Optical imaging of a CD44-based oral-rinse with fluorescent detection to visualize oral cancer

口腔鳞状细胞癌（包括口腔和口咽部的鳞状细胞癌）占影响这些组织的所有癌症的90%以上。在所有主要癌症中，口腔癌的5年生存率最低，只有62.7%。从历史上看，与这种癌症相关的死亡率特别高，因为它是在晚期（III期或IV期）发现的，通常是当它扩散到淋巴结或更深的结构时。晚期癌症死亡率高，治疗费用高，发病率高。目前口腔癌诊断的“金标准”是身体和视觉检查，然后进行活检。不幸的是，由于诊断的延误，大多数病例被诊断为晚期，即使经过积极的治疗方案，包括放射、手术和化疗的组合，生存率也在20-40%左右。然而，如果早期诊断（I/II期），5年生存率可高达80-90%，这意味着可节省大量医疗费用。因此，迫切需要一种改进的口腔癌早期检测方法，以挽救生命，改善临床结果并降低治疗费用。我们开发了一种独特的，易于使用的，针对口腔癌的，基于肿瘤起始标记CD44和总蛋白的快速侧流点护理（POC）测试，可以准确评估患者患口腔癌的风险。OncAlert的横向血流测试条从8500万烟酒使用者中识别出那些最有可能患有早期口腔癌或发展为口腔癌的人。然而，这些早期病变通常是肉眼看不见的，所以在非常早期的疾病中，单靠试纸不能显示异常细胞的位置。为了满足这一迫切的医疗需求，在这项应用中，我们将开发一种成像设备，该设备将包括一个简单的冲洗和唾液溶液，其中含有一个与不可吸收的微球结合的antiCD44探针，该微球将与表达异常高水平CD44的细胞结合。然后，结合的探针可以通过照射特定波长的光来可视化，这样就可以识别出非常早期的病变。如果成功，这有望在视觉症状出现之前就能早期发现和干预口腔癌，从而解决一个关键的临床未满足的需求。我们将进行以下具体目标：在具体目标#1中，我们将测试一个体外模型，用于使用膜上的荧光探针检测CD44升高的细胞。我们将确定这种潜在的显像剂是否能识别细胞表面的CD44。为了做到这一点，我们将培养具有良好特征的细胞系，这些细胞系被设计成过表达或过表达CD44。在特异性目标#2中，我们将在人类肿瘤移植体中测试该系统。我们将确定这种结合物在人体中识别致癌作用的能力。我们将在手术切除后立即使用人的体外空气消化道标本。我们将使用抗cd44荧光标记珠分析10例周围有正常边缘的口腔癌组织和10例因良性疾病（慢性扁桃体炎）切除的患者组织。

DESCRIPTION (provided by applicant): Oral squamous cell carcinomas (including those of the mouth and oropharynx) comprise more than 90% of all cancers affecting these tissues. Of all the major cancers, oral cancer has one of the worst five-year survival rates of 62.7%. Historically, the death rate associated with this cancer is particularly high because it is discovered late (stage III or IV), often when it has spread to the lymph nodes or deeper structures. Late stage cancers have high mortality, are expensive to treat and have high morbidity. The current "gold standard" for oral cancer diagnosis is a physical and visual examination followed by biopsy. Unfortunately, due to delays in diagnosis, most cases are diagnosed in late stage with a survival rate around 20-40% even after aggressive treatment regimens including combinations of radiation, surgery and chemotherapy. However, if diagnosed early (stage I/II), the five-year survival-rate can be as high as 80-90%, representing a significant healthcare cost savings. Thus, there is a pressing need for an improved oral cancer early detection test which will save lives, improve clinical outcome and reduce treatment costs. We have developed a unique, easy to use, oral cancer specific, rapid lateral flow point-of-care (POC) test based on the tumor initiating marker CD44 and total protein that accurately assesses patient's risk for oral cancer. The OncAlert lateral flow test strip identifies those individuals a most risk of harboring early or developing oral cancer from the 85 million tobacco and alcohol users. However, often these early lesions can be invisible to the naked eye, so the test strip alone will not indicate where the abnormal cells are in cases of very early disease. To answer this pressing medical need, in this application, we will develop an imaging device that will involve a simple rinse and spit solution with an antiCD44 probe bound to non-absorbable microspheres that will bind to cells expressing abnormally high levels of CD44. The bound probe can then be visualized by shining a light of specific wavelength so that very early lesions can be identified. If successful, this is expected to enable early detection and intervention of orl cancer even be- fore visual symptoms are revealed, thus addressing a crucial clinically unmet need. We will carry out the fol- lowing specific aims: in Specific Aim #1 we will test an in-vitro model for detecting CD44 elevated cells using fluorescent probes on membranes. We will determine whether this potential imaging agent recognizes CD44 on the surface of cells. To do this we will culture well-characterized cell lines that have been engineered to overexpress or underexpress CD44. In Specific Aim #2 we will test the system in tumor explants from humans. We will determine how well the conjugate identifies carcinogenesis in humans. We will use human up- per aero-digestive tract specimens ex vivo, immediately following surgical resection. We will analyze 10 oral cancer tissues with surrounding normal margin and 10 patient tissues that were excised for benign conditions (chronic tonsillitis) using the anti-CD44 fluorescently labeled beads.