髓外疾病（EMD）是多发性骨髓瘤（MM）的一个高风险特征，即使在新型免疫疗法时代仍然是一个不良的预后因素。在这里，我们将空间转录组学（tomo-seq [n=2] 和 10X Visium [n=12]）和单细胞 RNA 测序 (scRNAseq [n=3]) 应用于一组 14 个 EMD 活检，以剖析三个-肿瘤细胞及其微环境的维度结构。总体而言，浸润的免疫细胞和基质细胞显示出患者内部和患者之间的差异，并且在病灶上分布不均匀。我们观察到浆细胞内拷贝数水平的显着异质性，包括在肿瘤的限制区域中出现新的亚克隆，这与基因组不稳定性一致。我们进一步确定了双特异性抗体治疗的两种重要抗原 GPRC5D 和 TNFRSF17 的空间表达差异。 EMD 肿块被各种免疫细胞（包括 T 细胞）浸润。值得注意的是，耗尽的 TIM3 /PD-1 T 细胞与 MM 细胞广泛共定位，而功能性和活化的 CD8 T 细胞与其他无肿瘤区域的 M1 巨噬细胞一起显示出局灶性浸润模式。在对 T 细胞接合双特异性抗体产生反应的情况下，这种健康 T 细胞和耗尽 T 细胞的分离得到了解决。 MM细胞和微环境细胞嵌入影响免疫激活和血管生成的复杂网络中，氧化磷酸化代表EMD病变内的主要代谢程序。总之，空间转录组学揭示了 EMD 中的多细胞生态系统，具有检查点抑制和双重靶向作为潜在的新治疗途径。版权所有 © 2024 美国血液学会。

Extramedullary disease (EMD) is a high-risk feature of multiple myeloma (MM) and remains a poor prognostic factor even in the era of novel immunotherapies. Here we applied spatial transcriptomics (tomo-seq [n=2] and 10X Visium [n=12]), and single-cell RNA sequencing (scRNAseq [n=3]) to a set of 14 EMD biopsies to dissect the three-dimensional architecture of tumor cells and their microenvironment. Overall, the infiltrating immune and stromal cells showed both intra- and inter-patient variation with no uniform distribution over the lesion. We observed substantial heterogeneity at the copy number level within plasma cells, including the emergence of new subclones in circumscribed areas of the tumor, consistent with genomic instability. We further identified spatial expression differences of GPRC5D and TNFRSF17, two important antigens for bispecific antibody therapy. EMD masses were infiltrated by various immune cells, including T-cells. Notably, exhausted TIM3+/PD-1+ T-cells diffusely co-localized with MM cells, whereas functional and activated CD8+ T-cells showed a focal infiltration pattern along with M1 macrophages in otherwise tumor-free regions. This segregation of fit and exhausted T-cells was resolved in the case of response to T-cell engaging bispecific antibodies. MM cells and microenvironment cells were embedded in a complex network that influenced immune activation and angiogenesis, and oxidative phosphorylation represented the major metabolic program within EMD lesions. In summary, spatial transcriptomics has revealed a multicellular ecosystem in EMD with checkpoint inhibition and dual targeting as potential new therapeutic avenues.Copyright © 2024 American Society of Hematology.