Multiplexed and dynamically targeted photoimmunotherapy of heterogeneous, chemoresistant micrometastases guided by online in vivo optical imaging of cell-surface biomarkers

项目摘要 尽管在手术、化疗和靶向治疗方面取得了进展，但转移性上皮性卵巢癌的生存率仍然很低。 癌症仍然令人沮丧，部分原因是肿瘤的异质性和残留的显微镜下无法检测到的疾病。 传统的成像模式。这种恶性肿瘤涉及晚期腹膜癌病;也就是说， 盆腔及其周围器官广泛的肿瘤。为了解决这些“隐形”肿瘤，我们 介绍了一系列分子靶向和细胞活化的成像和治疗剂， 新开发的用于体内多路分子成像的小型化显微镜-细胞分辨率 高光谱荧光显微内窥镜-其独特地使显微沉积物可视化， 体内的肿瘤细胞多重生物标志物成像功能的动机是迫切需要 为了定量监测对治疗抗性和逃逸显著的癌细胞表型（例如，癌症干 细胞由多个细胞表面生物标志物限定）。同时，我们还开发了近红外 靶向癌细胞过表达的细胞膜分子的光细胞毒性免疫缀合物（PIC）， 包括表皮生长因子受体（EGFR），以产生组合的光动力学和受体 用于肿瘤靶向的可活化的光免疫疗法（taPIT）的拮抗剂治疗剂。光动力 并且荧光成分在癌细胞结合时变得去猝灭（激活），细胞 内化和溶酶体抗体蛋白水解。该策略克服了脱靶光毒性，包括 肠光毒性是复杂部位（如小肠）光活化治疗的主要临床障碍， 盆腔在此，我们建立在我们先前的工作基础上，该工作表明PIC在卵巢微转移中被激活。 在使用EGFR过表达的异种移植小鼠模型中， 人上皮性卵巢癌细胞，能够准确识别小至30 μm的肿瘤， 破坏播散性腹膜微转移。我们建议进一步发展这个平台， 解决了残留肿瘤沉积物中潜伏的肿瘤异质性和化疗耐药表型， 代表了癌症治疗的关键利基。目前的临床成像技术无法解决微观 手术和化疗留下的肿瘤沉积物，患者的治疗选择有限 复发性耐药肿瘤我们预计，这种新的模式，显微内窥镜引导的taPIT 将补充目前的治疗模式，为患者的晚期疾病和那些接受 挽救疗法，为个性化医疗开辟了新的途径。新功能将应用于 引导动态靶向的taPIT适应于响应于治疗的表型进化并用于监测 显微镜下残留病的治疗反应。

PROJECT SUMMARY Despite advances in surgery, chemotherapy and targeted therapies, survival of metastatic epithelial ovarian cancer remains dismal due in part to tumor heterogeneity and residual microscopic disease undetectable by traditional imaging modalities. This malignancy involves peritoneal carcinomatosis at advanced stages; that is, extensive tumor studding of the pelvic cavity and its resident organs. To address these “invisible” tumors, we introduce a series of molecular-targeted and cell-activatable imaging and therapeutic agents integrated with a newly developed miniaturized microscope for multiplexed molecular imaging in vivo—a cellular-resolution hyperspectral fluorescence microendoscope—that uniquely enable visualization of microscopic deposits of tumor cells deep inside the body. The multiplexed biomarker imaging feature is motivated by the critical need to quantitatively monitor cancer cell phenotypes salient to treatment resistance and escape (e.g., cancer stem cells are defined by multiple cell-surface biomarkers). In parallel, we have also developed near-infrared photocytotoxic immunoconjugates (PICs) that target cell membrane molecules overexpressed by cancer cells, including the epidermal growth factor receptor (EGFR), to create a combined photodynamic and receptor antagonist therapeutic agent for tumor-targeted, activatable photoimmunotherapy (taPIT). The photodynamic and fluorescence components become de-quenched (activated) upon cancer cell binding, cellular internalization and lysosomal antibody proteolysis. This strategy overcomes off-target phototoxicity, including bowel phototoxicity, the major clinical obstacle for photoactivated treatments in complex sites such as the pelvic cavity. Here, we build on our prior work that shows the PIC is activated within ovarian micrometastases with 93% sensitivity and 93% specificity in vivo, in a xenograft mouse model using EGFR overexpressing human epithelial ovarian cancer cells, enabling accurate recognition of tumors as small as 30 μm and selective destruction of disseminated peritoneal micrometastases. We propose to further develop this platform to address tumor heterogeneity and chemoresistant phenotypes lurking within residual tumor deposits, which represents a critical niche in cancer therapy. Current clinical imaging technologies cannot resolve microscopic tumor deposits left behind by surgery and chemotherapy, and there are limited treatment options for patients with recurrent, chemoresistant tumors. We anticipate that this new paradigm for microendoscopy-guided taPIT will complement current treatment modalities for patients with advanced-stage disease and those receiving salvage therapies, opening new avenues for personalized medicine. The new capabilities will be applied to guide dynamically targeted taPIT adaptive to phenotype evolution in response to therapy and for monitoring treatment response of microscopic residual disease.