Spectral Imaging of Melanoma Cell Adhesion & Metastasis

描述(申请人提供)：黑色素瘤患者的预后与诊断时原发灶的侵袭深度直接相关。因此，当在早期诊断时，患者通常通过广泛而深入地切除黑色素瘤来治愈。然而，一旦原发黑色素瘤转移到不同的部位，预后是严重的，因为转移性黑色素瘤对传统治疗方法难以奏效。根据这一发现，与黑色素瘤前病变、原发和转移性人类黑色素瘤以及从晚期人类黑色素瘤标本建立的细胞系相比，高水平表达细胞黏附分子N-钙粘素、M-CAM和β3整合素，我们推测这三个基因/蛋白单独或联合在黑色素瘤细胞侵袭和转移形成中发挥重要作用。为了验证这一假设，我们建议将反义基因打靶和光学生物成像相结合，在体内确定这三种黏附分子是否控制黑色素瘤细胞的黏附、侵袭和转移形成。为了获得这个重要但尚未回答的问题的答案，在裸鼠体内生长为皮下肿瘤的原发人类黑色素瘤将单独和联合注射在人类酪氨酸酶启动子控制下产生N-钙粘素、M-CAM和/或β3整合素反义转录的载体构建物，该载体仅在人类黑色素瘤细胞中表达。然后，这些肿瘤将被注射不同的针对人N-钙粘蛋白、M-CAM、β3整合素和S100人黑色素瘤细胞表面抗原的花菁荧光偶联抗体。因此，非侵入性的宏观光谱成像将在体内记录反义靶向黑色素瘤中可能丢失的细胞-细胞和细胞-间质相互作用，这些都不应该在非靶向对照肿瘤中观察到。在对黑色素瘤进行体内成像分析后，将处死动物，并对其切除的肿瘤组织切片和裸鼠不同器官进行定性和定量显微光谱成像。这些体外光学成像分析有望证实体内捕获的动态肿瘤内变化，并提供关于与非靶向肿瘤相比，反义靶向肿瘤组小鼠的肺部、腹部和脑中黑色素瘤转移的缺失或存在的重要信息。

DESCRIPTION (provided by applicant): The prognosis for patients with melanoma is directly related to the depth of invasion of the primary lesion at the time of diagnosis. Thus, when diagnosed at an early stage, the patient is often cured by a wide and deep excision of the melanoma. However, once primary melanoma metastasizes to different sites, the prognosis is grave because metastatic melanoma is refractory to conventional therapy. Based upon the finding that compared to melanoma precursor lesions, primary and metastatic human melanomas, and cell lines established from advanced-stage human melanoma specimens, express high levels of the cell adhesion molecules N-cadherin, M-CAM, and beta3 integrin, we postulate that these three genes/proteins, alone or in concert, play important roles in melanoma cell invasion and formation of metastases. To test this hypothesis, we propose to combine antisense gene targeting and optical bioimaging to determine, in vivo, whether these three adhesion molecules govern melanoma cell adhesion, invasion, and metastasis formation. To obtain an answer for this important but unanswered question, primary human melanomas, grown as subcutaneous tumors in nude mice, will be injected, alone and in combination, with vector constructs generating N-cadherin, M-CAM, and/or beta3 integrin antisense transcripts under the control of the human tyrosinase promoter, which is only expressed in human melanoma cells. The tumors will then be injected with different cyanine fluorochrome-conjugated antibodies specific for human N-cadherin, M-CAM, beta3 integrin, and the S100 human melanoma cell surface antigen. Thereupon, non-invasive, macroscopic Spectral Imaging will be performed to document, in vivo, possible loss of cell-cell and cell-stroma interactions in the antisense-targeted melanomas, none of which should be observed in non-targeted control tumors. Following the in vivo imaging analyses of the melanomas, the animals will be sacrificed and tissue sections of their resected tumors and different organs of the nude mice will be subjected to qualitative and quantitative microscopic Spectral Imaging. These ex vivo optical imaging analyses are expected to substantiate the dynamic intratumoral changes captured in vivo, and to provide important information regarding absence versus presence of melanoma metastases in the lungs, abdomen, and brain of mice that carded the antisense-targeted tumors compared to mice that carded the non-targeted tumors.