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Abstract In multiple myeloma spatial differences in the subclonal architecture, molecular signatures and composition of the microenvironment remain poorly characterized. To address this shortcoming, we perform multi-region sequencing on paired random bone marrow and focal lesion samples from 17 newly diagnosed patients. Using single-cell RNA- and ATAC-seq we find a median of 6 tumor subclones per patient and unique subclones in focal lesions. Genetically identical subclones display different levels of spatial transcriptional plasticity, including nearly identical profiles and pronounced heterogeneity at different sites, which can include differential expression of immunotherapy targets, such as CD20 and CD38. Macrophages are significantly depleted in the microenvironment of focal lesions. We observe proportional changes in the T-cell repertoire but no site-specific expansion of T-cell clones in intramedullary lesions. In conclusion, our results demonstrate the relevance of considering spatial heterogeneity in multiple myeloma with potential implications for models of cell-cell interactions and disease progression.

Abstract Virtually all patients with multiple myeloma become unresponsive to treatment over time. Relapsed/refractory multiple myeloma (RRMM) is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations and profound changes of the bone marrow microenvironment (BME). However, the molecular mechanisms that drive drug resistance remain elusive. Here, we analyze the heterogeneous tumor cell population and its complex interaction network with the BME of 20 RRMM patients by single cell RNA-sequencing before/after treatment. Subclones with chromosome 1q-gain express a specific transcriptomic signature and frequently expand during treatment. Furthermore, RRMM cells shape an immune suppressive BME by upregulation of inflammatory cytokines and close interaction with the myeloid compartment. It is characterized by the accumulation of PD1+ γδ T-cells and tumor-associated macrophages as well as the depletion of hematopoietic progenitors. Thus, our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming with implications for clinical decision-making.

Abstract Toll-like receptors (TLRs) in the liver compartment have repeatedly been attributed to the development of non-alcoholic fatty liver disease (NAFLD). Knowledge on TLR expression in blood cells and their relation to intestinal microbiota and NAFLD development is limited. Here, we determined TLR expression patterns in peripheral blood mononuclear cells (PBMCs) of NAFLD patients and controls, their relation to intestinal microbiota and the impact of TLRs found altered in NAFLD development. Markers of intestinal permeability in blood and TLR mRNA expression in PBMCs were determined in 37 NAFLD patients and 15 age-matched healthy controls. Fecal microbiota composition was evaluated in 21 NAFLD patients and 9 controls using 16S rRNA gene amplicon sequencing. Furthermore, TLR1 −/− and C57BL/6 mice (n = 5–6/group) were pair-fed a liquid control or a fat-, fructose- and cholesterol-rich diet. Intestinal microbiota composition and markers of intestinal permeability like zonulin and bacterial endotoxin differed significantly between groups with the latter markers being significantly higher in NAFLD patients. Expression of TLR1-8 and 10 mRNA was detectable in PBMCs; however, only TLR1 expression, being higher in NAFLD patients, were significantly positively correlated with the prevalence of Holdemanella genus while negative correlations were found with Gemmiger and Ruminococcus genera. TLR1 −/− mice were significantly protected from the development of diet-induced NAFLD when compared to wild-type mice. While intestinal microbiota composition and permeability differed significantly between NAFLD patients and healthy subjects, in PBMCs, only TLR1 expression differed between groups. Still, targeting these alterations might be a beneficial approach in the treatment of NAFLD in some patients.