Idelalisib

喜欢我们，设为星标，接收最新类器官进展与资讯！知识星球搜索“肠道类器官”获取更加专业的培养基配方、标准方案、AI类器官工具和技术手册。  传播科学知识 促进同行交流 推动产业发展 服务国家战略  本公众号由Organoid Agent智能体强力赋能，让您的类器官研究始终站在潮头浪尖！ 推荐语       该研究系统揭示了胰腺导管腺癌（PDAC）中磷脂酰肌醇-3-激酶（PI3K）α/δ亚型与小泛素样修饰（SUMO）通路之间的互为依赖关系。通过CRISPR筛选、多组学分析及体内模型验证，研究者发现单独抑制PI3K或SUMO通路会诱导对方代偿性激活，而联合靶向PI3Kα/δ与SUMO E1酶可触发协同致死效应，显著抑制肿瘤生长并增强免疫细胞浸润。该策略不仅克服了既往PI3K单药治疗无效的瓶颈，还揭示了肿瘤应激反应中的新脆弱性，为PDAC这一高度耐药肿瘤提供了具有临床转化潜力的理性联合疗法。研究兼具机制深度与治疗前瞻性，对推动靶向治疗与免疫微环境调控的整合具有重要启示。 技术路线图 科学背景与研究现状 一、胰腺癌临床治疗的严峻挑战与研究紧迫性​ 胰腺导管腺癌（Pancreatic Ductal Adenocarcinoma, PDAC）作为胰腺癌的主要病理亚型，以高度侵袭性、早期转移特性和极差预后著称，其 5 年生存率长期低于 13%，部分研究甚至显示不足 12%，预计到 2030-2040 年将攀升为全球癌症死亡的第二大原因。临床治疗面临多重困境：首先，化疗作为基础治疗手段，虽以 FOLFIRINOX 或吉西他滨联合白蛋白紫杉醇为标准方案，但 80% 以上患者会在短期内产生耐药，且副作用显著影响生活质量；其次，靶向治疗进展缓慢，尽管 KRAS 基因突变在 PDAC 中发生率高达 90%，但 KRAS 抑制剂易引发继发耐药，且仅对部分突变亚型有效；2024 年 NCCN 指南（2024.V2）新增的 HER2 阳性患者靶向方案（德曲妥珠单抗），在胰腺癌队列中中位总生存期（mOS）仍仅 5.0 个月，疗效远未达临床需求。这些现状凸显了 PDAC 治疗新策略探索的迫切性。​ 二、核心信号通路的研究进展与现存空白​ （一）PI3K 通路：从致癌机制到靶向困境​ 磷脂酰肌醇 3 - 激酶（Phosphatidylinositol 3-Kinase, PI3K）通路是调控 PDAC 发生、增殖及耐药的核心信号网络，其通过催化磷脂酰肌醇（PI）磷酸化生成 PIP3，激活下游 AKT 等分子，调控细胞生长、代谢及存活信号。研究证实，PDAC 中 PI3K 通路异常激活主要源于两点：一是肿瘤抑制蛋白 PTEN（Phosphatase and Tensin Homolog）功能缺失，其可通过去磷酸化 PIP3 拮抗 PI3K 信号；二是上游调控因子异常，如角鲨烯环氧化酶（SQLE）过表达可激活 Src/PI3K/Akt 通路，促进肿瘤生长。​ 尽管 PI3K 通路的致癌作用明确，但靶向治疗遭遇瓶颈：早期单靶点 PI3K 抑制剂（如泛 PI3K 抑制剂 buparlisib）在 PDAC 临床试验中未显示显著生存获益，原因在于通路代偿激活及肿瘤异质性导致的耐药。这提示单一阻断 PI3K 通路难以突破治疗壁垒，需探索通路间协同调控机制。​ （二）SUMO 通路：从蛋白修饰机制到癌症关联​ 类泛素化修饰（SUMOylation）是由小泛素相关修饰蛋白（Small Ubiquitin-like Modifier, SUMO）介导的可逆性翻译后修饰，通过 SUMO 激活酶（E1）、结合酶（E2）和连接酶（E3）的级联反应，调控靶蛋白的定位、活性及稳定性，参与转录调控、DNA 损伤修复及应激响应等关键生物学过程。​ 近年研究揭示 SUMO 通路与 PDAC 密切相关：Wang 等通过 TCGA 数据库分析发现，PDAC 组织中 SUMO1、SUMO2/3 蛋白水平显著高于正常胰腺组织，SUMO E1 抑制剂 TAK-981 可显著抑制 PDAC 细胞增殖；余健秀实验室更发现 SUMO1 可通过修饰 PTEN 的 C2 结构域，增强其膜结合能力，直接拮抗 PI3K-AKT 信号活化。这些发现证实 SUMO 通路在 PDAC 中的促癌作用，但现有研究存在明显空白：SUMO 通路与 PI3K 通路在 PDAC 中是否存在直接互依性？这种互依性能否成为治疗靶点？尚无研究给出明确答案。​ （三）类器官模型：临床转化研究的技术突破​ 患者源性肿瘤类器官（Patient-Derived Organoids, PDO）是由肿瘤组织体外培养形成的三维结构，保留了原肿瘤的病理亚型、基因特征及异质性，在药物敏感性预测和治疗策略开发中展现独特优势。高栋团队构建的 239 例 PDAC 类器官库证实，PDO 的多组学特征与临床疗效高度关联，基于 PDO 的药敏筛选可鉴定出 79.6% 患者携带的可靶向基因变异；另一项前瞻性研究显示，PDO 对药物反应的预测敏感性达 83.3%，特异性达 92.9%，且与患者无进展生存期显著相关。​ 但 PDO 模型的应用仍存探索空间：不同病理亚型（如上皮型、间质型）PDAC 的 PDO 对靶向治疗的响应差异及分子机制尚未明确，这限制了精准治疗方案的个性化实施。​ 三、核心科学问题的提出与研究必要性​ 综合上述研究现状，PDAC 治疗研究面临三重关键瓶颈：其一，单一通路靶向治疗易触发代偿耐药，PI3K 或 SUMO 通路的单靶点抑制均无法实现持续疗效；其二，两条通路在 PDAC 中的互作机制尚未阐明，缺乏基于通路协同调控的治疗策略；其三，不同亚型 PDAC 对靶向治疗的响应差异不明，难以实现精准分层治疗。​ 由此引出核心科学问题：PI3K 与 SUMO 通路在 PDAC 中是否存在可靶向的互依性？针对这种互依性的联合靶向治疗能否诱导合成致死效应，并有效克服耐药？不同亚型 PDAC 对该联合方案的响应差异及机制是什么？​ 本研究的开展具有强烈必要性：一方面，其可填补 PI3K-SUMO 通路互作机制的研究空白，为理解 PDAC 耐药本质提供新视角；另一方面，通过类器官模型验证联合治疗的有效性及亚型特异性，可为临床转化提供直接依据，有望突破 PDAC 治疗的现有困境，为患者带来新的生存希望。​ 实验技术方法1. PDO 样本来源与基础特征样本来源 ：选取 5 例 PDAC 患者的肿瘤组织，分为两类亚型 —— 上皮样（epi-PDO：PAN41T、PAN74T）和间质样（mes-PDO：PAN2T、PAN17T、PAN70T），均进行 KRAS、CDKN2A、TP53、SMAD4 基因突变检测及转录组谱分析，确保模型与原肿瘤遗传背景和表型一致。核心目的 ：对比不同亚型 PDO 对标准治疗（SOC）和 PI3Kα/δ-SUMO 联合治疗的响应差异，验证联合治疗对间质样 PDAC（临床中更耐药）的针对性疗效。2. PDO 分离与培养关键步骤（补充材料详述，正文核心信息提取）预处理 ：将新鲜 PDAC 肿瘤组织剪碎后，用胶原酶消化获得单细胞悬液，过滤去除杂质以富集肿瘤细胞；培养基配置 ：采用含 EGF、Noggin、R-Spondin-1 等生长因子的类器官专用培养基（模拟体内肿瘤微环境），维持 PDO 三维结构与增殖能力；培养条件 ：接种于 Matrigel 基质胶中（提供细胞黏附与三维支撑），置于 37℃、5% CO₂培养箱，每 3-4 天更换一次培养基，待 PDO 直径达 100-200μm 时进行药物处理。3. PDO 药物敏感性检测与分析检测药物与分组 ： 标准治疗组：FOLFIRINOX（亚叶酸 + 氟尿嘧啶 + 伊立替康 + 奥沙利铂，PDAC 临床一线化疗方案）； 联合治疗组：pictilisib（PI3Kα/δ 抑制剂，浓度梯度设置）+ subasumstat（SUMO E1 抑制剂，固定浓度 10nmol/L）； 单药对照组：单独 pictilisib 或 subasumstat，以及空白对照（含 DMSO 的培养基）。检测步骤 ： 将对数生长期 PDO 接种于 96 孔板（每孔含等量 Matrigel），培养 24h 使其适应环境； 加入对应浓度药物，继续培养 96h； 加入 CellTiter-Glo 试剂（Promega，通过检测 ATP 含量反映细胞活力），室温孵育 30min 后，用 FLUOstar-OPTIMAmicroplate-reader（BMG-Labtech）检测发光值； 计算各组相对活力（以空白对照为 100%），判断 PDO 对药物的敏感性。后续分析 ：对药物响应差异显著的 PDO 进行转录组测序，通过基因集富集分析（GSEA）验证间质样 PDO 中 SUMOylation 信号的高富集特征，解释其对联合治疗的高响应机制。二、细胞水平核心实验技术1. 药理筛选实验（发现 PI3K 与 SUMO 通路互依性）实验目的 ：筛选与 SUMO 抑制剂（subasumstat）存在合成致死效应的通路抑制剂，聚焦 PI3K 通路。关键试剂与材料 ： 药物库：≥99 种靶向 PDAC 相关通路的抑制剂（购自 Selleckchem），其中 PI3K 亚型抑制剂包括 alpelisib（PI3Kα）、GSK2636771（PI3Kβ）、eganelisib（PI3Kγ）、idelalisib/parsaclisib（PI3Kδ），copanlisib/pictilisib（PI3Kα/δ 双抑制剂，购自 MedChemExpress，货号分别为 HY-15346R、HY50094）；subasumstat（SUMO E1 抑制剂，由 Takeda 赠送）； 细胞系：人 PDAC 细胞系 MiaPaCa-2、PSN-1，小鼠 PDAC 细胞系 53631PPT。详细步骤 ： 细胞接种：将对数生长期细胞以 5×10³ 细胞 / 孔接种于 96 孔板，培养 24h； 药物处理：设置 subasumstat 的 20% 生长抑制浓度（GI₂₀）为基础，联合不同浓度的 PI3K 抑制剂（4×5 浓度矩阵），单独药物组与空白对照组同步设置； 活力检测：培养 72h 后，加入 25μL CellTiter-Glo 试剂，室温孵育 15min，检测发光值； 数据分析：用 RStudio（Posit Software）的 GRmetrics 脚本计算药物浓度 - 效应曲线下面积（AUC）和半数抑制浓度（IC₅₀），通过 SynergyFinder 软件的 Zero Interaction Potency（ZIP）方法计算协同得分，判断是否存在合成致死效应。2. CRISPR/Cas9 基因编辑实验（验证 PI3Kα/δ 亚型必要性）实验目的 ：通过基因敲除明确 PI3Kα/δ 亚型在联合治疗中的特异性作用，排除其他亚型干扰。关键试剂与材料 ： 编辑工具：sgRNA（针对 PI3Kα 的外显子 2、PI3Kδ 的外显子 3，序列见补充材料）、Cas9 蛋白（购自 PNA-Bio）； 转染系统：Neon-Transfection-System（Thermo Fisher/Invitrogen）； 验证试剂：Terra PCR Direct Card Kit（Takara Bio，用于检测基因切割效率）、PI3Kα/δ 特异性一抗（用于免疫印迹验证蛋白敲除效果）。科普知识 ：CRISPR/Cas9 是一种定点基因编辑技术，核心是 “sgRNA 引导 + Cas9 切割”——sgRNA 通过碱基互补结合目标基因的特定序列，引导 Cas9 核酸酶在该位点切割 DNA，造成双链断裂；细胞修复断裂时易产生移码突变，导致基因功能丧失（敲除），可精准验证特定基因的生物学功能。详细步骤 ： 细胞转染：取 150,000 个 MiaPaCa-2 细胞，与 500ng sgRNA、1μg Cas9 蛋白混合，用 Neon 转染系统进行转染； 切割效率验证：转染 72h 后，提取细胞基因组 DNA，用 Terra PCR Direct Card Kit 扩增目标外显子区域，通过电泳或测序判断切割效率； 单克隆筛选：将转染后细胞进行梯度稀释（单克隆化），接种于 96 孔板，培养 2-3 周； 蛋白验证：挑取单克隆细胞，提取蛋白裂解液，通过 Western Blot 检测 PI3Kα/δ 蛋白表达，筛选纯合敲除克隆用于后续实验。3. Western Blot 实验（检测蛋白表达与修饰）实验目的 ：检测 PI3K 通路抑制效果（下游靶蛋白磷酸化水平）、SUMOylation 修饰水平（SUMO1/2/3 蛋白）及核心通路蛋白（如 SAE1、UBA2）的表达变化。关键试剂与材料 ： 抗体：PI3Kα/δ 一抗、磷酸化 AKT（p-AKT）一抗、SUMO1/2/3 一抗、SAE1/UBA2 一抗、β-Actin 一抗（内参）、辣根过氧化物酶（HRP）偶联二抗； 显影系统：OdysseyM imager（LI-COR Biotech）、EmpiriaStudio 软件（用于灰度分析）。详细步骤 ： 蛋白提取：细胞经药物处理后，用 RIPA 裂解液（含蛋白酶抑制剂和磷酸酶抑制剂）裂解，4℃离心 15min（12000g），取上清； 蛋白定量：用 BCA 法测定蛋白浓度，确保上样量一致； SDS-PAGE 电泳：将蛋白样品与 loading buffer 混合，煮沸变性后上样，进行聚丙烯酰胺凝胶电泳； 转膜与孵育：将凝胶上的蛋白转移至 PVDF 膜，5% 脱脂牛奶封闭 1h，加入一抗（4℃孵育过夜），TBST 洗膜后加入二抗（室温孵育 1h）； 显影与分析：用 OdysseyM imager 显影，EmpiriaStudio 软件分析目标蛋白与内参蛋白的灰度比值，量化蛋白表达 / 修饰水平。4. 活细胞成像与原位耐药实验（评估长期治疗效果）活细胞成像（生长曲线检测） ： 目的：实时监测药物对细胞增殖的长期影响； 步骤：96 孔板接种 5×10³ 细胞 / 孔，培养 24h 后加药，用 Incucyte 活细胞成像仪（2024A 版本软件）实时拍摄，通过细胞特异性汇合度图像掩码计算汇合度，绘制生长曲线。原位耐药实验 ： 目的：模拟临床长期治疗中的耐药发生过程，对比单药与联合治疗的耐药风险； 步骤：96 孔板接种 250 细胞 / 孔（低密度模拟肿瘤克隆形成），第 1 天加药，每周换液并刷新药物，用 Incucyte 监测汇合度，将≥50% 汇合度定义为耐药，绘制 Kaplan-Meier 耐药曲线。三、组学分析技术（解析联合治疗分子机制）1. 多组学样本制备与检测实验目的 ：从转录（RNA）、蛋白、磷酸化修饰三个层面，解析 PI3Kα/δ-SUMO 联合抑制的分子机制（如程序性细胞死亡 RCD、代谢通路变化）。关键材料与方法 ： RNA 测序（RNAseq）：提取总 RNA，构建 cDNA 文库，进行高通量测序，分析差异表达基因； 蛋白质组 / 磷酸蛋白质组：提取蛋白后进行胰酶酶解，磷酸化肽段用 TiO₂富集，通过液相色谱 - 串联质谱（LC-MS/MS）检测，使用 MaxQuant 软件匹配数据库并定量。 样本：MiaPaCa-2 细胞，分为 4 组（DMSO 对照、subasumstat 单药、copanlisib 单药、联合治疗），药物处理 24h（转录组 / 蛋白组）或 6/24h（磷酸蛋白质组）； 组学检测：科普知识 ：多组学联合分析是 “从基因到功能” 的系统研究手段 ——RNAseq 反映基因转录水平变化，蛋白质组反映实际功能蛋白的表达，磷酸蛋白质组则聚焦蛋白翻译后修饰（如磷酸化，常调控蛋白活性），三者结合可避免 “转录 - 蛋白表达不一致” 导致的误判，全面揭示药物对细胞通路的调控网络。2. 单细胞水平组学（解析肿瘤微环境 TME 重塑）实验目的 ：通过单细胞核 RNA 测序（snRNAseq）分析联合治疗对 TME 中细胞类型（如 T 细胞、巨噬细胞）的影响；关键步骤 ： 样本获取：取免疫健全小鼠原位移植肿瘤（9091PPT 细胞），经药物处理后解离为单细胞悬液，提取细胞核； 测序与分析：构建 snRNA 文库并测序，用 Seurat 软件进行细胞聚类（识别 T 细胞、巨噬细胞、肿瘤细胞等类型），通过 Uniform Manifold Approximation and Projection（UMAP）降维可视化，分析各组细胞类型比例及差异基因表达（如 T 细胞的 Xcl1、Il18r1，巨噬细胞的 M1/M2 标志物）。四、动物模型实验技术1. 体内疗效评估（验证联合治疗抗瘤效果）实验模型 ： 异种移植模型：裸鼠皮下接种 MiaPaCa-2 细胞，监测肿瘤体积； 原位移植模型（免疫健全）：C57BL/6 小鼠胰腺原位接种 9091PPT 细胞（间质样 PDAC），模拟临床肿瘤生长环境。药物处理与检测 ： 给药方案：subasumstat（SUMO E1 抑制剂）与 copanlisib（PI3Kα/δ 抑制剂）采用剂量递增方案，对照组给予溶媒（DMSO + 生理盐水）； 疗效检测：每 3 天测量肿瘤体积（原位模型通过活体成像或解剖后称重），绘制肿瘤生长曲线，实验终点取肿瘤组织进行免疫组化和 cycIF 检测。2. 毒性分析（评估治疗安全性）实验目的 ：检测联合治疗对小鼠重要器官（肝、肾）及血液系统的毒性；关键步骤 ： 样本获取：实验终点通过心脏穿刺取小鼠血清，2000g 离心 10min 分离血清； 指标检测：用 Roche cobas 分析仪检测血清生化指标，包括肝功能（谷丙转氨酶 ALT、谷草转氨酶 AST、胆红素）、肾功能（尿素、肌酐）、血液指标（乳酸脱氢酶 LDH、白蛋白、钙、无机磷），对比对照组判断是否存在毒性。五、组织水平检测技术：Cyclic Immunofluorescence（cycIF）实验目的 ：在同一张肿瘤切片上检测多种免疫标志物，分析 TME 中免疫细胞浸润（如 CD3+T 细胞、CD8+CCL5+T 细胞）；科普知识 ：cycIF 是 “循环染色 - 成像 - 脱色” 技术 —— 传统免疫荧光一次仅能检测 3-4 种标志物，而 cycIF 通过多次重复 “一抗孵育 - 荧光二抗孵育 - 成像 - 脱色” 循环，可在同一样本上检测 10 种以上标志物，避免样本异质性干扰，精准分析细胞共表达特征（如 CD3+CD8+Ki67 + 增殖性细胞毒性 T 细胞）；关键步骤 ： 切片制备：肿瘤组织石蜡包埋切片，脱蜡至水； 循环染色：依次加入 CD3、CD4、CD8、PD-1、Ki67、CCL5 等一抗及对应荧光二抗，每次染色后用荧光显微镜成像，再用脱色液去除抗体； 数据分析：用 ImageJ 软件量化各标志物阳性细胞数量，计算其在肿瘤组织中的比例。 关键结果图表 本研究针对胰腺导管腺癌（Pancreatic Ductal Adenocarcinoma, PDAC）治疗困境提出核心研究问题：PDAC 作为高度侵袭性致死性癌症，5 年生存率 < 13%，现有标准治疗不足，KRAS 抑制剂易出现耐药，磷脂酰肌醇 3 - 激酶（Phosphatidylinositol-3-Kinase, PI3K）通路虽为 PDAC 发生维持的关键，但单靶点药理靶向无显著临床获益，而小泛素相关修饰蛋白（Small Ubiquitin-like Modifier, SUMO）通路在 PDAC 中具有促癌作用，两者在 PDAC 中的互作机制及能否作为联合治疗靶点尚不明确。基于此，研究提出假说：PI3K 与 SUMO 通路在 PDAC 中存在可靶向的双向互依性，同时抑制 PI3Kα/δ 亚型与 SUMO 信号可诱导肿瘤细胞合成致死，并重塑肿瘤微环境（Tumor Microenvironment, TME）以增强抗瘤效果。为验证该假说，研究采用多维度研究思路：通过全基因组 CRISPR/Cas9 耐药筛选、含≥99 种抑制剂的药理筛选挖掘通路互依性；利用转录组学、蛋白质组学、磷酸蛋白质组学解析分子机制；构建 PDAC 细胞系（MiaPaCa-2、PSN-1 等）、患者源性肿瘤类器官（Patient-Derived Organoids, PDO）及免疫健全小鼠原位移植模型，从细胞、类器官到动物水平验证联合治疗（PI3Kα/δ 抑制剂 copanlisib/pictilisib 与 SUMO E1 抑制剂 subasumstat）的疗效与安全性，同时通过单细胞核 RNA 测序（snRNAseq）、cyclic immunofluorescence（cycIF）分析 TME 重塑效应。1. PI3K 与 SUMO 通路存在双向互依性，为联合靶向提供理论基础 研究首先通过 CRISPR 筛选与药理实验证实 PI3K 与 SUMO 通路的互依性：如图 1D 所示，在 MiaPaCa-2 细胞的全基因组 CRISPR-PI3K 抑制剂（pictilisib）耐药筛选中，基因集富集分析（GSEA）显示 SUMOylation 相关通路为显著的合成致死通路，提示抑制 PI3K 时阻断 SUMO 通路可能诱导细胞死亡；进一步用 PI3Kα/δ 抑制剂 copanlisib 处理细胞，如图 1E、F 所示，随着处理时间延长（6-72h），SUMO1、SUMO2/3 及 SUMO 通路核心成分（SAE1、UBA2）的蛋白表达显著升高，且 copanlisib 耐药细胞中这些成分的转录与蛋白水平均进一步上调，证实 PI3K 抑制可激活 SUMO 通路。反之，研究用 SUMO E1 抑制剂 subasumstat 进行全基因组 CRISPR 耐药筛选，如图 2B 所示，KEGG 通路分析显示 PI3K-AKT 通路为 MiaPaCa-2 和 PSN1 细胞中显著的合成致死通路；同时在人（MiaPaCa-2、PSN1）与小鼠（53631PPT）PDAC 细胞系中开展 subasumstat 锚定的药理筛选，如图 2C 所示，仅 PI3K 抑制剂与 subasumstat 组合显示显著协同效应，且通过 ZIP 方法计算协同得分（图 2D、E），证实 pictilisib 或 copanlisib 与 subasumstat 联合时，在 3 种 PDAC 细胞系中均呈现浓度依赖性协同抑制效应，明确两者存在双向互依性：PI3K 抑制会激活 SUMO 通路，而 SUMO 抑制会诱导细胞对 PI3K 信号产生依赖，这种互依性为联合靶向提供了核心理论依据。2. 仅 PI3Kα/δ 双亚型抑制与 SUMO 抑制存在协同作用，单一亚型靶向无效 为明确 PI3K 家族中关键作用亚型，研究测试了多种 PI3K 亚型特异性抑制剂与 subasumstat 的联合效应：如图 3A 所示，PI3Kα 抑制剂 alpelisib、PI3Kβ 抑制剂 GSK2636771、PI3Kγ 抑制剂 eganelisib、PI3Kδ 抑制剂 idelalisib/parsaclisib 单独与 subasumstat 联合时，ZIP 协同得分均接近 0，无显著协同作用；仅 PI3Kα/δ 双抑制剂（copanlisib、pictilisib）与 subasumstat 联合时，协同得分显著升高（>10）。为进一步验证，研究通过 CRISPR/Cas9 敲除 MiaPaCa-2 细胞中的 PI3Kα（外显子 2）或 PI3Kδ（外显子 3），如图 3C 所示，免疫印迹证实 PI3Kα/δ 蛋白表达被有效敲除，但单一敲除 PI3Kα 或 PI3Kδ 后，与 subasumstat 联合处理仍无显著协同抑制效应（图 3D 左、右）；仅当 PI3Kα 敲除细胞再用 PI3Kδ 抑制剂 idelalisib 处理，或 PI3Kδ 敲除细胞再用 PI3Kα 抑制剂 alpelisib 处理时，与 subasumstat 联合才呈现显著协同效应（ZIP 得分分别为 12.613、14.746，图 3E），且如图 3F 所示，PI3Kα 或 PI3Kδ 敲除细胞中 SUMO1/2/3 蛋白水平显著升高，与 PI3K 抑制剂处理结果一致。这一结果表明，仅 PI3Kα/δ 双亚型抑制与 SUMO E1 抑制剂联合时可诱导显著的合成致死效应，单一 PI3K 亚型（α、β、γ、δ）靶向无法实现协同作用，明确了联合治疗的关键靶点特异性，为临床药物选择提供精准指导。3. 联合抑制诱导程序性细胞死亡（RCD）并调控代谢与 mRNA 代谢通路 为解析联合治疗的分子机制，研究对 MiaPaCa-2 细胞进行单药（subasumstat、copanlisib）与联合治疗后的多组学分析：如图 4A 所示，通过转录组、蛋白质组及磷酸蛋白质组的整合分析，发现联合治疗组与单药组及对照组相比，呈现独特的通路调控特征；如图 4D 所示，转录组与蛋白质组的重叠富集通路中，凋亡通路、血红素代谢通路等 6 个通路显著上调，提示细胞死亡相关通路被激活；进一步检测凋亡标志物，如图 4F 所示，联合治疗组 Annexin-V 阳性细胞比例显著升高（约 35%），且加入泛胱天蛋白酶抑制剂 zVAD-fmk 后，该比例降至约 12%，证实凋亡是 RCD 的组成部分，但单独凋亡不足以解释强烈的细胞杀伤效应；通过磷酸蛋白质组分析（图 4G），发现联合治疗特异性调控代谢通路（如三羧酸循环）、mRNA 代谢通路及有丝分裂调控通路，这些通路异常可能共同导致细胞活力丧失，最终证实联合抑制通过多模态诱导 RCD，而非单一凋亡途径，解释了其强效杀伤肿瘤细胞的机制。4. PDO 模型验证联合治疗对间质样 PDAC 的亚型特异性疗效 为模拟临床 PDAC 异质性，研究构建 5 例 PDAC 患者来源 PDO（2 例上皮样、3 例间质样），对比标准治疗（FOLFIRINOX）与联合治疗的疗效：如图 5C 所示，间质样 PDO（PAN2T、PAN17T、PAN70T）在 FOLFIRINOX 处理后仍保持较高细胞活力（相对活力 > 60%），而上皮样 PDO（PAN41T、PAN74T）活力显著降低（<40%），证实间质样 PDAC 对标准治疗耐药；反之，如图 5D 所示，联合治疗（pictilisib+subasumstat）对间质样 PDO 的抑制效应显著强于上皮样 PDO，当 pictilisib 浓度为 1000nmol/L 时，间质样 PDO 相对活力 < 20%，而上皮样 PDO 仍> 50%；通过 GSEA 分析（图 5E），发现间质样 PDO 中 SUMOylation 相关信号显著富集，这与前文 PI3K 抑制激活 SUMO 通路的机制一致，解释了其对联合治疗的高敏感性；此外，如图 5F 所示，在原位耐药实验中，单药（subasumstat 或 copanlisib）处理的 PDAC 细胞（9091PPT、PSN1、Panc1）在 3-4 周出现耐药（汇合度 > 50%），而联合治疗组在 4-5 周时仍无细胞汇合，细胞被判定为非存活，证实联合治疗可有效克服间质样 PDAC 的耐药性，且具有长期疗效稳定性。5. 免疫健全小鼠模型中联合治疗抑制肿瘤生长并重塑 TME 在免疫健全小鼠原位移植模型（9091PPT 细胞，间质样 PDAC）中，研究验证联合治疗的体内疗效与 TME 重塑效应：如图 6D 所示，联合治疗组肿瘤体积显著小于单药组与对照组（治疗 21 天时，联合组肿瘤体积约为对照组的 1/3，P<0.01），且无显著体重下降；通过 cycIF 与免疫组化分析（图 6E、F），发现联合治疗组 CD3+T 细胞浸润数量显著增加（约为对照组的 2.5 倍），CD4+、CD8+T 细胞数量无显著变化，但 snRNAseq 分析（图 6G、H）显示，联合治疗组 T 细胞中 Xcl1（T 细胞趋化因子）和 Il18r1（免疫激活受体）的转录水平显著升高，且 CD3+CD8+CCL5+（细胞毒性标志物）和 CD3+CD8+Ki67+（增殖标志物）细胞比例显著增加（图 6J），证实 T 细胞活性被激活；对于巨噬细胞，如图 6K、L 所示，联合治疗组 M2 型巨噬细胞（免疫抑制型）比例降低，M1 型巨噬细胞（促炎型）及抗原呈递型巨噬细胞比例升高，且如图 6M 所示，CD68 + 巨噬细胞中 CCL5（免疫细胞招募因子）表达上调，进一步促进免疫细胞浸润；同时，补充图 7 显示，联合治疗组小鼠血清中肝功能（ALT、AST）、肾功能（尿素）及血液指标（LDH、白蛋白）与对照组无显著差异，证实其体内安全性良好。 研究通过系统实验证实，PI3Kα/δ 与 SUMO 通路在 PDAC 中存在双向互依性，针对该互依性的联合抑制（PI3Kα/δ 抑制剂 + SUMO E1 抑制剂）可通过诱导合成致死效应杀伤肿瘤细胞，同时重塑 TME（激活 T 细胞、重编程巨噬细胞），且对临床耐药的间质样 PDAC 具有特异性疗效，在 PDO 与免疫健全动物模型中均展现出强效与安全性。该研究的核心结论在于：首次揭示 PI3Kα/δ 与 SUMO 信号的互依性机制，并提出基于该机制的联合治疗策略，为 PDAC（尤其是耐药亚型）提供了具有临床转化潜力的新方案，同时明确了 PI3K 亚型特异性的关键作用，避免了泛 PI3K 靶向的局限性。基于本研究结果，新的延伸问题可聚焦于：在临床 PDAC 样本中，PI3Kα/δ 与 SUMO 通路活性是否可作为联合治疗的疗效预测标志物？联合治疗与免疫检查点抑制剂（如 PD-1 抗体）是否存在协同效应以进一步增强 TME 重塑？此外，下一步实验可开展临床前大型 PDO 队列验证，并探索联合治疗在 KRAS 耐药 PDAC 中的疗效，为后续临床 trials 奠定基础。 特色与创新之处 该研究在胰腺导管腺癌（PDAC）治疗策略探索中展现出多项科学严谨且具有实质意义的创新： 首次揭示PI3Kα/δ与SUMO通路之间的双向适应性互作机制：研究通过CRISPR耐药筛选与多组学分析，明确PI3K抑制可诱导SUMO化通路代偿性激活，反之亦然，表明二者构成一个功能耦合的应激缓冲网络，而非独立信号轴。 确立PI3Kα与PI3Kδ亚型协同作用的不可替代性：通过系统比较多种亚型选择性PI3K抑制剂及CRISPR介导的单/双亚型敲除模型，证明仅当PI3Kα与PI3Kδ同时被抑制时，才能有效触发对SUMO通路的依赖，单一亚型抑制不足以产生合成致死效应，纠正了既往对PI3K亚型功能冗余性的简化认知。 提出并验证“PI3Kα/δ–SUMO E1”协同靶向的合成致死治疗范式：在免疫健全的原位PDAC小鼠模型中，联合使用临床级PI3Kα/δ抑制剂copanlisib与SUMO E1抑制剂subasumstat，不仅显著抑制肿瘤生长，还重塑免疫微环境，增强T细胞浸润与活性，突破了传统PI3K单药治疗在PDAC中疗效有限的瓶颈。 整合多维功能基因组学与药理学数据驱动靶点发现：研究融合全基因组CRISPR筛选、药理协同筛选、转录组、蛋白组及磷酸化蛋白组，构建从遗传依赖到药物响应的完整证据链，为理性设计联合疗法提供了方法论范例。 将基础机制发现与临床转化潜力紧密结合：所用抑制剂均为临床级或已进入临床试验阶段，联合方案在动物模型中显示可控毒性与显著疗效，具备直接推进至临床试验的可行性，尤其针对当前缺乏有效靶向手段的KRAS突变型PDAC。 可以改进的地方[AI基于大数据总结，仅供参考] 该研究在机制探索与治疗策略构建方面具有显著价值，但仍存在若干科学与转化层面的局限，需在后续工作中加以完善： 缺乏对SUMO通路激活机制的深入解析研究观察到PI3K抑制后SUMO核心组分（如SAE1、SUMO1/2）表达上调，但未阐明其调控层级（转录、翻译后稳定性或酶活性变化）及关键中介分子。建议通过ChIP-seq、启动子报告基因实验或激酶-底物磷酸化网络分析，明确PI3K-AKT信号如何调控SUMO化机器的表达或活性，例如是否通过FOXO、MYC或NF-κB等下游转录因子介导。 类器官模型未用于功能验证尽管研究引用了患者来源类器官（PDO）的转录组数据以支持PI3K通路在化疗后激活，但未在PDO平台中测试PI3Kα/δ与SUMO联合抑制的疗效。PDO能更好保留患者肿瘤的异质性与微环境特征，建议在多例PDO（尤其是KRAS突变、PIK3CA野生型）中进行药敏测试，评估该策略的普适性与生物标志物潜力。 免疫微环境变化的机制阐释不足研究发现联合治疗促进免疫细胞浸润，但未解析其因果机制：是肿瘤细胞死亡释放抗原所致，还是SUMO/PI3K抑制直接调控肿瘤细胞的免疫原性（如MHC-I、PD-L1表达）或趋化因子分泌？建议结合体外共培养体系（如PDO与T细胞）及单细胞/单核RNA测序（snRNA-seq），明确免疫激活的细胞来源与信号通路。 毒性评估较为初步小鼠毒性数据仅基于血清生化指标，未系统评估对免疫系统、肠道稳态或代谢器官的长期影响。鉴于PI3Kδ在淋巴细胞中的关键作用及SUMO通路的广泛调控性，建议开展更全面的毒理学研究，包括外周血免疫分型、组织病理学评分及体重/行为监测，以评估临床应用的安全窗口。 未探索耐药机制与应对策略虽然研究展示了联合治疗的初始疗效，但未模拟或预测可能的获得性耐药机制（如SUMO通路旁路激活、药物外排泵上调等）。建议建立长期药物暴露诱导的耐药细胞系或PDO模型，结合全外显子组测序与功能回补实验，提前识别耐药驱动因素并设计三联干预策略。 上述改进方向均基于现有技术平台可行，将显著提升该联合疗法的机制深度、临床预测性和转化稳健性。 求点赞 求分享 求推荐 如果你想在此显示您的广告信息，请联系我们。 与万千同行共同关注类器官！ 关注我们  肠道类器官（Organoid Bulletin） 本公众号由AI智能体ORGANOID AGENT驱动赋能，您可以直接与公众号对话，获得最专业的类器官知识指导与信息。      我们致力于发布本领域优质资讯与评述，推送国内外类器官与干细胞相关领域最新研究论文、综述与技术进展。发布科普文章、专家观点评论、行业分析、招聘信息和会议通知。您可以在微信、小红书、百家号、企鹅号、今日头条、知乎和Bilibili关注我们。秉承干细胞与类器官创新计划组织(ISCO)愿景，我们积极传播科学知识，促进同行交流，推动产业发展，服务国家战略，积极为类器官与干细胞技术发展提供助力。欢迎各位关注、分享、转载、投稿！原创内容欢迎转载，直接私信即可，完全免费授权。内容多来自学术论文或网络公开资源，如有侵权还请联系删除。 编辑部联系邮箱：zj@organoid.ac.cn 广告赞助 欢迎找到组织 类器官创新计划组织 类器官创新计划组织（Innovations in organoids organization）是由来自中国科学院、北京大学、清华大学和复旦大学等多个顶级研究机构的头部研究团队组织发起的，完全公益的学术与研究技术交流合作组织，成员主要由来自类器官行业内的企业高管、技术经理、高级PI等构成。加入“类器官创新计划”后，您将以极低的成本实现类器官资源的互换与共享，基于肠道类器官和类器官创新计划组织微信公众号平台，您将获得来自全行业的赋能与指导，进行标准化的类器官制备与检测，保证您的研究顺利且高质量开展。我们会将你纳入本组织“技术研发”、“开源公益”和“产品服务”等多个微信群，这样您能够与所有参与计划的成员及时沟通，您所有的类器官问题都能最及时的得到解答和帮助，互换类器官品系和干细胞研究工具资源，解决技术难题，获得技术支持。我们已有上万个来自全国顶级类器官研究机构、大学和企业的注册认证会员，大家一起为类器官事业发展点燃星星之火，推动类器官产业发展壮大。我们还建立了AI知识问答和开源类器官分析工具等多个公益项目为类器官研究提供帮助，构建开放包容的领域生态。在这里,我们敢为人先，始终积极寻求更新类器官更优的解决方案，勇于突破经验与预想的界限，致力打造科学卓越的行业新标准。 官方网站：www.organoid.ac.cn 联系邮箱：info@isco.ac.cn 组织愿景 1.共建开放协作生态：搭建行业资源共享平台，促进技术互通与经验共享，降低类器官研究门槛。 2.引领标准与规范：推动制定行业与国家工程标准，实现类器官培养与应用的规范化、全流程标准化。 3.打造国际级资源枢纽：推动建设全球认可的类器官库与智库平台，加速技术向精准医学与药物研发转化。 4.驱动技术融合创新：推动整合生物材料、智能装备等工程技术创新，突破类器官规模化制备与功能重建瓶颈。 5.强化技术自主可控：推进国产化试剂、仪器研发与技术体系替代，保障核心研发链安全与战略主动权。 加入交流       ISCO面向类器官全领域同行提供交流平台和会员身份认证服务，可以加入“技术研发”、“开源公益”、“产品服务”和“专属领域”等多个类型的交流群，涵盖科学研究技术、开源工具和公益、销售产品与服务、细分领域如类器官芯片、生物材料等专属群等平台，参与讨论。高级研究员或独立PI可以申请成为荣誉发起人，成为类器官创新计划贡献者与受益者。本公益组织目前已有8000+会员，期待您加入讨论分享，让我们共同促进类器官产业发展。      现在，您只需添加发起人微信，备注自己的真实姓名与单位例如“李四-中国科学院大学”，说明申请加入类器官创新计划，提供有效身份证件如学生证、身份证、工作证或其他任何可以证明身份的真实性的证件，即可加入对应社群。而这一切都是公益的，免费的，不收取任何费用。 欢迎您找到组织！~ 扫描二维码添加发起人微信以进群交流 专业收费资源 面向专业科普需求和企业级资源共享，现在扫描二维码加入“肠道类器官”知识星球，就能获得来自顶级研究机构分享的类器官培养基配方和方案，类器官创新计划组织发布的官方标准指南、AI类器官分析工具等更加专业的资料。与1000+位行业精英共同交换类器官技术资料和资源，向行业专家提问，免费参与国内领先的类器官品牌产品内测试用，获得类器官工程师认证等。全行业最专业的类器官知识社区和公益平台，让您的类器官研究少走弯路！ #为什么要加入知识星球？ 1.最新最专业的培养基配方和技术资料即时获取。 2.标准类器官培养方案和技术标准材料发布唯一渠道。 3.最强AI智能体和类器官分析软件和工具解析和下载。 4.类器官领域TOP专家一对一沟通，权威资料触手可及。 5.类器官企业新产品评测和免费试用特权通道。 6.定期发放免费会议入场券和资料，提供演讲机会。  更多福利，请移步知识星球内部了解。 开源公益  类器官能模拟人体器官功能与结构，为疾病研究、药物研发提供有力模型。但分析类器官数据困难复杂，开源算法项目汇聚前沿科学家智慧，让科研人员共享代码，优化数据处理与分析，加速成果转化，推动类器官研究迈向新高度。我们的管理团队和多个科研团队合作，依托类器官创新计划组织，率先开源基于大模型的类器官AI算法工具，未来类器官创新计划组织将不断提供开源免费的类器官分析与报告工具，助力行业快速发展。 #公益知识问答项目Organoid Agent, 采用最新的AI智能体技术，提供超越博士级类器官研究水平和教授级实验技术知识储备。 https://www.coze.cn/store/agent/7376069505418280970?bot_id=true&bid=6fck970447g1k 您还以直接在公众号对话框与它对话，获取帮助。 #开源AI图形算法项目地址 https://github.com/zjdevo/ISCO https://gitee.com/zjdevo/ISCO 开源项目自发起以来，持续受到企业公益赞助,我们感谢以下企业的支持： 上海捷方凯瑞生物科技有限公司  吉诺生命健康控股集团有限公司  如您有意通过赞助公益项目，实现品牌推广与发展，欢迎与我们联系。 扫描二维码添加微信以咨询赞助政策 注册会员      注册会员是面向所有人的公益身份识别项目。如需以组织会员身份获取各种资源与免费福利。您只需访问www.organoid.ac.cn注册会员，并补充个人真实有效身份信息，经人工审核后，即可获得注册会员认证。通过“类器官创新计划组织”和“肠道类器官公众号”的“身份认证”菜单就能找到查询入口。也可以下载专属的身份二维码用于各种需要出示本组织身份的场合如学术会议、合作商提供的免费产品申请。由PI或课题组长及以上级别提供实名信息可注册为“注册会员”，加入“注册认证会员群”优先享受本组织提供的学术界与产业界资源。特别的，您所有的信息仅用于身份识别，不会被进行任何形式的商业用途，     欢迎监督举报（邮箱：isco@organoid.ac.cn）。未进行注册的，不被认为是我组织正式会员，合作方有权拒绝提供任何服务和或产品。 具体注册方式点击下列文章了解： 如何注册成为注册会员？ 扫描二维码快速进入 身份认证系统

Product Sales Excluding Veklury Increased 4% Year-Over-Year to $7.1 billion Biktarvy Sales Increased 6% Year-Over-Year to $3.7 billion FOSTER CITY, Calif.--(BUSINESS WIRE)-- Gilead Sciences, Inc. (Nasdaq: GILD) announced today its third quarter 2025 results of operations. “We continue to deliver on Gilead's robust portfolio with a strong start for Yeztugo, rapidly growing uptake of Biktarvy, Descovy and Livdelzi, and positive data for Trodelvy in 1L metastatic triple negative breast cancer,” said Daniel O’Day, Gilead’s Chairman and Chief Executive Officer. “With multiple potential product launches in 2026, the strongest clinical pipeline in Gilead’s history, and no major loss of exclusivity expected until 2036, we are well-positioned to drive positive impact for patients and continued growth of our business.” Third Quarter 2025 Financial Results Total third quarter 2025 revenues increased 3% to $7.8 billion compared to the same period in 2024, broken down as follows: Total third quarter 2025 product sales decreased 2% to $7.3 billion compared to the same period in 2024, primarily driven by lower Veklury® (remdesivir) and Cell Therapy sales, partially offset by higher HIV and Livdelzi® (seladelpar) sales. Total third quarter 2025 royalty, contract and other revenues increased by approximately $400 million compared to the same period in 2024, primarily driven by revenue related to a previous sale of intellectual property not expected to reoccur. Diluted earnings per share (“EPS”) was $2.43 in the third quarter 2025 compared to $1.00 in the same period in 2024. The increase was primarily driven by a prior year pre-tax in-process research and development (“IPR&D”) impairment charge of $1.75 billion that did not repeat in the current period, as well as the $400 million increase in other revenue mentioned above, lower acquired IPR&D expenses and higher net unrealized gains on equity investments in the current period, partially offset by higher tax expense. Non-GAAP diluted EPS of $2.47 in the third quarter 2025 compared to $2.02 in the same period in 2024. The increase was primarily driven by the $400 million increase in other revenue mentioned above and lower acquired IPR&D expenses. As of September 30, 2025, Gilead had $9.4 billion of cash, cash equivalents and marketable debt securities compared to $10.0 billion as of December 31, 2024. During the third quarter 2025, Gilead generated $4.1 billion in operating cash flow. During the third quarter 2025, Gilead paid dividends of $1.0 billion and repurchased $435 million of common stock. Total third quarter 2025 product sales decreased 2% to $7.3 billion compared to the same period in 2024, primarily driven by lower Veklury® (remdesivir) and Cell Therapy sales, partially offset by higher HIV and Livdelzi® (seladelpar) sales. Total third quarter 2025 royalty, contract and other revenues increased by approximately $400 million compared to the same period in 2024, primarily driven by revenue related to a previous sale of intellectual property not expected to reoccur. Third Quarter 2025 Product Sales Total third quarter 2025 product sales decreased 2% to $7.3 billion compared to the same period in 2024. Total third quarter 2025 product sales excluding Veklury increased 4% to $7.1 billion compared to the same period in 2024, primarily due to higher HIV and Livdelzi sales, partially offset by lower Cell Therapy sales. HIV product sales increased 4% to $5.3 billion in the third quarter 2025 compared to the same period in 2024, primarily driven by higher demand and favorable inventory dynamics, partially offset by lower average realized price. Biktarvy®(bictegravir 50mg/emtricitabine (“FTC”) 200mg/tenofovir alafenamide (“TAF”) 25mg) sales increased 6% to $3.7 billion in the third quarter 2025 compared to the same period in 2024, primarily driven by higher demand and favorable inventory dynamics, partially offset by lower average realized price. Descovy®(FTC 200mg/TAF 25mg) sales increased 20% to $701 million in the third quarter 2025 compared to the same period in 2024, primarily driven by higher demand. The Liver Disease portfolio sales increased 12% to $819 million in the third quarter 2025 compared to the same period in 2024, primarily driven by higher demand for Livdelzi. Veklury sales decreased 60% to $277 millionin the third quarter 2025 compared to the same period in 2024, primarily driven by lower rates of COVID-19-related hospitalizations. Cell Therapy product sales decreased 11% to $432 million in the third quarter 2025 compared to the same period in 2024, reflecting ongoing competitive headwinds. Yescarta® (axicabtagene ciloleucel) sales decreased 10% to $349 million in the third quarter 2025 compared to the same period in 2024, primarily driven by lower demand. Tecartus® (brexucabtagene autoleucel) sales decreased 15% to $83 million in the third quarter 2025 compared to the same period in 2024, primarily reflecting lower demand. Trodelvy® (sacituzumab govitecan-hziy) sales increased 7% to $357 million in the third quarter 2025 compared to the same period in 2024, primarily driven by higher demand. Third Quarter 2025 Product Gross Margin, Operating Expenses and Effective Tax Rate Product gross margin remained relatively flat at 78.6% in the third quarter 2025 compared to 79.1% in the same period in 2024. Non-GAAP product gross margin also remained relatively flat at 86.5% in the third quarter 2025 compared to 86.8% in the same period in 2024. Research and development (“R&D”) expenses and non-GAAP R&D expenses were $1.3 billion in the third quarter 2025 compared to $1.4 billion in the same period in 2024, decreasing primarily due to lower study-related and clinical manufacturing expenses. Acquired IPR&D expenses were $170 million in the third quarter 2025, primarily related to a $120 million upfront payment related to our collaboration with Shenzhen Pregene Biopharma Co., Ltd. (“Pregene”). Selling, general and administrative (“SG&A”) expenses and non-GAAP SG&A expenses of $1.4 billion in the third quarter 2025 remained relatively flat compared to the same period in 2024, with lower corporate expenses being largely offset by higher HIV promotional expenses. The effective tax rate (“ETR”) was 16.2% in the third quarter 2025 compared to (31.1)% in the same period in 2024, primarily driven by the prior year impact of a legal entity restructuring and the aforementioned IPR&D impairment charge that did not repeat in the current period. The non-GAAP ETR was 17.5% in both the third quarter 2025 and the same period in 2024. Guidance and Outlook For the full-year, Gilead expects: (in millions, except per share amounts) October 30, 2025 Guidance Low End High End Comparison to Prior Guidance Product sales $ 28,400 $ 28,700 Previously $28,300 to $28,700 Product sales excluding Veklury $ 27,400 $ 27,700 Previously $27,300 to $27,700 Veklury $ 1,000 $ 1,000 Unchanged Diluted EPS $ 6.65 $ 6.85 Previously $5.85 to $6.15 Non-GAAP diluted EPS $ 8.05 $ 8.25 Previously $7.95 to $8.25 Additional information and a reconciliation between GAAP and non-GAAP financial information for the 2025 guidance is provided in the accompanying tables. The financial guidance is subject to a number of risks and uncertainties. See the Forward-Looking Statements section below. Key Updates Since Our Last Quarterly Release Virology Announced settlement agreements to resolve Biktarvy patent litigation with generic manufacturers Lupin Ltd., Cipla Ltd. and Laurus Labs Ltd. Under the agreements, the earliest date the three generic manufacturers can market a generic version of full dose Biktarvy in the U.S. is April 1, 2036, subject to standard acceleration provisions. This is more than two years later than our previous loss of exclusivity projection for Biktarvy (December 2033). Received a strong recommendation for the use of twice-yearly injectable Yeztugo® (lenacapavir) for HIV pre-exposure prophylaxis (“PrEP”) in the new U.S. Centers for Disease Control and Prevention guidelines. Announced a partnership with the U.S. State Department and the U.S. President’s Emergency Plan for AIDS Relief (“PEPFAR”) to deliver lenacapavir for HIV PrEP for up to two million people over three years in countries supported by both PEPFAR and the Global Fund. Received European Commission marketing authorization for Yeytuo® (lenacapavir) for use as PrEP to reduce the risk of sexually acquired HIV-1 in adults and adolescents with increased HIV-1 acquisition risk. Oncology Presented Phase 3 ASCENT-03 data for Trodelvy® in 1L metastatic triple-negative breast cancer (“mTNBC”) patients who are not candidates for PD-1/PD-L1 checkpoint inhibitors at the 2025 European Society for Medical Oncology (“ESMO”) Congress. Trodelvy is not approved in this setting. Presented overall survival results at ESMO from Arm A1 of the Phase 2 EDGE-Gastric study evaluating combination treatment of the Fc-silent anti-TIGIT domvanalimab plus the anti-PD-1 zimberelimab and chemo in people with advanced gastric or esophageal cancer that has spread or cannot be removed with surgery. Domvanalimab and zimberelimab are investigational and not approved in this setting. Cell Therapy Announced the acquisition of Interius BioTherapeutics, Inc. (“Interius”), a privately held biotechnology company developing in vivo therapeutics. Corporate The Board declared a quarterly dividend of $0.79 per share of common stock for the fourth quarter of 2025. The dividend is payable on December 30, 2025, to stockholders of record at the close of business on December 15, 2025. Future dividends will be subject to Board approval. Moody’s has affirmed Gilead’s A3 senior unsecured rating and upgraded the company’s outlook to positive from stable, citing the momentum in the product pipeline. Announced ground-breaking on a new Pharmaceutical Development and Manufacturing Technical Development Center in Foster City, California as part of a planned $32 billion investment in the U.S. through 2030. Certain amounts and percentages in this press release may not sum or recalculate due to rounding. Conference Call At 1:30 p.m. Pacific Time today, Gilead will host a conference call to discuss Gilead’s results. A live webcast will be available on http://investors.gilead.com and will be archived on www.gilead.com for one year. Non-GAAP Financial Information The information presented in this document has been prepared in accordance with U.S. generally accepted accounting principles (“GAAP”), unless otherwise noted as non-GAAP. Management believes non-GAAP information is useful for investors, when considered in conjunction with Gilead’s GAAP financial information, because management uses such information internally for its operating, budgeting and financial planning purposes. Non-GAAP information is not prepared under a comprehensive set of accounting rules and should only be used to supplement an understanding of Gilead’s operating results as reported under GAAP. Non-GAAP financial information generally excludes acquisition-related expenses including amortization of acquired intangible assets and other items that are considered unusual or not representative of underlying trends of Gilead’s business, fair value adjustments of equity securities and discrete and related tax charges or benefits associated with such exclusions as well as changes in tax-related laws and guidelines, transfers of intangible assets between certain legal entities, and legal entity restructurings. Although Gilead consistently excludes the amortization of acquired intangible assets from the non-GAAP financial information, management believes that it is important for investors to understand that such intangible assets were recorded as part of acquisitions and contribute to ongoing revenue generation.Non-GAAP measures may be defined and calculated differently by other companies in the same industry. Reconciliations of the non-GAAP financial measures to the most directly comparable GAAP financial measures are provided in the accompanying tables. About Gilead Sciences Gilead Sciences, Inc. is a biopharmaceutical company that has pursued and achieved breakthroughs in medicine for more than three decades, with the goal of creating a healthier world for all people. The company is committed to advancing innovative medicines to prevent and treat life-threatening diseases, including HIV, viral hepatitis, COVID-19, cancer and inflammation. Gilead operates in more than 35 countries worldwide, with headquarters in Foster City, California. Forward-Looking Statements Statements included in this press release that are not historical in nature are forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Gilead cautions readers that forward-looking statements are subject to certain risks and uncertainties that could cause actual results to differ materially. These risks and uncertainties include those relating to: Gilead’s ability to achieve its full year 2025 financial guidance, including as a result of the uncertainty of the amount and timing of Veklury revenues, the impact of the Inflation Reduction Act, changes in U.S. regulatory or legislative policies, and changes in U.S. trade policies, including tariffs; Gilead’s ability to make progress on any of its long-term ambitions or priorities laid out in its corporate strategy; Gilead’s ability to accelerate or sustain revenues for its virology, oncology and other programs; Gilead’s ability to realize the potential benefits of acquisitions, collaborations or licensing arrangements, including the acquisitions of Interius, and the arrangements with Pregene and PEPFAR; the risk that Gilead’s U.S. manufacturing and R&D investment may not achieve their intended benefits; patent protection and estimated loss of exclusivity for our products and product candidates, including with respect to Biktarvy; Gilead’s ability to initiate, progress or complete clinical trials within currently anticipated timeframes or at all, the possibility of unfavorable results from ongoing and additional clinical trials, including those involving Trodelvy, domvanalimab and zimberelimab (such as the ASCENT-03, and EDGE-Gastric studies), and the risk that safety and efficacy data from clinical trials may not warrant further development of Gilead’s product candidates or the product candidates of Gilead’s strategic partners; Gilead’s ability to resolve the issues cited by the FDA in pending clinical holds to the satisfaction of the FDA and the risk that FDA may not remove such clinical holds, in whole or in part, in a timely manner or at all; Gilead’s ability to submit new drug applications for new product candidates or expanded indications in the currently anticipated timelines; Gilead’s ability to receive or maintain regulatory approvals in a timely manner or at all, including for additional approvals for lenacapavir for HIV PrEP, and the risk that any such approvals, if granted, may be subject to significant limitations on use and may be subject to withdrawal or other adverse actions by the applicable regulatory authority; Gilead’s ability to successfully commercialize its products; the risk of potential disruptions to the manufacturing and supply chain of Gilead’s products; pricing and reimbursement pressures from government agencies and other third parties, including required rebates and other discounts; a larger than anticipated shift in payer mix to more highly discounted payer segments; market share and price erosion caused by the introduction of generic versions of Gilead products; the risk that physicians and patients may not see advantages of Gilead’s products over other therapies and may therefore be reluctant to prescribe the products, including Yeztugo/Yeytuo; Gilead’s ability to effectively manage the access strategy relating to lenacapavir for HIV PrEP, subject to necessary regulatory approvals; and other risks identified from time to time in Gilead’s reports filed with the SEC, including annual reports on Form 10-K, quarterly reports on Form 10-Q and current reports on Form 8-K. In addition, Gilead makes estimates and judgments that affect the reported amounts of assets, liabilities, revenues and expenses and related disclosures. Gilead bases its estimates on historical experience and on various other market specific and other relevant assumptions that it believes to be reasonable under the circumstances, the results of which form the basis for making judgments about the carrying values of assets and liabilities that are not readily apparent from other sources. There may be other factors of which Gilead is not currently aware that may affect matters discussed in the forward-looking statements and may also cause actual results to differ significantly from these estimates. Further, results for the quarter ended September 30, 2025 are not necessarily indicative of operating results for any future periods. Gilead directs readers to its press releases, annual reports on Form 10-K, quarterly reports on Form 10-Q and other subsequent disclosure documents filed with the SEC. Gilead claims the protection of the Safe Harbor contained in the Private Securities Litigation Reform Act of 1995 for forward-looking statements. The reader is cautioned that forward-looking statements are not guarantees of future performance and is cautioned not to place undue reliance on these forward-looking statements. All forward-looking statements are based on information currently available to Gilead and Gilead assumes no obligation to update or supplement any such forward-looking statements other than as required by law. Any forward-looking statements speak only as of the date hereof or as of the dates indicated in the statements. Additional information is available on our Investor Relations website, https://investors.gilead.com. Among other things, an estimate of Acquired IPR&D expenses is expected to be made available on the Quarterly Results page within the first ten (10) days after the end of each quarter. Gilead owns or has rights to various trademarks, copyrights and trade names used in its business, including the following: GILEAD®, GILEAD SCIENCES®, KITE®, AMBISOME®, ATRIPLA®, BIKTARVY®, CAYSTON®, COMPLERA®, DESCOVY®, DESCOVY FOR PREP®, EMTRIVA®, EPCLUSA®, EVIPLERA®, GENVOYA®, HARVONI®, HEPCLUDEX®, HEPSERA®, JYSELECA®, LIVDELZI®/LYVDELZI®, LETAIRIS®, ODEFSEY®, SOVALDI®, STRIBILD®, SUNLENCA® , TECARTUS®, TRODELVY®, TRUVADA®, TRUVADA FOR PREP®, TYBOST®, VEKLURY®, VEMLIDY®, VIREAD®, VOSEVI®, YESCARTA®, YEZTUGO®/YEYTUO® and ZYDELIG®. Other trademarks and trade names are the property of their respective owners. For more information on Gilead Sciences, Inc., please visit www.gilead.com or call the Gilead Public Affairs Department at 1-800-GILEAD-5 (1-800-445-3235). GILEAD SCIENCES, INC. CONDENSED CONSOLIDATED STATEMENTS OF OPERATIONS (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except per share amounts) 2025 2024 2025 2024 Revenues: Product sales $ 7,345 $ 7,515 $ 21,013 $ 21,074 Royalty, contract and other revenues 424 30 505 111 Total revenues 7,769 7,545 21,518 21,185 Costs and expenses: Cost of goods sold 1,569 1,574 4,610 4,670 Research and development expenses 1,346 1,395 4,215 4,266 Acquired in-process research and development expenses 170 505 485 4,674 In-process research and development impairments — 1,750 190 4,180 Selling, general and administrative expenses 1,357 1,433 3,980 4,184 Total costs and expenses 4,442 6,657 13,480 21,975 Operating income (loss) 3,327 888 8,038 (790 ) Interest expense 256 238 769 728 Other (income) expense, net (569 ) (306 ) (449 ) (41 ) Income (loss) before income taxes 3,641 956 7,718 (1,477 ) Income tax expense (benefit) 589 (297 ) 1,391 (174 ) Net income (loss) 3,052 1,253 6,327 (1,303 ) Net income attributable to noncontrolling interest — — — — Net income (loss) attributable to Gilead $ 3,052 $ 1,253 $ 6,327 $ (1,303 ) Basic earnings (loss) per share attributable to Gilead $ 2.46 $ 1.00 $ 5.08 $ (1.04 ) Diluted earnings (loss) per share attributable to Gilead $ 2.43 $ 1.00 $ 5.04 $ (1.04 ) Shares used in basic earnings (loss) per share attributable to Gilead calculation 1,243 1,247 1,245 1,247 Shares used in diluted earnings (loss) per share attributable to Gilead calculation 1,254 1,254 1,256 1,247 Supplemental Information: Cash dividends declared per share $ 0.79 $ 0.77 $ 2.37 $ 2.31 Product gross margin 78.6 % 79.1 % 78.1 % 77.8 % Research and development expenses as a % of revenues 17.3 % 18.5 % 19.6 % 20.1 % Selling, general and administrative expenses as a % of revenues 17.5 % 19.0 % 18.5 % 19.8 % Operating margin 42.8 % 11.8 % 37.4 % (3.7 )% Effective tax rate 16.2 % (31.1 )% 18.0 % 11.8 % GILEAD SCIENCES, INC. TOTAL REVENUE SUMMARY (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages) 2025 2024 Change 2025 2024 Change Product sales: HIV $ 5,277 $ 5,073 4 % $ 14,952 $ 14,160 6 % Liver Disease 819 733 12 % 2,372 2,302 3 % Oncology 788 816 (3 )% 2,395 2,446 (2 )% Other 184 201 (8 )% 594 705 (16 )% Total product sales excluding Veklury 7,068 6,823 4 % 20,313 19,613 4 % Veklury 277 692 (60 )% 700 1,461 (52 )% Total product sales 7,345 7,515 (2 )% 21,013 21,074 — % Royalty, contract and other revenues 424 30 NM 505 111 NM Total revenues $ 7,769 $ 7,545 3 % $ 21,518 $ 21,185 2 % GILEAD SCIENCES, INC. NON-GAAP FINANCIAL INFORMATION(1) (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages) 2025 2024 Change 2025 2024 Change Non-GAAP: Cost of goods sold $ 992 $ 995 — % $ 2,875 $ 2,933 (2 )% Research and development expenses $ 1,334 $ 1,382 (3 )% $ 4,123 $ 4,120 — % Acquired IPR&D expenses $ 170 $ 505 (66 )% $ 485 $ 4,674 (90 )% Selling, general and administrative expenses $ 1,351 $ 1,405 (4 )% $ 3,931 $ 4,051 (3 )% Other (income) expense, net $ (87 ) $ (48 ) 80 % $ (251 ) $ (189 ) 33 % Diluted earnings per share attributable to Gilead $ 2.47 $ 2.02 22 % $ 6.29 $ 2.72 NM Shares used in non-GAAP diluted earnings per share attributable to Gilead calculation 1,254 1,254 — % 1,256 1,254 — % Product gross margin 86.5 % 86.8 % -27 bps 86.3 % 86.1 % 24 bps Research and development expenses as a % of revenues 17.2 % 18.3 % -115 bps 19.2 % 19.4 % -29 bps Selling, general and administrative expenses as a % of revenues 17.4 % 18.6 % -123 bps 18.3 % 19.1 % -85 bps Operating margin 50.5 % 43.2 % 729 bps 47.0 % 25.5 % NM Effective tax rate 17.5 % 17.5 % -2 bps 17.6 % 30.0 % NM NM - Not Meaningful (1) Refer to Non-GAAP Financial Information section above for further disclosures on non-GAAP financial measures. A reconciliation between GAAP and non-GAAP financial information is provided in the tables below. GILEAD SCIENCES, INC. RECONCILIATION OF GAAP TO NON-GAAP FINANCIAL INFORMATION (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages and per share amounts) 2025 2024 2025 2024 Cost of goods sold reconciliation: GAAP cost of goods sold $ 1,569 $ 1,574 $ 4,610 $ 4,670 Acquisition-related – amortization(1) (577 ) (579 ) (1,735 ) (1,737 ) Restructuring — — — 1 Non-GAAP cost of goods sold $ 992 $ 995 $ 2,875 $ 2,933 Product gross margin reconciliation: GAAP product gross margin 78.6 % 79.1 % 78.1 % 77.8 % Acquisition-related – amortization(1) 7.9 % 7.7 % 8.3 % 8.2 % Restructuring — % — % — % — % Non-GAAP product gross margin 86.5 % 86.8 % 86.3 % 86.1 % Research and development expenses reconciliation: GAAP research and development expenses $ 1,346 $ 1,395 $ 4,215 $ 4,266 Acquisition-related – other costs(2) (4 ) (9 ) (41 ) (78 ) Restructuring (8 ) (5 ) (52 ) (68 ) Non-GAAP research and development expenses $ 1,334 $ 1,382 $ 4,123 $ 4,120 IPR&D impairment reconciliation: GAAP IPR&D impairment $ — $ 1,750 $ 190 $ 4,180 IPR&D impairment — (1,750 ) (190 ) (4,180 ) Non-GAAP IPR&D impairment $ — $ — $ — $ — Selling, general and administrative expenses reconciliation: GAAP selling, general and administrative expenses $ 1,357 $ 1,433 $ 3,980 $ 4,184 Acquisition-related – other costs(2) — (5 ) — (88 ) Restructuring (5 ) (23 ) (49 ) (45 ) Non-GAAP selling, general and administrative expenses $ 1,351 $ 1,405 $ 3,931 $ 4,051 Operating income (loss) reconciliation: GAAP operating income (loss) $ 3,327 $ 888 $ 8,038 $ (790 ) Acquisition-related – amortization(1) 577 579 1,735 1,737 Acquisition-related – other costs(2) 4 13 41 167 Restructuring 14 28 101 112 IPR&D impairment — 1,750 190 4,180 Non-GAAP operating income $ 3,921 $ 3,258 $ 10,104 $ 5,406 Operating margin reconciliation: GAAP operating margin 42.8 % 11.8 % 37.4 % (3.7 )% Acquisition-related – amortization(1) 7.4 % 7.7 % 8.1 % 8.2 % Acquisition-related – other costs(2) — % 0.2 % 0.2 % 0.8 % Restructuring 0.2 % 0.4 % 0.5 % 0.5 % IPR&D impairment — % 23.2 % 0.9 % 19.7 % Non-GAAP operating margin 50.5 % 43.2 % 47.0 % 25.5 % Other (income) expense, net reconciliation: GAAP other (income) expense, net $ (569 ) $ (306 ) $ (449 ) $ (41 ) Gain (loss) from equity securities, net 483 258 198 (148 ) Non-GAAP other (income) expense, net $ (87 ) $ (48 ) $ (251 ) $ (189 ) Income (loss) before income taxes reconciliation: GAAP income (loss) before income taxes $ 3,641 $ 956 $ 7,718 $ (1,477 ) Acquisition-related – amortization(1) 577 579 1,735 1,737 Acquisition-related – other costs(2) 4 13 41 167 Restructuring 14 28 101 112 IPR&D impairment — 1,750 190 4,180 (Gain) loss from equity securities, net (483 ) (258 ) (198 ) 148 Non-GAAP income before income taxes $ 3,752 $ 3,068 $ 9,586 $ 4,866 GILEAD SCIENCES, INC. RECONCILIATION OF GAAP TO NON-GAAP FINANCIAL INFORMATION - (Continued) (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages and per share amounts) 2025 2024 2025 2024 Income tax expense (benefit) reconciliation: GAAP income tax expense (benefit) $ 589 $ (297 ) $ 1,391 $ (174 ) Income tax effect of non-GAAP adjustments: Acquisition-related – amortization(1) 120 121 360 363 Acquisition-related – other costs(2) — 2 — 39 Restructuring 3 4 18 21 IPR&D impairment — 440 51 1,051 Gain from equity securities, net (43 ) (46 ) (33 ) (52 ) Discrete and related tax charges(3) (11 ) 314 (101 ) 214 Non-GAAP income tax expense $ 657 $ 538 $ 1,686 $ 1,461 Effective tax rate reconciliation: GAAP effective tax rate 16.2 % (31.1 )% 18.0 % 11.8 % Income tax effect of above non-GAAP adjustments and discrete and related tax adjustments(3) 1.3 % 48.6 % (0.4 )% 18.2 % Non-GAAP effective tax rate 17.5 % 17.5 % 17.6 % 30.0 % Net income (loss) attributable to Gilead reconciliation: GAAP net income (loss) attributable to Gilead $ 3,052 $ 1,253 $ 6,327 $ (1,303 ) Acquisition-related – amortization(1) 457 458 1,374 1,374 Acquisition-related – other costs(2) 4 11 41 128 Restructuring 11 24 83 92 IPR&D impairment — 1,310 139 3,129 (Gain) loss from equity securities, net (440 ) (212 ) (165 ) 200 Discrete and related tax charges(3) 11 (314 ) 101 (214 ) Non-GAAP net income attributable to Gilead $ 3,095 $ 2,531 $ 7,901 $ 3,405 Diluted earnings (loss) per share reconciliation: GAAP diluted earnings (loss) per share $ 2.43 $ 1.00 $ 5.04 $ (1.04 ) Acquisition-related – amortization(1) 0.36 0.37 1.09 1.10 Acquisition-related – other costs(2) — 0.01 0.03 0.10 Restructuring 0.01 0.02 0.07 0.07 IPR&D impairment — 1.04 0.11 2.51 (Gain) loss from equity securities, net (0.35 ) (0.17 ) (0.13 ) 0.16 Discrete and related tax charges(3) 0.01 (0.25 ) 0.08 (0.17 ) Difference in shares used for GAAP vs. Non-GAAP $ — $ — $ — $ (0.01 ) Non-GAAP diluted earnings per share $ 2.47 $ 2.02 $ 6.29 $ 2.72 Non-GAAP adjustment summary: Cost of goods sold adjustments $ 577 $ 579 $ 1,735 $ 1,736 Research and development expenses adjustments 12 13 93 146 IPR&D impairment adjustments — 1,750 190 4,180 Selling, general and administrative expenses adjustments 5 28 49 133 Total non-GAAP adjustments to costs and expenses 594 2,370 2,067 6,196 Other (income) expense, net, adjustments (483 ) (258 ) (198 ) 148 Total non-GAAP adjustments before income taxes 112 2,113 1,868 6,343 Income tax effect of non-GAAP adjustments above (79 ) (521 ) (396 ) (1,421 ) Discrete and related tax charges(3) 11 (314 ) 101 (214 ) Total non-GAAP adjustments to net income attributable to Gilead $ 43 $ 1,278 $ 1,573 $ 4,708 (1) Relates to amortization of acquired intangibles. (2) Adjustments include integration expenses and contingent consideration fair value adjustments associated with Gilead’s recent acquisitions. (3) Represents discrete and related deferred tax charges or benefits primarily associated with acquired intangible assets and transfers of intangible assets from a foreign subsidiary to Ireland and the United States. GILEAD SCIENCES, INC. RECONCILIATION OF GAAP TO NON-GAAP 2025 FULL-YEAR GUIDANCE(1) (unaudited) (in millions, except percentages and per share amounts) Provided February 11, 2025 Updated April 24, 2025 Updated August 7, 2025 Updated October 30, 2025 Projected product gross margin GAAP to non-GAAP reconciliation: GAAP projected product gross margin 77.0% - 78.0% 77.0% - 78.0% ~ 78.0% ~ 78.0% Acquisition-related expenses ~ 8.0% ~ 8.0% ~ 8.0% ~ 8.0% Non-GAAP projected product gross margin 85.0% - 86.0% 85.0% - 86.0% ~ 86.0% ~ 86.0% Projected operating income GAAP to non-GAAP reconciliation: GAAP projected operating income $10,200 - $10,700 $10,200 - $10,700 $10,300 - $10,700 $10,300 - $10,600 Acquisition-related, IPR&D impairment and restructuring expenses ~ 2,500 ~ 2,500 ~ 2,700 ~ 2,800 Non-GAAP projected operating income $12,700 - $13,200 $12,700 - $13,200 $13,000 - $13,400 $13,100 - $13,400 Projected effective tax rate GAAP to non-GAAP reconciliation: GAAP projected effective tax rate(2) ~ 20% ~ 21% ~ 21% ~ 16% Income tax effect of above non-GAAP adjustments and fair value adjustments of equity securities, and discrete and related tax adjustments(2) (~ 1%) (~ 2%) (~ 2%) ~ 3% Non-GAAP projected effective tax rate ~ 19% ~ 19% ~ 19% ~ 19% Projected diluted EPS GAAP to non-GAAP reconciliation: GAAP projected diluted EPS $5.95 - $6.35 $5.65 - $6.05 $5.85 - $6.15 $6.65 - $6.85 Acquisition-related, IPR&D impairment and restructuring expenses, fair value adjustments of equity securities and discrete and related tax adjustments(2) ~ 1.75 ~ 2.05 ~ 2.10 ~ 1.40 Non-GAAP projected diluted EPS $7.70 - $8.10 $7.70 - $8.10 $7.95 - $8.25 $8.05 - $8.25 (1) Our full-year guidance excludes the potential impact of any (i) acquisitions or business development transactions that have not been executed, (ii) future fair value adjustments of equity securities and (iii) discrete tax charges or benefits associated with changes in tax related laws and guidelines that have not been enacted, as Gilead is unable to project such amounts. The non-GAAP full-year guidance includes non-GAAP adjustments to actual current period results as well as adjustments for the known future impact associated with events that have already occurred, such as future amortization of our intangible assets and the future impact of discrete and related deferred tax charges or benefits primarily associated with acquired intangible assets and in-process research and development, transfers of intangible assets from a foreign subsidiary to Ireland and the United States, and legal entity restructurings. (2) GAAP projected effective tax rate and tax adjustments for the October 30, 2025 update include an October 2025 settlement with a tax authority related to a prior year legal entity restructuring. GILEAD SCIENCES, INC. CONDENSED CONSOLIDATED BALANCE SHEETS (unaudited) September 30, December 31, (in millions) 2025 2024 Assets Cash, cash equivalents and marketable debt securities $ 9,354 $ 9,991 Accounts receivable, net 5,095 4,420 Inventories(1) 4,387 3,589 Property, plant and equipment, net 5,500 5,414 Intangible assets, net 17,970 19,948 Goodwill 8,314 8,314 Other assets 7,914 7,319 Total assets $ 58,533 $ 58,995 Liabilities and Stockholders’ Equity Current liabilities $ 12,298 $ 12,004 Long-term liabilities 24,780 27,744 Stockholders’ equity(2) 21,456 19,246 Total liabilities and stockholders’ equity $ 58,533 $ 58,995 (1) Includes current and long-term inventories, which are disclosed separately in the notes to our financial statements in Form 10-K and Form 10-Q. (2) As of September 30, 2025 and December 31, 2024, there were 1,242 and 1,246 shares of common stock issued and outstanding, respectively. GILEAD SCIENCES, INC. SELECTED CASH FLOW INFORMATION (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2025 2024 2025 2024 Net cash provided by operating activities $ 4,109 $ 4,309 $ 6,692 $ 7,853 Net cash used in investing activities (427 ) (710 ) (2,958 ) (3,224 ) Net cash used in financing activities (1,490 ) (1,379 ) (6,482 ) (5,693 ) Effect of exchange rate changes on cash and cash equivalents (5 ) 44 87 15 Net change in cash and cash equivalents 2,187 2,265 (2,661 ) (1,049 ) Cash and cash equivalents at beginning of period 5,144 2,772 9,991 6,085 Cash and cash equivalents at end of period $ 7,330 $ 5,037 $ 7,330 $ 5,037 Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2025 2024 2025 2024 Net cash provided by operating activities $ 4,109 $ 4,309 $ 6,692 $ 7,853 Purchases of property, plant and equipment (147 ) (140 ) (358 ) (376 ) Free cash flow(1) $ 3,962 $ 4,169 $ 6,335 $ 7,478 (1) Free cash flow is a non-GAAP liquidity measure. Please refer to our disclosures in the Non-GAAP Financial Information section above. GILEAD SCIENCES, INC. PRODUCT SALES SUMMARY Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2025 2024 2025 2024 HIV Biktarvy – U.S. $ 2,940 $ 2,826 $ 8,212 $ 7,726 Biktarvy – Europe 427 375 1,231 1,110 Biktarvy – Rest of World 320 272 922 814 3,686 3,472 10,366 9,649 Descovy – U.S. 652 534 1,791 1,339 Descovy – Europe 23 24 67 75 Descovy – Rest of World 25 28 81 82 701 586 1,939 1,496 Genvoya – U.S. 323 384 950 1,088 Genvoya – Europe 34 44 114 138 Genvoya – Rest of World 19 21 54 66 377 449 1,118 1,292 Odefsey – U.S. 206 248 642 705 Odefsey – Europe 61 69 184 217 Odefsey – Rest of World 10 9 30 30 277 326 857 952 Symtuza - Revenue share(1) – U.S. 95 103 265 338 Symtuza - Revenue share(1) – Europe 26 33 88 101 Symtuza - Revenue share(1) – Rest of World 3 3 9 9 124 139 362 448 Other HIV(2) – U.S. 82 65 198 190 Other HIV(2) – Europe 22 26 85 96 Other HIV(2) – Rest of World 9 9 28 36 112 100 310 322 Total HIV – U.S. 4,299 4,161 12,059 11,386 Total HIV – Europe 592 570 1,769 1,737 Total HIV – Rest of World 386 342 1,124 1,038 5,277 5,073 14,952 14,160 Liver Disease Sofosbuvir / Velpatasvir(3) – U.S. 146 222 497 737 Sofosbuvir / Velpatasvir(3) – Europe 65 67 227 230 Sofosbuvir / Velpatasvir(3) – Rest of World 97 96 273 299 309 385 996 1,266 Vemlidy – U.S. 136 126 358 338 Vemlidy – Europe 12 11 36 33 Vemlidy – Rest of World 132 95 389 328 280 232 783 699 Other Liver Disease(4) – U.S. 132 45 307 134 Other Liver Disease(4) – Europe 81 54 233 148 Other Liver Disease(4) – Rest of World 17 17 53 55 231 116 593 337 Total Liver Disease – U.S. 414 393 1,162 1,210 Total Liver Disease – Europe 158 132 496 411 Total Liver Disease – Rest of World 247 207 714 682 819 733 2,372 2,302 Veklury Veklury – U.S. 140 393 390 784 Veklury – Europe 43 81 84 204 Veklury – Rest of World 93 219 225 473 277 692 700 1,461 GILEAD SCIENCES, INC. PRODUCT SALES SUMMARY - (Continued) (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2025 2024 2025 2024 Oncology Cell Therapy Tecartus – U.S. 40 63 122 181 Tecartus – Europe 35 29 107 102 Tecartus – Rest of World 8 6 25 22 83 98 254 305 Yescarta – U.S. 123 145 444 502 Yescarta – Europe 151 182 455 509 Yescarta – Rest of World 75 60 228 170 349 387 1,127 1,181 Total Cell Therapy – U.S. 163 208 566 683 Total Cell Therapy – Europe 186 211 562 611 Total Cell Therapy – Rest of World 83 66 253 192 432 485 1,381 1,485 Trodelvy Trodelvy – U.S. 221 226 626 655 Trodelvy – Europe 89 80 259 217 Trodelvy – Rest of World 47 26 128 88 357 332 1,013 960 Total Oncology – U.S. 384 433 1,192 1,338 Total Oncology – Europe 275 291 821 828 Total Oncology – Rest of World 129 92 381 280 788 816 2,395 2,446 Other AmBisome – U.S. 2 6 15 37 AmBisome – Europe 69 71 201 210 AmBisome – Rest of World 52 52 175 176 123 130 391 424 Other(5) – U.S. 34 47 125 203 Other(5) – Europe 7 8 23 26 Other(5) – Rest of World 20 16 55 52 61 71 204 281 Total Other – U.S. 36 53 140 241 Total Other – Europe 76 80 225 236 Total Other – Rest of World 72 68 230 228 184 201 594 705 Total product sales – U.S. 5,274 5,433 14,943 14,958 Total product sales – Europe 1,144 1,154 3,395 3,416 Total product sales – Rest of World 928 928 2,674 2,700 $ 7,345 $ 7,515 $ 21,013 $ 21,074 (1) Represents Gilead’s revenue from cobicistat (“C”), FTC and TAF in Symtuza (darunavir/C/FTC/TAF), a fixed dose combination product commercialized by Janssen Sciences Ireland Unlimited Company. (2) Includes Atripla, Complera/Eviplera, Emtriva, Sunlenca, Stribild, Truvada, Tybost and Yeztugo/Yeytuo. (3) Includes Epclusa and the authorized generic version of Epclusa sold by Gilead’s separate subsidiary, Asegua Therapeutics LLC (“Asegua”). (4) Includes ledipasvir/sofosbuvir (Harvoni and the authorized generic version of Harvoni sold by Asegua), Hepcludex, Hepsera, Livdelzi/Lyvdelzi, Sovaldi, Viread and Vosevi. (5) Includes Cayston, Jyseleca, Letairis and Zydelig. Investors: Jacquie Ross, CFA investor_relations@gilead.com Media: Ashleigh Koss public_affairs@gilead.com