三陰性乳癌低氧外泌體於癌相關纖維細胞分化之影響

Abstract

三陰性乳癌（triple negative breast cancer, TNBC）因缺乏雌激素受體、黃體素受體及人類上皮生長因子2，導致傳統激素治療效果有限；且較其他乳癌亞型而言有較差的預後以及高轉移性。在腫瘤中，低氧（hypoxia）是普遍存在的特徵，其能透過影響下游轉錄促進腫瘤增生，並可依成因及時間長短分為急性（acute）與慢性（chronic）等低氧現象，不同時長的低氧處理也會造成hypoxia inducible factor (HIF)表現量產生切換的現象。低氧現象也會造成細胞的基因體產生不穩定性。腫瘤分泌的外泌體（exosomes）在轉移前利基（pre-metastatic niche, PMN）的形成中扮演關鍵角色，並能誘導其中重要的纖維母細胞轉化為癌相關纖維細胞（CAFs）。然而，目前對於三陰性乳癌低氧外泌體對於癌相關纖維細胞的影響及機制尚未明確。本研究旨在界定三陰性乳癌細胞株MDA-MB 231在低氧狀態下的兩種HIFs－HIF-1α 與 HIF-2α 表現切換的時間點，以界定急性低氧及慢性低氧的時間點，並探討急性與慢性低氧外泌體對NIH-3T3纖維細胞分化的影響。結果顯示，在 1% O₂ 濃度下，12 小時缺氧處理可視為類急性低氧（HIF-1α 高表現），而 72 小時則為類慢性低氧（HIF-2α 高表現）。除此之外，正常氧（Normoxia）、急性及慢性低氧TNBC外泌體均能誘導 NIH-3T3 表現 CAF 標誌蛋白，但正常氧與急性低氧外泌體誘導 α-SMA 及 γ-H2AX的程度高於慢性低氧外泌體。針對外泌體進行的蛋白質體學分析進一步顯示，慢性低氧外泌體具有更豐富的蛋白質種類，與外泌體標誌蛋白，且富集於缺氧訊號、糖解作用、蛋白質摺疊錯誤及免疫相關路徑。此外，慢性低氧外泌體在 DNA 修復路徑上呈現略微的上調，這可能解釋了其誘導受體細胞表現出相較急性低氧外泌體處理組別較低的 DNA 損傷的原因。總體而言，本研究確立了 TNBC 缺氧外泌體在 CAF 誘導中的差異性，並揭示了慢性缺氧外泌體在免疫調節中的潛在作用。

Triple negative breast cancer (TNBC) possessed poorer prognosis and higher metastasis rate compared to other breast cancer subtypes. Hypoxia is a hallmark of tumors that promotes tumorigenesis by influencing gene transcription; it can be categorized into acute and chronic forms based on etiology and induces genomic instability within cells. Tumor-derived exosomes play a pivotal role in the formation of the pre-metastatic niche (PMN) and is able to induce the differentiation of fibroblasts into cancer-associated fibroblasts (CAFs). However, the specific effects and underlying mechanisms of exosomes derived from hypoxic TNBC cells on CAFs remain unclear. This study aims to identify the time-dependent manner at which the expression of HIF-1α and HIF-2α switches in the TNBC cell line MDA-MB-231 under hypoxic conditions—thereby defining the onset of acute versus chronic hypoxia—and to investigate the effects of exosomes derived under these conditions on the differentiation of NIH-3T3 fibroblasts. The results demonstrated that, at 1% O₂, a 12 hour hypoxic treatment could be referred to as acute hypoxia-like phenotype, whereas a 72 hour treatment represented chronic hypoxia-like phenotype. Furthermore, exosomes derived from TNBC cells under normoxic, acute hypoxic, and chronic hypoxic conditions all induced the expression of CAF marker proteins in NIH-3T3 cells; however, different treatment of exosomes lead to distinct CAF marker expression. Proteomic analysis revealed that exosomes derived from chronic hypoxic conditions possessed a richer diversity of proteins and exosomal markers, with enrichment in pathways related to hypoxic signaling, glycolysis, protein misfolding, and immune regulation, while normoxic exosomes express higher enrichment in extracellular matrix constructions. Additionally, chronic hypoxia-derived exosomes demonstrated a slight upregulation in DNA repair pathways. In summary, this study establishes the differential effects of TNBC-derived hypoxic exosomes on CAF induction and reveals the potential role of chronic hypoxia-derived exosomes in immune regulation.