Drug tolerant persisters and immunotherapy persister cells exhibit cross-resistance and share common survival mechanisms

Persisters are a rare sub-population of tumor cells that survive anti-cancer therapy and are thought to be a major cause of recurrence. These cells have been identified following both drug- and immune-therapy but are generally considered to be distinct entities. Since both pharmacological agents and immune cells often kill via apoptosis, we tested a hypothesis that both types of cells survive based on reduced mitochondrial apoptotic sensitivity, which in turn would yield a similar and reciprocal multi-agent resistant phenotype. Supporting this hypothesis, we indeed observed that IPCs acquired a reduced sensitivity to multiple drug classes and radiotherapy, suggesting non-immune mechanisms are important in the survival of cancer cells after immunotherapy. Likewise, DTPs developed not only a reduced sensitivity to multiple drug classes and radiotherapy, but also acquired a reduced sensitivity to T cell killing. Both IPCs and DTPs developed a decreased sensitivity to mitochondrial apoptosis. A sub-population of IPCs downregulated antigen and upregulated PD-L1. Intriguingly, in the IPCs that didn't employ these mechanisms of resistance, a greater decrease in sensitivity to mitochondrial apoptosis was observed, suggesting that the presence or absence of a resistance mechanism can exert selective pressures over the emergence of others. Targeting anti-apoptotic dependencies in persisters increased sensitivity to chemotherapy or CAR T therapy. These results suggest that common biological mechanisms underly survival of persisters, whether derived from immune or drug therapy, and offer an explanation for the acquired cross-resistance to these two types of therapies often observed in the clinic. Overall design: RNA was extracted from parental HeLa cells, selected HeLa immune persister cells (IPC) and 3 different HeLa drug tolerant persister (DTP) cells following respective treatment with IC90 concentrations of 5-FU, Oxaliplatin and Etopiside for 7 days. RNA was extracted using the RNeasy Mini Kit (Qiagen). Prior to RNA sequencing, RNA was treated with DNase using RNase-Free DNase Set (Qiagen). rRNA depletion was performed from 100ng of purified RNA using QIAseq FastSelect rRNA HMR reagents according to manufacturer's protocol. Libraries were prepared using Roche Kapa Biosystems RNA HyperPrep sample preparation reagents on a Beckman Coulter Biomek i7. Finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced with paired-end 150bp reads on an Illumina NovaSeq 6000at the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Sequenced reads were aligned to the UCSC hg38 reference genome assembly and gene counts were quantified using STAR (v2.7.3a).

癌症持久性细胞（persister cells）是一类罕见的肿瘤细胞亚群，可在抗肿瘤治疗后存活，被认为是肿瘤复发的主要诱因之一。这类细胞既可在药物治疗后被鉴定到，也可在免疫治疗后被发现，但通常被认为是两类独立的细胞群体。由于药理药物与免疫细胞均多通过细胞凋亡（apoptosis）途径杀伤肿瘤细胞，我们提出假说：两类持久性细胞的存活均源于线粒体凋亡敏感性（mitochondrial apoptotic sensitivity）的降低，进而会产生相似且互为因果的多药耐药表型。

为验证该假说，我们观察到免疫持久性细胞（immune persister cells, IPC）对多种药物类别及放射治疗均产生了敏感性降低，这提示非免疫机制在免疫治疗后癌症细胞的存活中发挥重要作用。同样，药物耐受持久性细胞（drug tolerant persister cells, DTP）不仅对多种药物类别及放射治疗的敏感性降低，还获得了对T细胞杀伤的耐受性。两类细胞（IPC与DTP）均表现出线粒体凋亡敏感性的下降。其中一部分IPC出现了抗原表达下调与程序性死亡受体配体1（PD-L1）表达上调。有趣的是，未采用上述耐药机制的IPC群体，其线粒体凋亡敏感性的下降程度更为显著，这提示某一耐药机制的存在与否，可对其他耐药机制的出现施加选择压力。靶向持久性细胞的抗凋亡依赖通路，可增强其对化疗或嵌合抗原受体T细胞治疗（CAR T therapy）的敏感性。本研究结果表明，无论源于免疫治疗还是药物治疗，持久性细胞的存活均共享共同的生物学机制，这也为临床中常见的两类治疗获得性交叉耐药现象提供了解释。

实验整体设计：从亲本海拉细胞（HeLa）、分选得到的海拉免疫持久性细胞（IPC）以及3株海拉药物耐受持久性细胞（DTP）中提取总RNA，上述细胞均经对应IC90浓度的5-氟尿嘧啶（5-FU）、奥沙利铂（Oxaliplatin）与依托泊苷（Etopiside）处理7天。总RNA提取采用Qiagen公司的RNeasy Mini试剂盒。在进行RNA测序前，使用Qiagen公司的无RNase DNase试剂盒对RNA进行DNase处理。取100ng纯化后的RNA，按照制造商方案使用QIAseq FastSelect rRNA HMR试剂进行核糖体RNA（rRNA）去除。使用罗氏Kapa Biosystems的RNA HyperPrep样本制备试剂，结合贝克曼库尔特Biomek i7自动化工作站完成文库构建。最终的双链DNA文库通过Qubit荧光定量仪与安捷伦TapeStation 4200生物分析仪进行定量与质量评估。将唯一双索引文库以等摩尔比混合，在Illumina MiSeq测序仪上进行浅测序，以进一步评估文库质量与混合文库的平衡度。最终混合文库在丹娜法伯癌症研究院分子生物学核心实验室，使用Illumina NovaSeq 6000测序仪进行150bp双端测序。测序reads比对至UCSC hg38参考基因组组装版本，并使用STAR软件（v2.7.3a）进行基因计数定量。