carbocyanines and Head-and-Neck-Neoplasms

The overall objective of this study was to develop an optical imaging approach to simultaneously measure altered cell metabolism and changes in tissue extracellular pH with the progression of cancer using clinically isolated biopsies. In this study, 19 pairs of clinically normal and abnormal biopsies were obtained from consenting patients with head and neck cancer at University of California, Davis Medical Center. Fluorescence intensity of tissue biopsies before and after topical delivery of 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) and Alexa 647-pHLIP [pH (low) insertion peptide] was measured noninvasively by widefield imaging, and correlated with pathologic diagnosis. The results of widefield imaging of clinical biopsies demonstrated that 2-NBDG and pHLIP peptide can accurately distinguish the pathologically normal and abnormal biopsies. The results also demonstrated the potential of this approach to detect subepithelial lesions. Topical application of the contrast agents generated a significant increase in fluorescence contrast (3- to 4-fold) in the cancer biopsies as compared with the normal biopsies, irrespective of the patient and location of the biopsy within a head and neck cavity. This unpaired comparison across all the patients with cancer in this study highlights the specificity of the imaging approach. Furthermore, the results of this study indicated that changes in intracellular glucose metabolism and cancer acidosis are initiated in the early stages of cancer, and these changes are correlated with the progression of the disease. In conclusion, this novel optical molecular imaging approach to measure multiple biomarkers in cancer has a significant potential to be a useful tool for improving early detection and prognostic evaluation of oral neoplasia.

Topics: 4-Chloro-7-nitrobenzofurazan; Acidosis; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Carbocyanines; Contrast Media; Deoxyglucose; Disease Progression; Female; Glucose; Head and Neck Neoplasms; Humans; Hydrogen-Ion Concentration; Male; Microscopy, Fluorescence; Middle Aged; Mouth Neoplasms; Optical Imaging; Optics and Photonics; Prognosis

To evaluate cetuximab treatment in head and neck squamous cell carcinoma xenografts and cell lines, we investigated a preclinical model of head and neck squamous cell carcinoma. Head and neck squamous cell carcinoma cell lines SCC-1, FaDu, CAL27, UM-SCC-5 and UM-SCC-22A were used to generate subcutaneous flank xenografts in SCID mice. Mice were divided into control and cetuximab treatment groups, mice in the latter group received 250 μg cetuximab once weekly for four weeks. After completion of therapy, SCC-1 (p < 0.001), UM-SCC-5 (p < 0.001), UM-SCC-22A (p = 0.016) and FaDu (p = 0.007) tumors were significantly smaller than control, while CAL27 tumors were not different from controls (p = 0.90). Mice were systemically injected with 50 μg of the Cy5.5-cetuximab bioconjugate and imaged by stereomicroscopy to determine if tumor fluorescence predicted tumor response. Intact tumor fluorescence did not predict response. Tissue was harvested from untreated xenografts to evaluate ex vivo imaging. Cell lines were then evaluated in vitro for fluorescence imaging after Cy5.5-cetuximab bioconjugate labeling. The location of fluorescence observed in labeled cells was significantly different for cell lines that responded to treatment, relative to unresponsive cells. Tumors from cell lines that showed low internalized signal in vitro responded best to treatment with cetuximab. This preclinical model may aid in determining which cancer patients are best suited for cetuximab therapy.

Topics: Analysis of Variance; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carbocyanines; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Disease Models, Animal; ErbB Receptors; Fluorescent Antibody Technique; Fluorescent Dyes; Head and Neck Neoplasms; Humans; Mice; Mice, SCID; Microscopy, Fluorescence; Statistics, Nonparametric; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Removal of head and neck neoplasms, especially those of the parotid gland and those of the internal auditory canal and cerebellopontine angle, often requires microdissection of the facial nerve. Displacement or splaying of the nerve can make it difficult to identify facial nerve fibers and/or distinguish them from surrounding tissues. Here we tested a method of labeling the facial nerve with fluorescent lipophilic dyes as a method of providing intraoperative visual confirmation of nerve fibers.. The facial nerves of adult rats were retrogradely labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), or 3,3'-dilinoleyloxacarbocyanine perchlorate (Fast DiO) either by direct application to the nerve sheath or by microinjection into the facial muscles. The nerves were examined 30 days after dye application by means of a dissecting stereomicroscope equipped with epifluorescence filters.. Of the dyes tested, Fast DiO proved to be the most effective, providing labeling of the nerve sufficient to be seen with combined fluorescent and bright field stereomicroscopy. Nerve conduction studies indicated that fluorescent labeling did not adversely affect nerve function.. These results raise the possibility of using fluorescent lipophilic dyes to label nerves as a method of enhancing identification and distinguishing nerve fibers during surgery.

Topics: Animals; Carbocyanines; Disease Models, Animal; Electric Stimulation; Facial Nerve; Facial Nerve Injuries; Head and Neck Neoplasms; Humans; Monitoring, Intraoperative; Neural Conduction; Rats; Rats, Sprague-Dawley

Optical fluorescent technology has the potential to deliver real time imaging of cancer into the operating room and the clinic. To determine the efficacy of fluorescently labeled anti-vascular endothelial growth factor (VEGF) antibody to be used as a cancer specific optical contrast agent to guide surgical resections, we evaluated the sensitivity and specificity of this agent to detect microscopic residual disease in a preclinical model of head and neck squamous cell carcinoma (HNSCC). Using a flank murine model, mice were xenografted with SCC-1 tumor cells and injected with anti-VEGF antibody (bevacizumab) conjugated to an optically active fluorophore (Cy5.5). Tumors underwent sub-total resections and were assessed for the presence of residual disease by fluorescent stereomicroscopy. Expected positive and negative biopsies were taken according to the presence or absence of fluorescence, respectively. Histology was used to confirm the presence or absence of disease. Biopsies taken from areas of fluorescence within the wound bed (n=18) were found to be histologically malignant in all but one biopsy. Samples taken from a non-fluorescing tumor bed (n=15) were found to be histologically benign in 11 of 15. These findings correlated with a sensitivity and specificity of 80.9% and 91.7%, respectively. This data supports previous data presented by this group and supports further investigation of fluorescently labeled anti-tumor antibodies to detect disease in the surgical setting.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Carbocyanines; Head and Neck Neoplasms; Humans; Male; Mice; Mice, SCID; Neoplasm Transplantation; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

To determine whether Cy5.5-labeled antiepidermal growth factor (EGFR) antibody could be used to detect head and neck squamous cell carcinoma (HNSCC) xenografts in vivo.. AntiEGFR antibody (cetuximab) was labeled with Cy5.5, a fluorophore with emission in the near infrared range. The cetuximab-Cy5.5 conjugate was systemically administered in subtherapeutic doses (50 microg) to mice bearing orthotopically xenografted HNSCC cell lines (SCC1, CAL27, and FaDu). As a control, isotype-matched human immunoglobulin (Ig)G1k antibody labeled with Cy5.5 was systemically injected in parallel experiments. All tumor regions (n = 6) were imaged by fluorescent stereomicroscopy at 0, 6, 24, 48, or 72 hours. Tumor size was measured by high-frequency ultrasonography at 72 hours. Transcervical partial and near-total resections were then performed with stereomicroscopic imaging after each resection. The mandible and associated structures were then resected, paraffin embedded, and then serial sectioned for analysis.. Tumors could be clearly visualized by near infrared fluorescent stereomicroscopy at 48 and 72 hours after systemic administration of cetuximab-Cy5.5 but not after administration with the labeled isotype control antibody, IgG1k-Cy5.5. Ultrasound measurement of tumors (n = 5) correlated with fluorescent measurements of tumor (Spearman's coefficient, 0.92, P

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Carbocyanines; Cell Line, Tumor; Cetuximab; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Immunoglobulin G; Mice; Mice, SCID; Microscopy, Fluorescence; Transplantation, Heterologous; Ultrasonography

We have recently reported that apoptosis of T cells induced by squamous cell carcinoma of the head and neck (SCCHN) is partly Fas dependent. This tumor-induced T-cell death is mediated by the activities of caspase-8 and caspase-3 and is partially inhibited by antibodies to either Fas or Fas ligand. We report here that in contrast to apoptosis induced by agonistic anti-Fas antibody (Ab), the tumor-induced apoptotic cascade in Jurkat cells is significantly amplified by a mitochondrial loop. The involvement of mitochondria in tumor-induced apoptosis of T cells was demonstrated by changes in mitochondrial permeability transition as assessed by 3,3'-dihexiloxadicarbocyanine staining, by cleavage of cytosolic BID and its translocation to the mitochondria, by release of cytochrome c to the cytosol, and by the presence of active subunits of caspase-9 in Jurkat T cells cocultured with tumor cells. To further elucidate the significance of mitochondria in tumor-induced T-cell death, we investigated the effects of various inhibitors of the mitochondrial pathway. Specific antioxidants, as well as two inhibitors of mitochondria permeability transition, bongkrekic acid and cyclosporin A, significantly blocked the DNA degradation induced in Jurkat T cells by SCCHN cells. However, these inhibitors had no effect on cells triggered by anti-Fas Ab. Furthermore, a cell-permeable inhibitor of caspase-9, Ac-LEHD.CHO, which did not inhibit T-cell apoptosis induced by anti-Fas Ab, markedly inhibited apoptosis induced by etoposide or by coculture of Jurkat with SCCHN cells. These findings demonstrate that apoptotic cascades induced in Jurkat T lymphocytes by anti-Fas Ab or tumor cells are differentially susceptible to a panel of inhibitors of mitochondrial apoptotic events. It appears that besides the Fas-mediated pathway, additional mitochondria-dependent cascades are involved in apoptosis of tumor-associated lymphocytes. Inhibition of mitochondria-dependent cascades of caspase activation should be considered to enhance the success of immunotherapy or vaccination protocols in cancer.

Topics: Anti-Bacterial Agents; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Bongkrekic Acid; Carbocyanines; Carcinoma, Squamous Cell; Carrier Proteins; Caspase 9; Caspases; Coculture Techniques; Cyclosporine; Cytochrome c Group; Cytosol; DNA; Enzyme Activation; Enzyme Inhibitors; Etoposide; fas Receptor; Fluorescent Antibody Technique; Fluorescent Dyes; Head and Neck Neoplasms; Humans; Jurkat Cells; Lymphocytes; Microscopy, Fluorescence; Mitochondria; Oligopeptides; Protein Transport; Subcellular Fractions; T-Lymphocytes; Tumor Cells, Cultured