Multi-scale Molecular Imaging of the Degradome in Breast Tumors

描述(申请人提供)：溶酶体是乳腺癌侵袭、转移和细胞外基质(ECM)重塑过程中最重要的细胞器之一，因为它们含有几种降解酶，可以影响ECM的完整性和结构。溶酶体在体内乳腺癌降解体中的作用几乎没有被研究过，部分原因是缺乏可用于可视化溶酶体的非侵入性成像能力。我们最近开发并验证了新型近红外荧光(NIRF)探针，用于对细胞培养和体内肿瘤中的溶酶体进行非侵入性成像。使用这些探针获得的初步数据支持溶酶体在乳腺癌侵袭和转移中的重要性。因此，我们打算使用这些探针来了解和定义癌细胞中的溶酶体对侵袭、转移和细胞外基质重塑的影响。I型胶原纤维是细胞外基质的重要组成部分，可促进乳腺肿瘤的形成、侵袭和转移。在这项应用中，我们将进行多尺度的分子和功能成像研究，以表征原发肿瘤和转移性结节中的Col1纤维基质，以了解癌症降解体如何塑造Col1纤维，以及这如何促进侵袭和转移。我们将使用创新和临床可转换的光学二次谐波产生(SHG)显微镜对COL1光纤进行显微成像，该显微镜检测来自COL1光纤的本征光信号。我们将利用溶酶体特异性的NIRF探针IR-2在体内对乳腺肿瘤模型中的溶酶体进行成像。我们将使用MMPSense来检测基质金属蛋白酶的活性，并使用ProSense来检测组织蛋白酶的活性，来光学成像体内的蛋白酶活性。我们将重点了解溶酶体在AIM 1中体内乳腺肿瘤侵袭中的作用，以及在AIM 2中使用多尺度光学成像方法在乳腺癌转移中的作用。在目标3中，我们将确定溶酶体和溶酶体酶对体内实体瘤和转移性结节中Col1纤维结构和完整性的影响。这三个目标将为溶酶体和溶酶体酶在乳腺癌模型中整体降解体中的作用提供新的见解。这项拟议的研究将进一步加深我们对溶酶体在癌症侵袭、转移和Col1纤维重塑中的作用的理解，这最终可能导致基于溶酶体的治疗癌症的新策略。了解溶酶体和溶酶体酶在侵袭、转移和Col1纤维重塑中的作用可能会导致创新的诊断成像技术的发展，这种技术可以非侵入性地检测溶酶体探针作为替代标记来评估这些肿瘤的侵袭性、转移潜力和Col1纤维结构。 公共卫生相关性：拟议的研究将进一步加深我们对乳腺癌降解组中溶酶体的重要性的理解，以及它们在侵袭、转移和细胞外基质重塑中的作用。这一结果可能会导致基于溶酶体的治疗乳腺癌的新策略。用近红外探测器IR-2成像溶酶体隔室大小可能提供癌症侵袭性的替代标记物。

DESCRIPTION (provided by applicant): Lysosomes are one of the most important cellular organelles in breast cancer invasion, metastasis, and extracellular matrix (ECM) remodeling, because they contain several degradative enzymes that can influence the integrity and structure of the ECM. The roles of lysosomes in the breast cancer degradome in vivo are virtually unexplored, due, in part, to a lack of noninvasive imaging capabilities available to visualize lysosomes. We recently developed and validated novel near-infrared fluorescent (NIRF) probes for noninvasive imaging of lysosomes in cell culture and tumors in vivo. Preliminary data obtained using these probes support the importance of lysosomes in breast cancer invasion and metastasis. We therefore intend to use these probes to understand and define the influence of lysosomes in cancer cells on invasion, metastasis, and ECM remodeling. Collagen type I (Col1) fibers are an important component of the ECM that can facilitate breast tumor formation, invasion, and metastasis. In this application, we will perform multi-scale molecular and functional imaging studies to characterize the Col1 fiber matrix in primary tumors and metastatic nodules to understand how Col1 fibers are shaped by the cancer degradome, and how this promotes invasion and metastasis. We will microscopically image Col1 fibers using innovative and clinically translatable optical second harmonic generation (SHG) microscopy, which detects an intrinsic optical signal from Col1 fibers. We will utilize the lysosome-specific NIRF probe IR-2 to image lysosomes in breast tumor models in vivo. We will optically image protease activities in vivo using MMPSense to detect matrix metalloprotease activities and ProSense to detect cathepsin activities. We will focus on understanding the role of lysosomes in breast tumor invasion in vivo in Aim 1, and in breast cancer metastasis in Aim 2 using multi-scale optical imaging approaches. In Aim 3, we will determine the influence of lysosomes and lysosomal enzymes on Col1 fiber structure and integrity in solid tumors and metastatic nodules in vivo. These three Aims will provide novel insights into the role of lysosomes and lysosomal enzymes within the overall degradome in breast cancer models. The proposed research will further our understanding of the role of lysosomes in cancer invasion, metastasis, and Col1 fiber remodeling, which may eventually lead to novel lysosome-based therapeutic strategies to treat cancer. Understanding the role of lysosomes and lysosomal enzymes in invasion, metastasis, and Col1 fiber remodeling may lead to the development of innovative diagnostic imaging techniques that noninvasively detect lysosomal probes as surrogate markers to assess invasiveness, metastatic potential, and Col1 fiber structure in these tumors. PUBLIC HEALTH RELEVANCE: The proposed research will further our understanding of the importance of lysosomes within the breast cancer degradome, and their role in invasion, metastasis, and extracellular matrix remodeling. The resulting findings may lead to novel lysosome-based therapeutic strategies to treat breast cancer. Imaging the lysosomal compartment size with the near-infrared probe IR-2 may provide a surrogate marker of cancer aggressiveness.