Department of Bioengineering, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA 94305, USA.
Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA 94305, USA.
J Control Release. 2021 Jul 10;335:281-289. doi: 10.1016/j.jconrel.2021.05.026. Epub 2021 May 21.
Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection reagent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection reagent. Secreted GLuc correlated with HSV-1 sr39TK expression with R = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.
早期癌症检测可以显著增加患者的治疗选择和存活率，但早期肿瘤的检测仍然很困难。在这里，我们展示了一种两步策略，通过将编码血液和成像报告基因组合的肿瘤激活迷你环 (MC) 质粒递送到肿瘤细胞中，来检测和定位癌性病变。我们通过控制泛肿瘤特异性 Survivin 启动子对 MC 进行基因工程改造，使其编码：1）Gaussia 荧光素酶 (GLuc)，一种可以轻松在血液样本中检测到的分泌生物标志物；2）单纯疱疹病毒 1 胸苷激酶突变体 (HSV-1 sr39TK)，一种 PET 报告基因，可用于肿瘤位置的高灵敏度和定量成像。我们评估了两种 MC 递呈方法，即用化学转染试剂 jetPEI 复合 MC 或用源自人宫颈癌 HeLa 细胞系的细胞外囊泡 (EV) 包裹 MC。与没有转染试剂的 MC 相比，EV 或 jetPEI 递呈的 MC 在细胞培养中产生了报告基因的显著表达。分泌的 GLuc 与 HSV-1 sr39TK 表达呈高度相关，R = 0.9676。与 jetPEI 复合的 MC 传递了更大质量的 MC，从而增强了转染，这对于体内动物研究至关重要，其中通过 jetPEI 递呈 MC 导致 GLuc 和 HSV-1 sr39TK 的表达水平明显高于对照。据我们所知，这是首次报道通过肿瘤激活的 MC 递呈 PET 报告基因 HSV-1 sr39TK 用于早期癌症检测策略的报告。这项工作探索了早期癌症检测策略中内源性血液生物标志物和分子成像的局限性的解决方案，并阐明了 EV 的递呈能力和局限性。
