Pre-clinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma. Pre-clinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma

Multiple myeloma (MM) is a neoplasia of bone marrow (BM) plasma cells that remains largely incurable with current treatment. Advancing therapeutic discoveries has been hampered by the lack of genetically heterogeneous models of MM. To circumvent this limitation, we engineered fifteen mouse models carrying combinations of eight MM genetic drivers, which fulfilled the pathogenesis of human disease including progression from premalignant states under immune surveillance. Integrative analyses of ⁓500 mice and ⁓1,000 patients revealed a MAPK-MYC genetic pathway that regulates time to progression and immune escape mechanisms across genetically heterogeneous tumors. During progression, MYC-dependent and independent remodeling of the BM microenvironment divided MM into immune categories with predominance of selected T-cell subpopulations, which dictated immunotherapy responses. Experimental targeting of the cytotoxic or immunosuppressive T-cell states observed in refractory MM patients enhanced immunotherapy effectiveness. Our resource enabled characterization of MM cell-intrinsic and immunological traits at unprecedented levels, which will accelerate the translation of personalized immunotherapy. Overall design: scRNA-seq + scTCR-seq were performed in 13 bone marrow aspirates from 2 healthy (Ycɣ1), 3 MGUS (BIcɣ1), 3 MM (BIcɣ1), 3 MGUS (MIC) and 2 MM (MIC) bearing mice. Cells were FACS sorted (a mix of 0.8x10^5 T cells + 0.8x10^5 NK cells + 0.25x10^5 monocytes + 0.15x10^5 B cells) in 100 µL of PBS+0.05% BSA. Samples with at least 90% viability were processed using the 10X Genomics (CA, USA) scRNA/TCRseq kit, following the manufacturer’s instructions (Chromium Next GEM Single Cell V(D)J v1.1 protocol rev F for human samples and Chromium Next GEM Single Cell 5’ v2 Dual Index protocol rev B for mice samples). Quality control was performed with Qubit Fluorometric Quantification (ThermoFisher Scientific, MA, USA) using the double-stranded DNA high-sensitivity assay kit, and with TapeStation (Agilent, Santa Clara, CA) using high-sensitivity screentapes. Libraries were sequenced on a NextSeq 550 (Illumina, San Diego, CA). WES was performed in 71 BM samples isolated from GFP+ CD138+B220− PCs (purity, >99%), including 62 samples from the MM stage, 3 samples of pooled PCs from nine mice at the MGUS stage (3 mice with similar genotype were included on each pooled sample), and 6 samples from MM-derived cell lines. As MM reference controls, 5TGM1 and 12598Vk*Myc cell lines were also characterized.

多发性骨髓瘤（Multiple myeloma, MM）是一种骨髓（bone marrow, BM）浆细胞肿瘤，当前治疗手段下仍难以实现完全治愈。由于缺乏遗传异质性的MM模型，治疗相关研究进展长期受阻。为突破这一局限，我们构建了15种携带8种MM遗传驱动因子组合的小鼠模型，这些模型复现了人类疾病的发病进程，涵盖免疫监视下从癌前状态向恶性肿瘤进展的全过程。
通过对近500只小鼠和约1000例患者开展整合分析，我们发现了一条调控遗传异质性肿瘤进展时长与免疫逃逸机制的MAPK-MYC遗传通路。在疾病进展过程中，骨髓微环境的MYC依赖性与非依赖性重塑将MM划分为以特定T细胞亚群占优的免疫亚型，这些亚型决定了免疫治疗的响应效果。针对复发难治性MM患者中检出的细胞毒性或免疫抑制性T细胞状态进行实验性靶向干预，可显著提升免疫治疗的疗效。
本数据集资源首次实现了MM细胞内在特性与免疫学特征的高精度解析，将加速个性化免疫治疗的转化研究。
整体实验设计：我们对13份骨髓穿刺样本进行了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与单细胞T细胞受体测序（single-cell T cell receptor sequencing, scTCR-seq），样本来源包括2只健康小鼠（Ycɣ1组）、3例意义未明单克隆免疫球蛋白血症（monoclonal gammopathy of undetermined significance, MGUS）小鼠（BIcɣ1组）、3例MM小鼠（BIcɣ1组）、3例MGUS小鼠（MIC组）及2例MM小鼠（MIC组）。通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）将细胞按比例混合：0.8×10^5个T细胞 + 0.8×10^5个NK细胞 + 0.25×10^5个单核细胞 + 0.15×10^5个B细胞，重悬于100 μL含0.05%牛血清白蛋白（bovine serum albumin, BSA）的磷酸盐缓冲液（phosphate-buffered saline, PBS）中。将存活率≥90%的样本按照10X Genomics（美国加利福尼亚州）单细胞RNA/TCR测序试剂盒的操作说明进行处理：人类样本采用Chromium Next GEM单细胞V(D)J v1.1 rev F方案，小鼠样本采用Chromium Next GEM单细胞5’ v2双索引方案rev B。
质控环节采用Qubit荧光定量系统（ThermoFisher Scientific, 美国马萨诸塞州）的双链DNA高灵敏度检测试剂盒，以及TapeStation生物分析仪（Agilent, 美国加利福尼亚州圣克拉拉市）的高灵敏度屏幕胶带完成质检。构建好的文库在NextSeq 550测序平台（Illumina, 美国加利福尼亚州圣地亚哥市）上完成测序。
全外显子测序（whole exome sequencing, WES）针对71份源自GFP阳性CD138+B220−浆细胞（plasma cells, PCs，纯度>99%）的骨髓样本开展，其中包括62份MM阶段样本、3份MGUS阶段的混合浆细胞样本（每个混合样本纳入3只基因型一致的小鼠），以及6份MM来源细胞系样本。作为MM参考对照，我们同时对5TGM1与12598Vk*Myc细胞系进行了测序分析。