Development of an apoptosis biosensor for monitoring of breast cancer

成功监测肿瘤对新辅助化疗（NAC）的反应可以提供机会， 为乳腺癌患者量身定制的个性化治疗。目前乳腺癌的治疗一般适用于 不管治疗成功与否都是“一刀切”。监测“治疗中”反应的能力对于 1）具有完全响应并且可以从减少NAC以降低发病率中受益的患者， 2)对目前NAC无反应的患者建议改变治疗方法， 抗肿瘤反应。因此，通过基于成像的免疫组织化学技术可靠地监测肿瘤对NAC治疗的反应的能力是非常重要的。 系统是实现患者定制治疗的重要一步，将使我们能够进一步摆脱 乳腺肿瘤学中的“一刀切”模式。因此，我们建议开发一种新的基于方酸的 生物传感器（SAB）使用新的成像技术，多光谱光声识别可检测的细胞凋亡 断层扫描（MSOT）。由于MSOT是一种新的成像技术，外源性报告染料仅限于2 FDA 批准的染料，异硫烷蓝和吲哚氰绿绿色，它们都不能与肽缀合。我们 目的：（1）开发一种新的光声优化的可偶联的报告染料，作为凋亡的一部分 生物传感器和（2）在体内测试Squaraine凋亡生物传感器（SAB）以鉴定细胞凋亡， 体内化疗治疗。 我们建议建立在我们最近使用多光谱光声断层扫描识别肿瘤的成功基础上 结合我们在近红外染料化学和分子生物学方面的专业知识。为了提高细胞摄取的特异性， 在原型SAB中，我们已经包括了细胞穿透肽的一部分，其对细胞外的 我们假设我们的方酸细胞凋亡生物传感器（SAB）的主要原型将具有 改进的细胞穿透肽（TS-CPP）和“打开”的凋亡序列，DEVD，以分离 从QC 1染料中提取方酸染料，最终鉴定细胞凋亡。为了验证我们的假设，我们提出了 以下目标：1）开发和表征原型Squaraine的衍生物，作为报告者， 2）评估优化的Squaraine和SAB作为光声生物传感器的性能， 3）评估优化的SAB，以促进体内乳腺肿瘤细胞凋亡的检测， 多光谱光声层析成像这些特定目标的成功完成将导致细胞凋亡 生物传感器，最终可用于识别体内细胞凋亡和临床监测肿瘤反应， 使用光声成像可检测的新报告染料。最终，这种凋亡生物传感器将很好地 适合用作多光谱造影剂混合物的一部分，用于识别疾病的分子特征。

Successful monitoring tumor response to neo-adjuvant chemotherapy (NAC) could offer the opportunity to tightly- tailored individualized therapy in patients with breast cancer. Current treatment of breast cancer generally applies a “one-size fits all” regardless of treatment success. The ability to monitor “on-treatment” response is critical for both 1) the patients that have complete response and could benefit from reduced NAC to reduce morbidity and 2) to the patients who are not responding to current NAC to suggest a change in treatments which induce greater anti-tumor response. Thus, the ability to reliably monitor tumor response to NAC treatment via an imaging-based system is a vital step toward realizing patient-tailored therapy and would enable us to further move away from a “one size fits all” paradigm in breast oncology. Therefore, we propose to develop a new Squaraine-based biosensor (SAB) to identify apoptosis detectable using a new imaging technology, Multispectral Optoacoustic Tomography (MSOT). As MSOT is a new imaging technology, exogenous reporter dyes are limited to 2 FDA approved dyes, isosulfane blue and indocynaine green, neither of which can be conjugated to peptides. Our objective is to: (1) develop a new optoacoustically optimized conjugatable reporter dye as part of the apoptosis biosensor and (2) test the Squaraine apoptosis biosensor (SAB) in vivo to identify apoptosis following chemotherapy treatment in vivo. We propose to build upon our recent success of identifying tumors using multispectral optoacoustic tomography combined with our expertise in NIR dye chemistry and molecular biology. To improve specificity of cell uptake of the prototype SAB, we have included a portion of a cell penetrating peptide that is responsive to extracellular acidic pH. We hypothesize that our lead prototype for the Squaraine Apoptosis Biosensor (SAB) will have improved cell penetrating peptide (TS-CPP) and “turn on” at the apoptosis sequence, DEVD, to separate the Squaraine dye from the QC1 dye, ultimately identifying apoptosis. To test our hypothesis, we propose the following aims: 1) develop and characterize derivatives of prototype Squaraine to serve as the reporter for the Squaraine apoptosis biosensor (SAB); 2) assess performance of optimized Squaraine and SAB as optoacoustic agents in vitro; and 3) assess optimized SAB to facilitate detection of apoptosis in breast tumors in vivo using multispectral optoacoustic tomography. Successful completion of these specific aims will develop an apoptosis biosensor that ultimately could be used to identify apoptosis in vivo and clinically to monitor tumor response and a new reporter dye detectable using optoacoustic imaging. Ultimately, this apoptosis biosensor would be well suited for use as part of a multispectral contrast agent cocktail for identifying molecular features of disease.