Functional analysis of BK-type oncogenic K+ channels with nanobody-based probes in 2D and 3D HER2-positive human breast cancer cells and mouse models

肿瘤微环境（TME）中的细胞外钾离子（K+ex）和K+通道的活性已被证明影响实体癌的发生和发展。在这方面，钙离子（Ca ~（2+））和电压激活的大电导K ~+通道（BK）似乎是最重要的。BK通道的单个亚基的过表达与人乳腺癌（BC）的恶性程度较高和预后较差相关，而我们最近发现，BK的缺乏减弱了相关临床前小鼠模型中BC的发生和发展。基于BK状态和HER 2+肿瘤之间的正相关性，我们假设BK活性的调节和由此产生的K+交换在调节恶性BC细胞行为中起重要作用。然而，BK的确切作用，特别是（i）其在BC细胞中的亚基分布和相互作用，（ii）在TME内提高局部或全局K+ex池的调节作用，以及（iii）与HER 2依赖性信号通路的功能相互作用，仍然知之甚少。为了解决这些问题，我们将开发针对BK癌症相关亚基的新型纳米抗体（Nbs），用于在2D和3D BC细胞模型中对BK及其亚基进行高级光学分析。此外，这些工具将与我们最近设计的专门针对人类HER 2的基于Nb融合FRET的K+生物传感器相结合。为了研究BK在调节K+ex的功能相关变化中的推定作用，将用基于Nb的FRET成像探针监测BK阳性和BK阴性2D和3D人HER 2 + BC模型，并且将获得的K+信号与BC恶性肿瘤的多个标志相关联。同时，HER 2信号传导对BK的影响的分子和蛋白质组学分析，反之亦然，将提供这些致癌因子之间的机制联系及其对BC中K+ex的贡献的信息。为了研究这种相互作用在体内的相关性，我们将生成、重复操作和活体内成像自发产生和异种移植的BK-熟练和BK-缺陷的人HER 2 + BC小鼠模型。我们预计，该项目中开发的基于Nb的探针，结合疾病相关的细胞和体内模型，将引导新的机会来阐明K+通道在癌症发展中的作用，并作为HER 2+相关恶性肿瘤新治疗方法的潜在靶点。

Extracellular potassium ions (K+ex) in the tumor microenvironment (TME) and the activity K+ channels have been shown to impact initiation and progression of solid cancer. In this respect the calcium ion (Ca2+)-and voltage activated K+ channel of big conductance (BK), seems to be of utmost importance. Overexpression of individual subunits of the BK channel has been associated with higher malignancy and poorer prognosis of human breast cancer (BC), while we recently showed that the absence of BK attenuates BC onset and development in relevant preclinical mouse models. Based on a positive correlation between BK status and HER2+ tumors, we hypothesize that modulation of BK activity and the resulting K+ex changes play an important role in regulating malignant BC cell behaviors. However, the precise role of BK, particularly (i) its subunit distribution and interaction in BC cells, (ii) modulatory roles in raising local or global K+ex pools within the TME, and (iii) the functional interplay with HER2-depdendent signaling pathways, remains poorly understood. To address these questions, we will develop novel nanobodies (Nbs) against cancer-associated subunits of BK for advanced optical analyses of BK and its subunits in 2D and 3D BC cell models. In addition, these tools will be combined with our recently engineered Nb-fused FRET-based K+ biosensors specifically targeting human HER2. To investigate the putative effect of BK in modulating functionally relevant changes of K+ex, BK-positive and BK-negative 2D and 3D human HER2+ BC models will be monitored with Nb-based FRET imaging probes and the obtained K+ signals will be correlated with multiple hallmarks of BC malignancy. In parallel, molecular and proteomic analysis of the effects of HER2 signaling on BK and vice versa will provide information on the mechanistic link between these oncogenic factors and their contribution to K+ex in BC. To investigate the relevance of this interplay in vivo, we will generate, pharmacologically manipulate, and intravitally image spontaneously arising and xenografted BK-proficient and BK-deficient human HER2+ BC mouse models. We anticipate that the Nb-based probes developed in this project, in combination with disease-relevant cellular and in vivo models, will guide new opportunities to elucidate the role of K+ channels in cancer development and as potential targets for novel therapeutic approaches in HER2+-related malignancies.