Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA, USA.
Deprtment of Population Sciences, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA, USA.
J Nanobiotechnology. 2024 Sep 28;22(1):589. doi: 10.1186/s12951-024-02858-x.
Patients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy - such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids.
Using Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls.
SEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.
HER2 阳性乳腺癌患者可从 HER2 靶向治疗中显著获益，如单克隆抗体曲妥珠单抗。然而，部分患者会出现治疗耐药或 HER2 状态改变。因此，我们迫切需要新的非侵入性策略来频繁监测患者。肿瘤细胞分泌的细胞外囊泡（EVs）正成为有潜力的生物标志物候选物。这些膜限定的纳米颗粒携带有源细胞的分子特征；快速报告细胞状态的变化；并可从可及的生物体液中频繁取样。
我们使用结合 EV 亲和分离与超分辨率显微镜的单 EV 纳米显微镜（SEVEN）平台，以 8nm 的精度和分子灵敏度提供了 EV 的多参数特征分析。我们首先研究了在四跨膜蛋白 CD9、CD63 和 CD81 上亲和富集的细胞培养 EV；这些跨膜蛋白通常存在于 EV 膜上。SEVEN 可稳健地提供单个、四跨膜蛋白富集 EV 的关键参数：浓度、大小、形状、分子货物含量和异质性。与曲妥珠单抗敏感细胞相比，曲妥珠单抗耐药细胞分泌的 EV 更多。此外，耐药细胞来源的 EV 四跨膜蛋白密度更低，HER2 密度更高。我们还评估了在 HER2 上亲和富集的 EV；发现这些 EV（与四跨膜蛋白富集 EV 相比）更大且更狭长。我们进一步优化了分析样本处理，以评估来自患者血浆的稀有 HER2 富集 EV 群体。在 HER2 蛋白表达升高的乳腺癌患者（与对照组相比）中，HER2 富集 EV 具有独特特征，包括通常增加的四跨膜蛋白分子数量和更大的尺寸。重要的是，与无癌对照组相比，这些 EV 的平均丰度高 25 倍。
SEVEN 揭示了培养细胞和复杂生物体液中 HER2 富集 EV 的独特特征。与当前的临床方法相结合，该方法有望支持精准的治疗决策。
