Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma.

OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

Case-specific three-dimensional hologram with a mixed reality technique for tumor resection in otolaryngology.

OBJECTIVE: We report our first experience of using a case-specific three-dimensional (3D) hologram for tumor resection in otolaryngology to show the proof of concept. In addition, a questionnaire was administered to assess the usefulness of the mixed reality technique in otolaryngology. METHODS: A case-specific 3D hologram was developed from enhanced images of dynamic computed tomography, with reference to contrast-enhanced magnetic resonance images, and used for preoperative planning and intraoperative image reference. To evaluate the usefulness of the 3D hologram with head mount displays (HMDs), 18 attendings and resident otolaryngologists completed a questionnaire with the Likert scale. RESULTS: The case-specific 3D hologram on HMDs was successfully used by means of easy gesture-handling without any monitors preoperatively and intraoperatively. The experience of picturing the tumor localization and evaluating the surgical approach was statistically better using the 3D hologram on HMDs than using the computer images (P < .01). Similarly, the holograms were observed to be better for intraoperative application and surgical education than computer images (P < .01). CONCLUSION: We demonstrated the use of a case-specific 3D hologram for tumor resection in otolaryngology. The technology may be useful for preoperative planning and intraoperative image reference, especially for challenging cases, and surgical education. LEVEL OF EVIDENCE: NA.

Elevated exosomal lysyl oxidase like 2 is a potential biomarker for head and neck squamous cell carcinoma.

OBJECTIVES: The secretory enzyme lysyl oxidase like 2 (LOXL2) is speculated to contribute to tumor progression through its functions in the remodeling of extracellular matrix and epithelial-mesenchymal transition. We previously identified elevated expression of LOXL2 in metastatic human head and neck squamous cell carcinoma (HNSCC) cells in a mouse lymph node metastases model. Here we performed a case series study examining LOXL2 expression levels in human serum from HNSCC patients to evaluate whether LOXL2 is worth evaluation in a large cohort study. METHODS: LOXL2 protein levels in three serum samples from HNSCC patients were assessed by immunoblotting and LOXL2 tissue expression was examined in one human tongue squamous cell carcinoma (SCC) tissue by immunohistochemistry as a representative of HNSCC tissue. Serum samples were further fractionated in exosomes and supernatants by ultracentrifugation, which were then subjected to immunoblot and in vitro LOX activity analyses. Exosomal LOXL2 levels of 36 serum samples from HNSCC patients and seven healthy volunteers were measured using polymer sedimentation exosome preparation followed by ELISA measurement and subjected to statistical analyses. RESULTS: Immunoblot analyses revealed that LOXL2 was present in serum exosomal fractions from three HNSCC patients, and we observed approximately threefold higher levels of LOXL2 in HNSCC patients compared with three healthy volunteers. Immunohistochemical LOXL2 staining was detected in HNSCC cells in addition to non-cancerous lipid tissues and some muscles in human tongue HNSCC tissue. Further measurements of exosomal LOXL2 by ELISA showed over ninefold higher mean LOXL2 levels in patients compared with controls. Statistical analysis revealed a correlation between elevated serum exosomal LOXL2 levels and low-grade, but not high-grade, HNSCC. CONCLUSIONS: Our case series study that elevated serum exosomal LOXL2 levels exhibited a correlation with low-grade HNSCCs. A follow-up large cohort clinical study will be required to determine the potential clinical utility of LOXL2 as a new biomarker and/or therapy target for HNSCCs. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E327-E334, 2020.

Development of proficiency-based knot-tying and suturing curriculum for otolaryngology residents: A pilot study.

OBJECTIVE: Basic surgical skills such as knot-tying and suturing are important for all otolaryngologists, regardless of subspecialty. The present study was undertaken in order to assess basic surgical techniques such as knot-tying and suturing required for novice otolaryngology residents with taking the variety of subspecialties into consideration, and evaluate the impact of a proficiency-based training curriculum based on these techniques. METHODS: A prospective study was performed for developing of proficiency-based knot-tying and suturing curriculum for otolaryngology residents in the third post-graduate year (PGY-3). The proficiency-based training curriculum was developed based on the tasks selected by RAND/UCLA method with expert panel, which is an iterative and anonymous survey used to establish consensus among participants. Expert panelists were selected from various divisions to reflect variety of their subspecialties. PGY-3 residents trained with the developed curriculum that included proctored pre-test, self-training to proficiency, and proctored post-test. Visual analogue scale (VAS) of trainees' overall competence in the operating room was self-assessed by each resident, before and after completing the training curriculum. RESULTS: Nine PGY-3 residents were enrolled as trainees. Eleven experts chosen as panelists had various subspecialty, including 2 from otology, 2 from rhinology, 2 from laryngology, 2 from head and neck surgery, and 3 from general otolaryngology. Seven tasks were selected from RAND/UCLA method and used to develop the curriculum. Trainee scores at pre-test were significantly lower than expert scores for all 7 tasks (p < 0.01) and each coefficient of variation of trainee score was larger than that of expert score (p < 0.05), supporting construct validity. The mean of composite scores between pre-test and post-test had statistical significance (68.6 ± 11.6 vs 95.9 ± 3.6, p < 0.01), documenting substantial improvement after training. Self-assessment VAS was also improved pre- to post-training (1.2 ± 0.9 vs 4.5 ± 1.4, p < 0.01). A follow-up questionnaire showed that trainees felt the educational curriculum to be beneficial. CONCLUSION: In the present study, seven basic technical skills were selected using the RAND/UCLA method and used to create a proficiency-based training curriculum. Our results indicate that this curriculum significantly improves proficiency of basic surgical skills of junior otolaryngology residents.

Elevated Na+/H+ exchanger-1 expression enhances the metastatic collective migration of head and neck squamous cell carcinoma cells.

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na+/H+ exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy.