修回日期: 2021-07-30
接受日期: 2021-09-29
在线出版日期: 2021-11-08
胰腺癌五年生存率低于5%, 现有的治疗手段治疗效果有限, 针对胰腺癌的治疗仍面临巨大挑战. 肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte, TIL)是肿瘤微环境的重要组成部分, 起到主导肿瘤微环境局部免疫反应的作用, 是许多癌症的有利预后因素. TIL能够特异性地识别肿瘤细胞, 通过细胞免疫对这些肿瘤细胞进行杀伤. TIL疗法已经被证明在黑色素瘤、宫颈癌、乳腺癌等实体瘤中具有良好的治疗效果, 但是应用TIL治疗胰腺癌仍然存在一些难点, 包括新生抗原的筛选、TIL快速扩增、导向到肿瘤部位及细胞衰竭. 本文将就近年来人们对TIL在胰腺癌中的研究进行概述, 挖掘TIL在胰腺癌治疗中的潜在应用价值.
核心提要: 胰腺癌肿瘤微环境的特异性, 即所谓的冷肿瘤, 是影响免疫治疗主要因素, 由冷转热, 结合肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte, TIL)疗法等免疫疗法可能会显著改善胰腺癌治疗现状.
引文著录: 祝文君, 胡湛斐, 袁周. 肿瘤浸润淋巴细胞在胰腺癌中研究进展. 世界华人消化杂志 2021; 29(21): 1207-1214
Revised: July 30, 2021
Accepted: September 29, 2021
Published online: November 8, 2021
The 5-year survival rate of pancreatic cancer is less than 5%, and the only available treatments, surgery, chemotherapy, and chemoradiation, have shown limited effectiveness. Therefore, alternative treatment strategies are urgently needed. In recent years, tumor infiltrating lymphocyte (TIL) therapy has shown promising successes in the treatment of some types of solid tumors because of its diverse TCR clonality, superior tumor-homing ability, and low off-target toxicity. The significant association between a high TIL density in pancreatic cancer tissue and a good clinical outcome and success of pancreatic cancer-specific TIL expansion ex vivo potentiates the rationality of the TIL therapy in pancreatic cancer. However, there are still many challenges ahead, such as neoantigen screening, rapid cell expansion, and low cytotoxicity. This article reviews the recent advances and limitations of TIL therapy in pancreatic cancer and discusses its future directions.
- Citation: Zhu WJ, Hu ZF, Yuan Z. Progress in research of tumor infiltrating lymphocytes in pancreatic cancer. Shijie Huaren Xiaohua Zazhi 2021; 29(21): 1207-1214
- URL: https://www.wjgnet.com/1009-3079/full/v29/i21/1207.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v29.i21.1207
胰腺癌是常见的消化道恶性肿瘤之一. 据中国国家癌症中心统计[1], 预估全国2015年胰腺癌发病率为6.92人/10万人, 位居人类实体肿瘤第十位; 死亡率6.16人/10万人, 位居人类实体肿瘤第六位. 而根据美国癌症研究协会的调查, 2020年美国胰腺癌死亡率高居肿瘤相关死亡的第四位, 胰腺癌的防控形势非常严峻.
胰腺导管腺癌(pancreatic ductal adenocarcinoma, PDAC)是胰腺癌的最常见组织学类型, 是最具侵袭性的消化道恶性肿瘤, 预后极差, 中位生存期仅6个月, 5年生存率9%[2]. 手术是唯一可能根治胰腺癌的方法. 但由于缺乏有效的早期诊断方法及肿瘤的高侵袭性, 80%-85%的PDAC患者在发现时已处于晚期, 仅有约20%的患者在确诊后适宜接受根治性切除术治疗, 这部分患者5年生存率也只有20%[3]. 尽管近年来有关胰腺癌的治疗取得一定的进步, 但其预后没有得到明显改善.
正常情况下, 免疫系统可以识别并清除肿瘤细胞, 但为了生存和生长, 肿瘤细胞能够采用不同策略, 使人体的免疫系统受到抑制, 不能正常的杀伤肿瘤细胞, 从而在抗肿瘤免疫应答的各阶段得以幸存. 近年来, 人们利用各种手段重新激活或者增强机体的肿瘤免疫, 如免疫检查点抑制剂(checkpoint inhibitors, ICIs)和细胞治疗等, 获得了较好的疗效, 显著改善了癌症患者的生存状况.
免疫治疗在部分肿瘤治疗中有一定突破, 但单一使用一种免疫治疗, 并不能显著改善胰腺癌患者的生存情况. 本文将结合胰腺癌微环境的特点以及免疫治疗进展, 对肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte, TIL)在胰腺癌中研究进展进行概述, 为胰腺癌的治疗提供新的思路.
一些致癌因素导致基因突变或者部分蛋白异常表达, 会导致细胞发生癌变. 在肿瘤的发展过程中, 癌细胞被免疫系统识别后, 第一阶段, 也就是细胞癌变早期, 癌细胞被免疫细胞识别清除; 下一阶段, 当致癌因素持续存在刺激的情况下, 癌变细胞类型和数量增多, 癌症和免疫力达到动态平衡; 最后阶段, 肿瘤细胞释放一些免疫抑制因子, 诱导机体免疫失活, 形成免疫逃逸, 肿瘤得以在体内生长, 肿瘤的微环境就此产生. 机体与肿瘤双方博弈的主要阵地就是肿瘤微环境[4].
胰腺癌肿瘤微环境的特征是广泛致密的增生间质, 包括细胞外基质成分和异种细胞群, 其中含有多种类型的免疫细胞, 但这些免疫细胞多处于数量与功能的失衡状态, 通常表现为具有抗肿瘤作用的CD4+、CD8+效应性T细胞、NK细胞和DC细胞数量减少, 并呈现无功能的或不成熟的表型和状态, 而具有免疫抑制作用的Tregs、髓源性抑制细胞(myeloid derived suppressor cells, MDSC)及肿瘤相关的巨噬细胞(tumor-associated macrophages, TAMs)功能活跃且大量存在[5-9]. 研究表明[10-12], 肿瘤相关的成纤维细胞、间充质基质细胞以及MDSC对肿瘤的浸润、增殖、转移等都具有促进作用. 此外, 微环境中的免疫抑制分子粘着斑激酶(FAK)、透明质酸和吲哚胺2, 3-双加氧酶(IDO)等[13-15]也对机体的免疫起负调控作用. 肿瘤微环境中的Treg细胞通过分泌抑制性细胞因子介导了效应T细胞对胰腺癌肿瘤相关抗原产生免疫耐受, 严重影响CD8+ CTL的功能, 从而使肿瘤细胞逃避免疫监视[13-17]. 冷肿瘤(如胶质母细胞瘤)中的肿瘤微环境(TME)含有较多抑制性免疫细胞以及丰富的癌相关成纤维细胞(CAFs)和细胞外基质(ECM), 细胞毒性T细胞很少浸润[18]. 相反, "热肿瘤"(如黑色素瘤和非小细胞肺癌)TME中有较多T细胞浸润, 对ICIs较为敏感[19]. PDAC被认为是冷肿瘤, 对包括免疫检查点抑制剂在内的免疫疗法不敏感[20,21].
肿瘤微环境不仅与胰腺癌细胞诱导的免疫抑制有关, 而且可能与疾病的严重程度密切相关. 有研究表明[22], 肿瘤微环境中Treg细胞的数量与胰腺癌肿瘤进展及不良的预后效果成正相关. 另外, 肿瘤微环境中的肥大细胞通过促进血管生成、癌细胞增殖和侵袭影响肿瘤生长[23]. 结直肠癌、肝细胞癌、结直肠癌肝转移患者中, 肿瘤组织肥大细胞浸润预测长期预后较差[24-26].
TIL是存在于肿瘤实质与间质内的免疫细胞, 是机体免疫系统对肿瘤局部免疫应答的直接反应, 也是肿瘤微环境的重要组成部分, 在肿瘤免疫机制中起免疫应答和调控作用, 其主要包括CD3+T淋巴细胞(75%的TILs)、CD20+B淋巴细胞(<20%的TILs)、单核细胞(<l0%的TILs)和NK细胞(<5%的TILs)等. TIL中的T淋巴细胞介导的获得性免疫是有效且持续的抗肿瘤反应的关键性基础. TIL可用于对PDAC的预后评价[27-30], 研究表明在PDAC患者中TIL的高水平浸润与更好的预后相关, TIL可能通过分泌TNF-α抑制PDAC的生长[31]. 肿瘤组织T细胞中IL-22的高表达, 与肿瘤分期和不良预后显著相关[32]. 有研究发现, 胰腺癌组织TIL分泌的膜结合或分泌蛋白semaphorin-4D参与抗肿瘤免疫调节[33].
理论上, PDAC细胞周围的T细胞密度越高, T细胞发挥的功能越强. PDAC微环境中CTL的高度浸润才能有效攻击肿瘤细胞.肿瘤微环境中其它免疫细胞如肥大细胞、Treg等对胰腺癌患者预后评价都有一定的意义. 肿瘤微环境中免疫状态可帮助评估PDAC患者术后的预后情况. 同时, 有针对性的选择治疗方法, 才能更有效地攻克胰腺癌治疗难题.
TIL是存在于肿瘤组织、肿瘤引流淋巴结、癌性胸腹水中的一群高度异质性细胞, 相比于外周血淋巴细胞, TIL细胞中含有较高比例的活化的肿瘤特异性T淋巴细胞, 这一类淋巴细胞经过体外分离、扩增、活化后, 对肿瘤细胞的杀伤活性提高.
TIL细胞治疗癌症的机理是这些T细胞实际上已经浸润在组织内部, 可以识别肿瘤多抗原靶点, 并识别并杀死癌细胞. 与嵌合抗原受体T细胞免疫疗法(chimeric antigen receptor T-cell immunotherapy, CAR-T)和T细胞受体嵌合型T细胞免疫疗法(chimeric T cell receptor T-cell immunotherapy, TCR-T)相比, TIL疗法存在着一系列优势: (1)TIL疗法由靶向癌细胞中多种抗原的T细胞组成, 因此可以通过多个靶点激发对癌细胞的细胞毒性反应, 而CAR-T和TCR-T细胞疗法通常只能靶向一个抗原, 这让癌细胞可能更容易对它们产生抗性, 这跟肿瘤的异质性有关, TIL可以杀死更多群体的肿瘤细胞; (2)CAR-T疗法在治疗实体瘤方面进展有限, 而TIL疗法已经在多种癌症类型中产生了可喜的疗效[34]; (3)TIL疗法使用的细胞是迁移到肿瘤中的天然TIL细胞, 一般不进行基因改造, 目前还未观察到在其它细胞治疗中常见的脱靶效应和细胞因子释放综合征等毒副作用, 如表1所示.
| TIL疗法 | TCR-T疗法 | CAR-T疗法 | |
| 特点 | 多个肿瘤抗原靶点 | 单个肿瘤抗原靶点 | 单个肿瘤抗原靶点 |
| 疗效 | 多种实体瘤中有效[34] | 少数实体瘤应用 | 暂无实体瘤成功案例 |
| 副作用 | 极少发现副作用 | 脱靶效应 | 细胞因子释放综合症 |
TIL疗法的应用史可以追溯至上世纪80年代, 1986年由Rosenberg等[35]首次尝试用TIL细胞治疗癌症. 1988年研究表明[36], 从黑色素瘤患者的肿瘤组织中分离的TIL, 经过离体培养扩增后回输至患者体内, 有效缓解了患者的病情恶化. Rosenberg在2002年报道化疗联合TIL治疗13例黑色素瘤患者, 其中10例表现出不同程度的缓解[37]; 从1994年到2008年间, Rosenberg研究组结合放化疗和过继性T细胞疗法, 将黑色素瘤的客观缓解率(objective response rate, ORR)从34%提高到72%[38]. 2013年报道CD4+/CD8+混合TIL群体自体输注疗效优于CD8+富集TIL群体[39]. 随着二代测序技术的发展, Rosenberg将外显子深度测序技术与TIL技术相结合, 开发出更为个体化的TIL精准医疗手段[40-43]. 2014年报道一名携带ERBB2IP突变的黑色素瘤患者经特异性CD4+ TH1 TIL输注后, 病情缓解, 实现一年半的带瘤生存, 经第二次95%CD4+ TH1 TIL输注, 病情再度缓解, 并且在其后6个月随访期内肿瘤持续性缩小[44]. 2019年ASCO公布的INNOVA TIL-04宫颈癌临床II期研究数据[45], 27名复发、转移或持续性宫颈癌患者, 平均年龄45岁, 之前平均接受2.4种治疗方案, 平均注射TIL细胞量: 2.8×1010个, 中位随访时间7.4个月, 没有发现任何严重副作用, 而且客观缓解率达到44%, TIL疗法已获FDA"突破性疗法"认定(ClinicalTrials. gov Identifier: NCT03108495), 表明TIL疗法应用于实体瘤以正在成为可能.
近年来TIL治疗的出色表现得益于技术的进步: (1)输注前清淋处理, 杀死抑制性免疫细胞并给输注的TIL提供空间; (2)输注幼稚细胞(Young TIL), 新工艺快速扩增技术(rapid expand protocol, REP)能在14天内将从肿瘤组织分离的TIL迅速扩增数千倍, 细胞产品中含更多的TCM, 其对肿瘤的杀伤作用优于长时间培养制备的细胞产品, 后者含有更多的疗效差一些的TEM; (3)TIL输注后同时大剂量用IL-2维持TIL体内增殖.
近年来, 单一使用树突状细胞疫苗、溶瘤病毒和免疫抑制剂等免疫治疗PDAC一直没有突破性进展. 导致胰腺癌失败的原因有很多, 免疫抑制性肿瘤微环境(TME)是关键因素, 其特征是效应T细胞浸润差[46-48], 以及较低的肿瘤突变负荷[49,50]. 近期研究显示[51-53], 部分肿瘤患者表现出效应T细胞高水平浸润, 这部分患者拥有更长的总生存期, 提示个体化治疗胰腺癌可能改善患者的生存状况. 通过将TME从"冷"变为"热"来突破PDAC中的免疫屏障, 可能与其他疗法联用增强疗效. 研究表明溶瘤病毒疗法可使TME由冷变热[54], 增加T细胞的浸润, 增强ICIs对晚期黑色素瘤患者的抗肿瘤作用[55]. 此外, 溶瘤病毒疗法诱导产生包括肿瘤抗原在内的肿瘤裂解物可以触发机体的先天性和获得性免疫反应[56]. 这种使胰腺癌由冷变热方法的出现, 为广泛利用TIL疗法治疗胰腺癌提供了契机.
机体具有抗癌活性的T淋巴细胞是使用过继细胞治疗的先决条件. TIL在胰腺癌治疗方面的研究还比较少. 2016年Meng[57]报道了从PDAC患者提取TIL的方法. 作者通过添加培养基中添加细胞因子(IL-2、IL-15、IL-21)来扩增TIL, 培养10天后, 补充CD3抗体(OKT3)和辐照过的PBMC, 17例胰腺癌来源的肿瘤组织块, 均可获得含大量CD8+T细胞的TIL, 并通过筛选可以得到高表达IFNγ的TIL. 这些都说明了TIL疗法在胰腺癌治疗中具有巨大潜力.
TIL疗法由于其在治疗实体瘤的优势, 特别是在黑色素瘤、宫颈癌等肿瘤方面的突出效果, 应用其治疗胰腺癌是一个很好发展方向. TIL细胞因其浸润在肿瘤组织内部, 在空间上与各种异质性肿瘤细胞接触, 随着基因重排, 表达丰富的TCR可以识别来自肿瘤细胞表面和内部的抗原, 应对多个肿瘤细胞群体, 这可能是人们利用TIL治疗实体瘤获得突破的原因之一, 但在胰腺癌的治疗中也存在一定的难点, 如表2所示. 通过对TIL细胞表面TCR进行测序可以分析, 并进行亲和力检测, 基因改造后筛选出亲和力更强的TCR, 通过转基因的方法装配到T细胞的表面可以更精准的靶向并识别胰腺癌, 或者直接筛选相应的TIL细胞群体, 也可能是TIL疗法应用的方向. Lu等人[58]从TIL获得的肿瘤特异性T淋巴细胞获得其T淋巴细胞受体(TCR)的遗传信息, 通过基因治疗将患者外周血中的T淋巴细胞转化, 也有可能改善肿瘤的治疗精准度和细胞制备时间. 另外, 使用胰腺癌肿瘤新生抗原(neoantigen)刺激TIL可以获得精准靶向更多细胞群体T细胞, 增强TIL对肿瘤杀伤的精准性. Tran等[44]通过这种方法已经临床治愈了一位肝肺转移的胆管癌患者. 不过, 当前基因测序所使用的肿瘤样本, 通常只是肿块中的一小部分, 无法代表整个肿瘤的基因突变状况, 这为肿瘤新抗原的选择增加了新的变量. 同时肿瘤新生抗原预测准确性较低, 难以实现较高的产品性价比. 还有就是开发和生产周期过长, 目前肿瘤新抗原药物开发、生产的时间在18周左右, 肿瘤患者愿不愿意等待以及能不能够等待这么长时间将是肿瘤新抗原药物应用的一大障碍. 除此之外, 通过筛选, 对大量同种癌症患者肿瘤组织进行高通量测序, 分析肿瘤相关抗原和新生抗原, 形成肿瘤抗原库, 最后用抗原库的抗原刺激体外培养的TIL治疗胰腺癌, 可能没有前一种精准, 但也很有可能会进一步提高效果, 这种方法仅仅是开始, 还没有临床数据. 因而, 目前临床上主要是采用已经比较成熟的针对多克隆TCR、未筛选(unselected)、幼稚细胞(young TIL)产品. 随着测序技术以及新抗原预测模型的进步, 将来也许可以改善当前的困境.
| TIL疗法治疗胰腺癌难点 | 可能解决方案 |
| 精准度相对较低 | (1)TIL测序, 筛选肿瘤特异性T细胞受体(TCR), 装配到TIL细胞上; (2)肿瘤新抗原筛选, 使用新抗原特异性TIL治疗. (需要进一步完善工艺, 缩短制备时间). |
| 免疫抑制微环境 | (1)与免疫检查点抑制剂联用; (2)转基因的方法使TIL细胞,同时表达免疫检查点抗体, 以降低免疫检查点抑制剂使用的副作用. |
| TIL中存在较多的耗竭T细胞以及大量不发挥肿瘤杀伤作用的T细胞 | 结合最新关于TIL表面标志物的研究,进一步筛选功能性的TIL,去除旁观者效应, 如CD39等. |
| 临床级TIL细胞生产难度大 | 以较小的肿瘤组织提取足够的TIL, 并将其在体外快速扩增到临床数量级, 需要工艺的优化, 如增加细胞因子、与滋养层细胞共培养等等. |
| 如何进一步增加TIL的归巢特性 | (1)联合其他肿瘤治疗方法, 如抗血管生成靶向药、清淋或者局部放疗; (2)TIL基因转导表达趋化因子或者特异性TCR等. |
虽然TIL出现在肿瘤的微环境中, 却不意味着癌细胞就可以被消灭, 具有免疫抑制作用的肿瘤微环境限制了它们的数量和活性, 削弱了有效攻击癌细胞的能力, 此外, TEM中还存在旁观T细胞. 假定扩增的TIL都具有杀伤活力, 回输后在外周血能证明起活性, 但TME会诱导其失活导致效果降低, 联合PD-1或者anti-PD1-TIL可能是更具有前景的一种策略.
体内数百种类型的T细胞, 并不是每个都能成为好的癌症战士. 目前的方法从胰腺癌肿瘤细胞中分离培养的TIL可能是耗竭的T细胞[59], 如果培养这些细胞后回输效果可能不理想, 所以也需要筛选并恢复T细胞的活性. TILs是高度异质性的细胞群, 不同患者和不同肿瘤之间均有较大差异. 研究表明[60], 表达CD39+的TIL细胞具有抗癌能力, 但其免疫活性受到肿瘤抑制, CD39-的TIL更多意义上是个旁观者. CD39有可能成为鉴别肿瘤特异性TIL、评估患者对基于TIL的免疫疗法敏感性的有效标志物, 或者作为筛选TIL的标志物, 成为多种免疫治疗方法的有效辅助手段.
当前限制TILs广泛应用的另一个主要问题是如何高效提取培养肿瘤微环境内的TILs细胞并使其扩增到临床治疗的数量级. 过去TIL的扩增大多需要较长的时间, 一些病程进展较快的患者根本无法使用TIL进行治疗. 较少的特异性TIL还不足以消除肿瘤. 因此, 需要扩大特异性TIL的数量, 以提高TIL治疗的疗效. Jin等人[61]提出了利用G-REX培养瓶对TIL进行原代培养和快速扩增的方法, 大大促进了TIL的生产, 用IL-2与4-1BB抗体和CD3抗体联合培养TIL可显著提高培养效率, 这种方法目前用于高分化浆液性卵巢癌. Poch等[62]通过添加4-1BB抗体, 优化原发性膀胱肿瘤中TIL的扩增, 为膀胱癌的治疗奠定了基础. 另外, Tavera等人[63]提出了一种基于CD137/4-1BB和白细胞介素-2刺激的TIL扩增新方法, 可以优先大量扩增CD8-TIL, 在这种方法中, TIL的扩增率迅速增加, 黑色素瘤中TIL的扩增时间不到三周.
TIL必须到达肿瘤部位才能发挥作用, 如何使T细胞归巢肿瘤, 也是提高TIL治疗胰腺癌的重大命题. 在过继性细胞治疗中, 回输的T细胞中仅有1%-2%真正浸润到肿瘤组织中. 活化的T细胞不能有效靶向穿透到肿瘤实质, 成为限制过继性细胞治疗发挥有效抗肿瘤作用的关键因素. 抗血管生成靶向药可以通过改善肿瘤血供而将胰腺癌TEM抑制状态恢复至正常状态, 并可能增加T细胞归巢数量, 因此, TIL联合抗血管生成靶向药可能会是一个比较好的方向. 放疗和清淋预处理可以创造有利于T细胞归巢的肿瘤局部微环境[34]. 趋化因子的不匹配、血管异常、内皮细胞无能、肿瘤局部抑制微环境等是阻碍T细胞高效归巢的主要因素. 为了进一步提高TIL杀伤肿瘤细胞的能力, 科学家们做了相关研究. 目前, 在黑色素瘤中有许多方法可以提高TIL到达肿瘤部位的效率, 相关实验证明, TIL中内源性CXCR1-TIL细胞迁移至黑色素瘤的数量较多, 这种方法有望扩展到其他肿瘤[64].
随着免疫治疗技术的发展, 特别是免疫检查点抑制剂以及DC疫苗, 溶瘤病毒等在胰腺癌治疗方面的研究, 胰腺癌的治疗也有了一定进步, 但是复发率和转移率居高不下, 难以显著改善患者的存活率.
治疗的主要难点是改善胰腺癌的肿瘤微环境, 增强肿瘤杀伤的能力. TIL因其特点有望改善胰腺癌治疗现状. 应用TIL治疗胰腺癌, 则需要筛选有效的TIL细胞, 改善TIL培养工艺, 提高TIL归巢到肿瘤组织的能力. TIL中有大量的旁观者细胞[65], 虽然浸润在肿瘤组织内部, 但是不发挥抗肿瘤活性. 因此, 改善TIL细胞的活性与精准度也是治疗胰腺癌必要的过程. 改变胰腺癌肿瘤微环境的免疫抑制特性, 转化冷肿瘤为热肿瘤(免疫检查点PARP抑制剂以及溶瘤病毒改善肿瘤微环境), 使之对免疫治疗更加敏感, 然后联合TIL疗法进行治疗, 有可能达到治愈胰腺癌的目的.
学科分类: 胃肠病学和肝病学
手稿来源地: 上海市
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科学编辑:张砚梁 制作编辑:张砚梁
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