本研究旨在探討受體外電脈衝刺激（EPS）活化之骨骼肌條件培養基（CM），是否能作為強效化療增敏劑，協同臨床一線化療藥物 Gemcitabine 抑制人類乳癌細胞（MDA-MB-231）之生長。實驗平台從常規二維（2D）單層細胞型態，橫跨至貼近真實病理之三維（3D）仿生骨轉移微組織模型。 二維平台之結果確立了骨骼肌分泌物之條件培養基（CM）作為化療增敏劑的分子可行性，乳癌細胞之半抑制濃度（IC50）隨電刺激電壓提升而呈現明確的電壓依賴性調降趨勢。然而，當研究推進至高度複雜的三維仿生骨支架環境時，乳癌微組織因立體空間幾何障壁與細胞間交互作用，展現出頑強的多細胞抗藥性（MCR）。在此組織層級環境下，相對低強度（15V）的電刺激 CM 已不再具備任何化療增敏有用性。相較之下，高強度（60V）的電刺激 CM 則展現出優秀的增敏效果，不僅能克服 3D 空間屏障引導藥物穿透至深層（250μm），更可促使中大型腫瘤團塊（>500μm）結構瓦解與小型化，成功逆轉三維組織之耐藥性並恢復 Gemcitabine 之化療毒殺效益。 綜上所述，本研究定性地證實了電刺激骨骼肌分泌物能協同化療藥物產生加成之組織毒殺效益，並揭示了環境維度與電刺激強度在抗癌療效上的關鍵交互作用。此成果為未來臨床上整合「電刺激運動處方」與「傳統腫瘤化療」之聯合抗癌策略提供了堅實的理論依據。;This study aims to investigate whether electrical pulse stimulation (EPS)-activated skeletal muscle conditioned medium (CM) can serve as a potent chemosensitizer to synergistically enhance the therapeutic efficacy of the frontline chemotherapeutic drug， Gemcitabine， against human breast cancer cells (MDA-MB-231). The experimental platform spans from conventional two-dimensional (2D) monolayers to a highly biomimetic three-dimensional (3D) scaffold model that recapitulates the breast cancer bone metastasis microenvironment at a tissue level. Results from the 2D platform established the molecular feasibility of secretions from myoblast as chemosensitizers， where the half-maximal inhibitory concentration (IC50) of breast cancer cells exhibited a distinct voltage-dependent decrease with increasing electrical stimulation intensity. However， when transitioned to the highly complex 3D biomimetic bone scaffold， the breast cancer micro-tissues displayed robust multicellular resistance (MCR) due to spatial geometric barriers and intricate cell-matrix interactions. Within this tissue-level microenvironment， conditioned medium generated from relatively low-intensity electrical stimulation (15V) lost its chemosensitizing utility completely. In contrast， high-intensity (60V) CM demonstrated superior spatial penetration capability， which not only overcomes the 3D spatial barrier to guide deep drug penetration (up to 250μm)， but also induces the structural disintegration and shrinkage of medium-to-large tumor aggregates (over 500μm). This successfully reverses the chemoresistance of 3D microtissues and restores the cytotoxic efficacy of Gemcitabine. In conclusion， this study qualitatively demonstrates that electrical stimulation-modulated skeletal muscle secretome can synergistically enhance chemotherapeutic tissue cytotoxicity， revealing a critical interaction between environmental dimensionality and electrical stimulation intensity in anticancer efficacy. These findings provide a solid theoretical foundation for future clinical translations combining "electrical stimulation exercise prescriptions" with "conventional oncological chemotherapy" as an innovative joint therapeutic strategy.
