MALDI Imaging of Cancer Signaling Signatures

描述(由申请人提供)：检测与致癌信号相关的细胞激酶的激活可以作为癌症诊断的有价值的分子标记物，并作为选择治疗的预测工具。在人头颈部鳞状细胞癌(SCCHN)中，EGFR的激活与治疗耐药、转移增加和预后不良有关。组织学和肿瘤结构提供了关于癌症分期和分级的补充和关键信息。通过适应和推广新兴的激酶传感器生物芯片技术，我们建议开发成像通过活组织检查或手术切除获得的肿瘤组织中癌症信号分布的能力。我们和其他人利用免疫检测、放射性核素掺入或MALDI-TOF MS分析，开发了可靠、灵敏和特定的基于生物芯片的细胞裂解物中激酶活性的分析方法。我们现在打算采用这些方法来创建多重分析，即作为参与致癌信号转导的激酶的特定底物的多个肽将可逆地连接到生物芯片的表面。这种多路生物传感器将通过“组织印迹”被暴露在肿瘤活检的厚片中，以允许多肽的磷酸化。细胞材料将被冲走，生物芯片将通过MALDI-TOF MS成像进行询问，以检测相对的多肽磷酸化，从而创建跨肿瘤切片的多种激酶活动的图像。这些激酶活性图像可以与常规组织学和免疫细胞化学相关联，以提高诊断和预后。为了在R21阶段建立原理证明，将开发使用人SCCHN组织培养细胞和在裸鼠体内生长的异种移植瘤的方法。我们将利用这种肿瘤的活化EGFR激酶特性以及特定的EGFR抑制剂吉非替尼和厄洛替尼的可用性来开发和验证我们的生物芯片传感器和成像能力。我们期望获得足够的灵敏度和分辨率来检测广泛间质中的SCCHN肿瘤岛。

DESCRIPTION (provided by applicant): Detection of the activation of cellular kinases associated with oncogenic signaling can serve as a valuable molecular marker for cancer diagnosis and as a predictive tool for selection of therapy. In human squamous cell carcinoma of the head and neck (SCCHN), EGFR activation is associated with therapeutic resistance, increased metastasis and poor outcomes. Histology and tumor architecture provide complementary and critical information about cancer stage and grade. By adapting and extending emerging technologies for kinase sensor biochips, we propose to develop the capability to image the distribution of cancer signaling in tumor tissue obtained by biopsy or surgical excision. We and others have developed robust, sensitive and specific biochip-based assays for kinase activity in cellular lysates, using immunodetection, radionuclide incorporation or MALDI-TOF MS analysis as a read-out. We now intend to adapt these methods to create multiplexed assays whereby multiple peptides, serving as specific substrates for kinase involved in oncogenic signaling, will be linked reversibly to the surface of a biochip. This multiplexed biosensor will be exposed to thick sections of tumor biopsies by "tissue print" to allow phosphorylation of the peptides. The cellular material will be washed away and the biochip will be interrogated by MALDI-TOF MS imaging to detect relative peptide phosphorylation, creating an image of the multiple kinase activities across the tumor section. These kinase activity images can be correlated with conventional histology and immunocytochemistry to enhance diagnosis and prognosis. To establish proof-of-principle in the R21 phase, methods will be developed using human SCCHN tissue culture cells and xenograft tumors of grown in athymic nude mice. We will take advantage of the activated EGFR kinase characteristic of this tumor and the availability of the specific EGFR inhibitors gefitinib and erlotinib to develop and validate our biochip sensor and imaging capabilities. We anticipate achieving sufficient sensitivity and resolution to detect SCCHN tumor islands embedded in extensive stroma.