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

总结 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 同时使用寿命分辨自发荧光和多谐波（OFT-SLAM）显微镜，以克服 缺乏在真实肿瘤微环境中同时可视化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、血管生成血管细胞和浸润性免疫细胞 参与正常生物功能，如胚胎/成人发育和炎症/免疫 反应（例如伤口愈合）。然而，在肿瘤微环境中， 从能量消耗转换到增殖性生物合成（瓦尔堡效应），这些正常（中性）细胞 最近都被认为是犯罪（癌症）的从犯。因此，拟议的发展 这种成像技术将询问转移性肿瘤细胞（主要）和各种 非肿瘤细胞（附件）密谋杀死癌症患者（犯罪）。这次审讯会比 比基于成像的癌症研究更全面，通常集中在一种特定的细胞类型上， 利益（犯罪的主犯或从犯）。如果没有像OFT-SLAM这样的无标签成像技术， 避免细胞特异性标记，同时观察各种细胞会扰乱肿瘤微环境 通过外源性染色、细胞/组织移植和遗传修饰。 我们将建立基于多模多光子显微镜和荧光寿命的OFT-SLAM的“SLAM” 成像，并调用细胞光学异质性和代谢活性的一般内在对比，以揭示 并区分肿瘤和非肿瘤细胞。然后，我们将授权“SLAM”与“OFT”灵活地访问 活体动物/临床前显微镜检查和离体人体/临床组织病理学中的不同解剖部位。 随后，我们将使用所得到的OFT-SLAM对福尔马林固定的人类乳腺标本进行成像 来自人类组织合作网络（CHTN）的癌症，包括原发性乳腺肿瘤、乳腺癌- 诱导的肺和脑转移，以及来自不同患者的不同阶段的肿瘤周围区域 （n > 200）。同时，我们将应用OFT-SLAM进行长期（成像窗口辅助）活体显微镜检查， 三种原型乳腺癌大鼠/小鼠模型，涵盖了整个侵袭-转移的所有已知步骤 级联。OFT-SLAM具有独特的能力，可桥接分离的离体人类组织病理学 （病理学家在临床环境中拍摄的快照）和活体动物显微镜检查（由 在实验室中的生物学家），我们将努力识别各种癌症相关细胞及其相互关系， 不断变化的肿瘤微环境及其对空间异质性和个体变异性的依赖性。的 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 这一技术可以在具有内置翻译能力的微环境中发挥作用，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.