Imaging tumor microenvironment by Optical Fiber-Tethered Simultaneous Lifetime-resolved Autofluorescence-Multiharmonic (OFT-SLAM) microscopy

摘要 提出了一种用于一般癌症研究的无标记成像技术，称为光纤系留成像技术 同时寿命分辨自体荧光和多次谐波(OFT-SLAM)显微镜，以克服 缺乏在真实的肿瘤微环境中同时显示肿瘤和非肿瘤细胞的通用工具。 非肿瘤细胞广泛地包括成纤维细胞、血管生成细胞和浸润性免疫细胞。 参与正常的生物功能，如胚胎/成人发育和炎症/免疫 反应(例如伤口愈合)。然而，在已知整体新陈代谢的肿瘤微环境中 为了从能量消耗转向增殖生物合成(Warburg效应)，这些正常(中性)细胞 最近都被认为是犯罪(癌症)的帮凶。因此，拟议的发展 这项成像技术将询问转移的肿瘤细胞(主要)和各种 合谋杀死癌症患者的非肿瘤细胞(附件)(犯罪)。这次审问将会有更多 比基于成像的癌症研究更全面，通常专注于一种特定类型的细胞 利息(犯罪的本金或从犯)。没有像OFT-SLAM这样的免标签成像技术 避免细胞特异性标记，同时显示各种细胞会扰乱肿瘤微环境 通过外源染色、细胞/组织移植和基因改造。 我们将建立基于多模多光子显微镜和荧光寿命的OFT-SLAM 成像，并引用细胞光学异质性和代谢活动的一般内在对比来揭示 分化肿瘤细胞和非肿瘤细胞。然后，我们将通过“OFT”赋予“SLAM”灵活的访问权限 活体动物/临床前显微镜和体外人类/临床组织病理学中的不同解剖部位。 随后，我们将使用产生的OFT-SLAM对福尔马林固定的人类乳房标本进行成像 来自合作人类组织网络(CHTN)的癌症，包括原发乳腺癌、乳腺癌- 不同时期不同患者诱发的肺和脑转移及其周围野 (N&gT；200)。同时，我们将OFT-SLAM应用于长期(成像窗口辅助)活体显微镜。 三个典型的乳腺癌大鼠/小鼠模型，涵盖了整个侵袭-转移的所有已知步骤 卡斯卡德。具有OFT-SLAM的独特能力来连接原本孤立的体外人类组织病理学 (病理学家在临床环境中拍摄的快照)和活体动物显微镜(电影由 生物学家)，我们将努力识别各种癌症相关细胞及其相互关系 不断演变的肿瘤微环境及其对空间异质性和个体变异性的依赖。这个 该项目的成功结果将展示一种多功能的可视化工具来询问肿瘤 具有内置翻译能力的微环境，从而改变癌症诊断和治疗。

Summary A label-free imaging technology is proposed for general cancer research, termed as Optical Fiber-Tethered Simultaneous Lifetime-resolved Autofluorescence and Multiharmonic (OFT-SLAM) microscopy, to overcome the lack of a versatile tool to simultaneously visualize tumor and non-tumor cells in authentic tumor microenvironment. The non-tumor cells broadly include the fibroblastic cells, angiogenic vascular cells, and infiltrating immune cells that engage normal biological functions such as embryonic/adult development and inflammatory/immune response (e.g. wound healing). However, in the tumor microenvironment where the overall metabolism is known to switch from energy consumption to proliferative biosynthesis (the Warburg effect), these normal (neutral) cells have all been recently recognized as the accessories to the crime (cancer). Thus, the proposed development of this imaging technology will interrogate the interrelations between metastatic tumor cells (principal) and various non-tumor cells (accessories) that conspire to kill a cancer patient (crime). This interrogation will be more comprehensive than imaging-based cancer research that has typically focused on one specific cell type of interest (the principal or one accessory of the crime). Without a label-free imaging technology like OFT-SLAM to avoid cell-specific labeling, simultaneous visualization of various cells would perturb the tumor microenvironment by exogenous staining, cell/tissue transplantation, and genetic modification. We will build the “SLAM” of OFT-SLAM based on multimodal multiphoton microscopy and fluorescence-lifetime imaging, and invoke general intrinsic contrasts of cellular optical heterogeneity and metabolic activity to reveal and differentiate tumor and non-tumor cells. We will then empower the “SLAM” with the “OFT” to flexibly access different anatomical sites in intravital animal/preclinical microscopy and ex vivo human/clinical histopathology. We will subsequently employ the resulting OFT-SLAM to image the formalin-fixed human specimens of breast cancer from Cooperative Human Tissue Network (CHTN), including the primary breast tumors, breast cancer- induced lung and brain metastases, and surrounding peri-tumoral fields at different stages from different patients (n > 200). In parallel, we will apply OFT-SLAM to long-term (imaging window-assisted) intravital microscopy of three prototypical breast cancer rat/mouse models, covering all known steps throughout the invasion-metastasis cascade. With the unique capability of OFT-SLAM to bridge otherwise isolated ex vivo human histopathology (snapshots taken by pathologists in a clinical setting) and intravital animal microscopy (movies acquired by biologists in a laboratory), we will strive to identify various cancer-associated cells and their interrelations in an evolving tumor microenvironment and their dependence on spatial heterogeneity and individual variability. The successful outcome of this project will demonstrate a versatile visualization tool to interrogate tumor microenvironment with built-in translational ability, and thus transform cancer diagnosis and therapy.