthy and cancerous tissue are fluorescent post-PPMMP application (A,vi). There is no visible fluorescence associated with either the negative control treatment (B,vi) or with PPMMPB applied to surgically-treated healthy tissue (C,vi). D) representative H&E (i) and confocal fluorescence images (ii) in tissue treated with PPMMP, showing non-specific fluorescence in healthy and cancerous tissue with limited penetration. E) corresponding images in tissue with negative control showing fluorescence. F) healthy cheek post-resection and treated with PPMMPB shows no beacon activation. N = 5 for each group. Note that, for spatial reference, hamster bottom teeth are1.5cm in length.
These control tissues were further imaged ex vivo using confocal microscopy. As shown in Fig. 4(D), the PPMMP-treated cheeks showed no tumor-specificity, with fluorescence equally distributed throughout both normal and tumor tissue, while the non-beacon negative control cheeks displayed negligible fluorescence in either tumor or normal tissues, Fig. 4(E). Consistently, the surgery-only normal tissues treated with PPMMPB showed no obvious fluorescence, Fig. 4(F).
Using HPLC to analyze the tumor extract, we observed that the PPMMPB-treated tumor clearly displayed two porphyrin-associated peaks at retention times 12.13 min and 12.72 min, characterized as the intact PPMMPB and the cleaved (pyro-GPLG) fragment, respectively [49], seen in Fig. 5(A). However, the recovered PPMMPB solution which remained outside of the tumor after topical incubation with the tumor showed only the intact PPMMPB peak by HPLC analysis, demonstrated in Fig. 5(B), further confirming tumor-associated cleavage of PPMMPB. This HPLC analysis was further used to quantify the cleavage rate of PPMMPB following in vivo fluorescence imaging of PPMMPB-treated tumors. Compared to the prescan images, in Fig. 5(C), following PPMMPB treatment, all tumors showed significant fluorescence increase, seen in Fig. 5(D) and the corresponding HPLC analysis confirmed PPMMPB cleavage to different degrees, in Fig. 5(E). The variability of PPMMPB cleavage between tumors, even within the same cheek, is most likely due to a combination of intrinsic tumor heterogeneity, differences in tumor size, and uneven application of PPMMPB solution which is technically challenging to control in this model. In contrast to the tumor tissue, surrounding tissue that had been treated with PPMMPB in the same way showed no detectable PPMMPB cleavage by HPLC analysis. This corresponds well with the fluorescence images, in Fig. 5(D), and provides further support for tumor-specific beacon activation.
Fig. 5 Representative HPLC traces A) from beacon extracted from tumor showing intact PPMMPB and PPMMP fragment, B) beacon applied to the tumor, where only intact PPMMPB is present, confirming PPMMPB specificity, not outside of it .C) images before PPMMPB incubation: i) color white light, ii) monochrome white light, iii) fluorescence image showing some background fluorescence, likely caused by bacteria. D) corresponding images after 15 min topical application of PPMMPB: the fluorescence images show that all tumors became fluorescent to different extent as indicated in (E). N = 3 in all cases.
4. Discussion
The study demonstrates tumor-specific activation of PPMMPB at a MMP cleavage site, resulting in enhanced fluorescence for identification of oral carcinoma tissue. Specific PPMMPB activation was validated in the mouse orthotopic xenograft model, seen in Fig. 2, and subsequently with topical application in the hamster cheek pouch model that more closely mimics the disease in patients and the clinical treatment procedures, Figs. 3 and 4. HPLC analysis further confirmed that oral carcinoma activates PPMMPB at a site characteristic of MMP-mediated cleavage, seen in Fig. 5. The lateral boundaries of the tumor could be identified within a short time (15 min) following topical application, allowing for ease of integration into surgical, and it is possible that this could be shortened further.
The small depth penetration of the topical beacon may be of concern and it will be worthwhile to investigate how this could be improved, for example by the use of a penetrating excipient. However, in the context of identifying residual tumor following maximal white light-guided resection, it does not prevent identification of the lateral boundaries of the disease. Oral carcinoma is characterized as being much greater in linear extent than depth [8,50], meaning that identification of lateral boundaries is the most critical metric of successful surgical resection. If the incubation time can be further reduced, it is also possible that the process of applying the beacon and then imaging could be repeated several times to ensure complete depth resection. The depth limitation could, of course, also be overcome by systemic administration of the beacon, although this introduces potential concerns of toxicity and cost as well as optimizing the time interval between administration and surgery to achieve maximum sensitivity and specificity.
A further potential limitation to this MB image-guided resection approach is that the MMP expression is heterogeneous, both across different tumors and within a given tumor. It will then be critical in human studies to determine the minimum level of beacon activation that can be detected by fluorescence imaging relative to the tissue autofluorescence background. One could also envisage combining AFI with MB-enabled fluorescence imaging to take advantage of their complementary sensitivity and specificity, by adjusting the camera gain and wavelength setting between the two modes. Determining the optimum concentration and incubation period of the beacon in the initial clinical studies will also be important. Studies using topical application of the beacon in human oral cancer tissues ex vivo are in progress to address these issues prior to first in vivo trials.
5. Conclusions
The present work supports the concept of using topically-applied MMP-specific molecular beacon to delineate oral carcinoma tissues. In both preclinical models, the results demonstrate high tumor specificity and, using realistic beacon concentration and a short incubation time, clear fluorescence imaging can be achieved in vivo, including imaging of residual tumor following (deliberately) incomplete but nevertheless substantial white-light resection. The findings were confirmed by fluorescence microscopy and HPLC of ex vivo tissues. Potential limitations have been identified but we believe that these should not prevent further translation of this approach into first-in-human studies. Thereby, this approach could make a significant contribution to improve the surgical outcome and quality of life of patients with this often devastating disease.
Acknowledgments
This work was supported by the Canadian Cancer Society Research Institute, the Canadian Institute of Health Research, the Joey and Toby Tanenbaum/Brazilian Ball Chair in Prostate Cancer Research, and Major International (Regional) Joint Research Project from National Science Foundation of China (61520106015). In addition, the authors would like to thank Sadiya Yousef for her animal care support and Dr. Thomas Carey from University of Michigan, USA for kindly providing human oral carcinoma cell line UM-SCC-1.
References and Links
1. N. Ramanujam, M. F. Mitchell, A. Mahadevan, S. Warren, S. Thomsen, E. Silva, and R. Richards-Kortum, “In vivo diagnosis of cervical intraepithelial neoplasia using 337-nm-excited laser-induced fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 91(21), 10193–10197 (1994). [CrossRef] [PubMed]
2. J. de Leeuw, N. van der Beek, W. D. Neugebauer, P. Bjerring, and H. A. Neumann, “Fluorescence detection and diagnosis of non-melanoma skin cancer at an early stage,” Lasers Surg. Med. 41(2), 96–103 (2009). [CrossRef] [PubMed]
3. S. Lam, T. Kennedy, M. Unger, Y. E. Miller, D. Gelmont, V. Rusch, B. Gipe, D. Howard, J. C. LeRiche, A. Coldman, and A. F. Gazdar, “Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy,” Chest 113(3), 696–702 (1998). [CrossRef] [PubMed]
4. D. C. G. De Veld, M. J. H. Witjes, H. J. C. M. Sterenborg, and J. L. N. Roodenburg, “The status of in vivo autofluorescence spectroscopy and imaging for oral oncology,” Oral Oncol. 41(2), 117–131 (2005). [CrossRef] [PubMed]
5. T. Mang, J. Kost, M. Sullivan, and B. C. Wilson, “Autofluorescence and Photofrin-induced fluorescence imaging and spectroscopy in an animal model of oral cancer,” Photodiagn. Photodyn. Ther. 3(3), 168–176 (2006). [CrossRef] [PubMed]
6. V. Jayaprakash, M. Sullivan, M. Merzianu, N. R. Rigual, T. R. Loree, S. R. Popat, K. B. Moysich, S. Ramananda, T. Johnson, J. R. Marshall, A. D. Hutson, T. S. Mang, B. C. Wilson, S. R. Gill, J. Frustino, A. Bogaards, and M. E. Reid, “Autofluorescence-guided surveillance for oral cancer,” Cancer Prev. Res. (Phila.) 2(11), 966–974 (2009). [CrossRef] [PubMed]
7. E. Svistun, R. Alizadeh-Naderi, A. El-Naggar, R. Jacob, A. Gillenwater, and R. Richards-Kortum, “Vision enhancement system for detection of oral cavity neoplasia based on auto fluorescence,” Head Neck 26(3), 205–215 (2004). [CrossRef] [PubMed]
8. P. M. Speight, P. M. Farthing, and J. E. Bouquot, “The pathology of oral cancer and precancer,” Curr. Diagn. Pathol. 3(3), 165–176 (1996). [CrossRef]
9. C. F. Poh, C. E. MacAulay, L. Zhang, and M. P. Rosin, “Tracing the “at-risk” oral mucosa field with autofluorescence: Steps toward clinical impact,” Cancer Prev. Res. (Phila.) 2(5), 401–404 (2009). [CrossRef] [PubMed]
10. C. F. Poh, S. P. Ng, P. M. Williams, L. Zhang, D. M. Laronde, P. Lane, C. Macaulay, and M. P. Rosin, “Direct fluorescence visualization of clinically occult high-risk oral premalignant disease using a simple hand-held device,” Head Neck 29(1), 71–76 (2007). [CrossRef] [PubMed]
11. M. Rana, A. Zapf, M. Kuehle, N. C. Gellrich, and A. M. Eckardt, “Clinical evaluation of an autofluorescence diagnostic device for oral cancer detection: a prospective randomized diagnostic study,” Eur. J. Cancer Prev. 21(5), 460–466 (2012). [CrossRef] [PubMed]
12. M. Scheer, J. Neugebauer, A. Derman, J. Fuss, U. Drebber, and J. E. Zoeller, “Autofluorescence imaging of potentially malignant mucosa lesions,” Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 111(5), 568–577 (2011). [CrossRef] [PubMed]
13. C. S. Farah, L. McIntosh, A. Georgiou, and M. J. McCullough, “Efficacy of tissue autofluorescence imaging (VELScope) in the visualization of oral mucosal lesions,” Head Neck 34(6), 856–862 (2012). [CrossRef] [PubMed]
14. W. Stummer, A. Novotny, H. Stepp, C. Goetz, K. Bise, and H. J. Reulen, “Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients,” J. Neurosurg. 93(6), 1003–1013 (2000). [CrossRef] [PubMed]
15. A. Scerrati, G. M. Della Pepa, G. Conforti, G. Sabatino, A. Puca, A. Albanese, G. Maira, E. Marchese, and G. Esposito, “Indocyanine green video-angiography in neurosurgery: A glance beyond vascular applications,” Clin. Neurol. Neurosurg. 124, 106–113 (2014). [CrossRef] [PubMed]
16. Q. T. Nguyen and R. Y. Tsien, “Fluorescence-guided surgery with live molecular navigation - a new cutting edge,” Nat. Rev. Cancer 13(9), 653–662 (2013). [CrossRef] [PubMed]
17. M. Junaid, M. M. Choudhary, Z. A. Sobani, G. Murtaza, S. Qadeer, N. S. Ali, M. J. Khan, and A. Suhail, “A comparative analysis of toluidine blue with frozen section in oral squamous cell carcinoma,” World J. Surg. Oncol. 10(1), 57 (2012). [CrossRef] [PubMed]
18. W. Stummer, U. Pichlmeier, T. Meinel, O. D. Wiestler, F. Zanella, H.-J. Reulen, and ALA-Glioma Study Group, “Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial,” Lancet Oncol. 7(5), 392–401 (2006). [CrossRef] [PubMed]
19. A. Leunig, C. S. Betz, M. Mehlmann, H. Stepp, S. Arbogast, G. Grevers, and R. Baumgartner, “Detection of squamous cell carcinoma of the oral cavity by imaging 5-aminolevulinic acid-induced protoporphyrin IX fluorescence,” Laryngoscope 110(1), 78–83 (2000). [CrossRef] [PubMed]
20. C. S. Betz, H. Stepp, P. Janda, S. Arbogast, G. Grevers, R. Baumgartner, and A. Leunig, “A comparative study of normal inspection, autofluorescence and 5-ALA-induced PPIX fluorescence for oral cancer diagnosis,” Int. J. Cancer 97(2), 245–252 (2002). [CrossRef] [PubMed]
21. C.-Y. Wang, T. Tsai, C.-P. Chiang, H.-M. Chen, and C.-T. Chen, “Improved diagnosis of oral premalignant lesions in submucous fibrosis patients with 5-aminolevulinic acid induced PpIX fluorescence,” J. Biomed. Opt. 14(4), 044026 (2009). [CrossRef] [PubMed]
22. W. Jerjes, T. Upile, Z. Hamdoon, C. A. Mosse, S. Akram, and C. Hopper, “Photodynamic therapy outcome for oral dysplasia,” Lasers Surg. Med. 43(3), 192–199 (2011). [CrossRef] [PubMed]
23. W. E. Grant, C. Hopper, A. J. MacRobert, P. M. Speight, and S. G. Bown, “Photodynamic therapy of oral cancer: photosensitisation with systemic aminolaevulinic acid,” Lancet 342(8864), 147–148 (1993). [CrossRef] [PubMed]
24. K. F. M. Fan, C. Hopper, P. M. Speight, G. Buonaccorsi, A. J. MacRobert, and S. G. Bown, “Photodynamic therapy using 5-aminolevulinic acid for premalignant and malignant lesions of the oral cavity,” Cancer 78(7), 1374–1383 (1996). [CrossRef] [PubMed]
25. X. Wang, L. Yang, Z. G. Chen, and D. M. Shin, “Application of nanotechnology in cancer therapy and imaging,” CA Cancer J. Clin. 58(2), 97–110 (2008). [CrossRef] [PubMed]
26. A. M. Wu and T. Olafsen, “Antibodies for molecular imaging of cancer,” Cancer J. 14(3), 191–197 (2008). [CrossRef] [PubMed]
27. R. Weissleder, C.-H. Tung, U. Mahmood, and A. Bogdanov Jr., “In vivo imaging of tumors with protease-activated near-infrared fluorescent probes,” Nat. Biotechnol. 17(4), 375–378 (1999). [CrossRef] [PubMed]
28. R. L. Scherer, J. O. McIntyre, and L. M. Matrisian, “Imaging matrix metalloproteinases in cancer,” Cancer Metastasis Rev. 27(4), 679–690 (2008). [CrossRef] [PubMed]
29. S. Tyagi and F. R. Kramer, “Molecular beacons: probes that fluoresce upon hybridization,” Nat. Biotechnol. 14(3), 303–308 (1996). [CrossRef] [PubMed]
30. W. Tan, K. Wang, and T. J. Drake, “Molecular beacons,” Curr. Opin. Chem. Biol. 8(5), 547–553 (2004). [CrossRef] [PubMed]
31. T. W. B. Liu, J. Chen, and G. Zheng, “Peptide-based molecular beacons for cancer imaging and therapy,” Amino Acids 41(5), 1123–1134 (2011). [CrossRef] [PubMed]
32. J. J. Li, R. Geyer, and W. Tan, “Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA,” Nucleic Acids Res. 28(11), E52 (2000). [CrossRef] [PubMed]
33. P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res. 32(6), e57 (2004). [CrossRef] [PubMed]
34. G. Zheng, J. Chen, K. Stefflova, M. Jarvi, H. Li, and B. C. Wilson, “Photodynamic molecular beacon as an activatable photosensitizer based on protease-controlled singlet oxygen quenching and activation,” Proc. Natl. Acad. Sci. U.S.A. 104(21), 8989–8994 (2007). [CrossRef] [PubMed]
35. T. W. Liu, M. K. Akens, J. Chen, L. Wise-Milestone, B. C. Wilson, and G. Zheng, “Imaging of specific activation of photodynamic molecular beacons in breast cancer vertebral metastases,” Bioconjug. Chem. 22(6), 1021–1030 (2011). [CrossRef] [PubMed]
36. J. Kusukawa, Y. Sasaguri, M. Morimatsu, and T. Kameyama, “Expression of matrix metalloproteinase-3 in Stage I and II squamous cell carcinoma of the oral cavity,” J. Oral Maxillofac. Surg. 53(5), 530–534 (1995). [CrossRef] [PubMed]
37. D. Muller, C. Wolf, J. Abecassis, R. Millon, A. Engelmann, G. Bronner, N. Rouyer, M. C. Rio, M. Eber, G. Methlin, and P. Basset, “Increased stromelysin 3 gene expression is associated with increased local invasiveness in head and neck squamous cell carcinomas,” Cancer Res. 53(1), 165–169 (1993). [PubMed]
38. M. Polette, C. Clavel, D. Muller, J. Abecassis, I. Binninger, and P. Birembaut, “Detection of mRNAs encoding collagenase I and stromelysin 2 in carcinomas of the head and neck by in situ hybridization,” Invasion Metastasis 11(2), 76–83 (1991). [PubMed]
39. S. T. Gray, R. J. Wilkins, and K. Yun, “Interstitial collagenase gene expression in oral squamous cell carcinoma,” Am. J. Pathol. 141(2), 301–306 (1992). [PubMed]
40. J. Kusukawa, Y. Sasaguri, I. Shima, T. Kameyama, and M. Morimatsu, “Expression of matrix metalloproteinase-2 related to lymph node metastasis of oral squamous cell carcinoma. A clinicopathologic study,” Am. J. Clin. Pathol. 99(1), 18–23 (1993). [PubMed]
41. M. J. Whitley, D. M. Cardona, A. L. Lazarides, I. Spasojevic, J. M. Ferrer, J. Cahill, C.-L. Lee, M. Snuderl, D. G. Blazer 3rd, E. S. Hwang, R. A. Greenup, P. J. Mosca, J. K. Mito, K. C. Cuneo, N. A. Larrier, E. K. O’Reilly, R. F. Riedel, W. C. Eward, D. B. Strasfeld, D. Fukumura, R. K. Jain, W. D. Lee, L. G. Griffith, M. G. Bawendi, D. G. Kirsch, and B. E. Brigman, “A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer,” Sci. Transl. Med. 8(320), 320ra4 (2016). [CrossRef] [PubMed]
42. T. W. Liu, J. Chen, L. Burgess, B. C. Wilson, G. Zheng, L. Zhan, W. K. Liu, and B. Y. Ha, “Activation Kinetics of Zipper Molecular Beacons,” J. Phys. Chem. B 119(1), 44–53 (2015). [CrossRef] [PubMed]
43. J. C. Brenner, M. P. Graham, B. Kumar, L. M. Saunders, R. Kupfer, R. H. Lyons, C. R. Bradford, and T. E. Carey, “Genotyping of 73 UM-SCC head and neck squamous cell carcinoma cell lines,” Head Neck 32(4), 417–426 (2010). [PubMed]
44. E. H. Moriyama, A. Kim, A. Bogaards, L. Lilge, and B. C. Wilson, “A Ratiometric Fluorescence Imaging System for Surgical Guidance,” Adv. Opt. Technol. 2008, 1–10 (2008). [CrossRef]
45. I. B. Gimenez-Conti and T. J. Slaga, “The hamster cheek pouch carcinogenesis model,” J. Cell. Biochem. Suppl. 53(S17F), 83–90 (1993). [CrossRef] [PubMed]
46. S. Nagini, P. V. Letchoumy, A. Thangavelu, and C.R. Ramachandran, “Of humans and hamsters: A comparative evaluation of carcinogen activation, DNA damage, cell proliferation, apoptosis, invasion, and angiogenesis in oral cancer patients and hamster buccal pouch carcinomas,” Oral Oncol. 45(6), e31–e37 (2009). [CrossRef] [PubMed]
47. W. J. Blot, J. K. McLaughlin, D. M. Winn, D. F. Austin, R. S. Greenberg, S. Preston-Martin, L. Bernstein, J. B. Schoenberg, A. Stemhagen, and J. F. Fraumeni Jr., “Smoking and drinking in relation to oral and pharyngeal cancer,” Cancer Res. 48(11), 3282–3287 (1988). [PubMed]
48. G. Shklar, J. Schwartz, D. Grau, D. P. Trickler, and K. D. Wallace, “Inhibition of hamster buccal pouch carcinogenesis by 13-cis-retinoic acid,” Oral Surg. Oral Med. Oral Pathol. 50(1), 45–52 (1980). [CrossRef] [PubMed]
49. C. G. Knight, F. Willenbrock, and G. Murphy, “A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases,” FEBS Lett. 296(3), 263–266 (1992). [CrossRef] [PubMed]
50. D. P. Slaughter, H. W. Southwick, and W. Smejkal, “Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin,” Cancer 6(5), 963–968 (1953). [CrossRef] [PubMed]
