Multiple Myeloma Microenvironment Research Articles

Prognostic marker CD27 and its micro-environmental in multiple myeloma

BackgroundThe Cluster of Differentiation 27 (CD27) is aberrantly expressed in multiple myeloma (MM) -derived. This expression facilitates the interaction between tumor and immune cells within TME via the CD27-CD70 pathway, resulting in immune evasion and subsequent tumor progression. The objective of this study is to investigate the correlation between CD27 expression and the prognosis of MM, and to elucidate its potential relationship with the immune microenvironment.MethodsIn this research, CD27 expression in T cells within the 82 newly diagnosed MM microenvironment was assessed via flow cytometry. We then examined the association between CD27 expression levels and patient survival. Subsequent a series of bioinformatics and in vitro experiments were conducted to reveal the role of CD27 in MM.ResultsClinical evidence suggests that elevated CD27 expression in T cells within the bone marrow serves as a negative prognostic marker for MM survival. Data analysis from the GEO database has demonstrated a strong association between MM-derived CD27 and the immune response, as well as the hematopoietic system. Importantly, patients with elevated levels of CD27 expression were also found to have an increased presence of MDSCs and macrophages in the bone marrow microenvironment. Furthermore, the PERK-ATF4 signaling pathway has been implicated in mediating the effects of CD27 in MM.ConclusionsWe revealed that CD27 expression levels serve as an indicative marker for the prognosis of MM patients. The CD27- PERK-ATF4 is a promising target for the treatment of MM.

Tumor integrin targeted theranostic iron oxide nanoparticles for delivery of caffeic acid phenethyl ester: preparation, characterization, and anti-myeloma activities.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells preferentially in the bone marrow. Currently, emerging chemotherapy drugs with improved biosafety profiles, such as immunomodulatory agents and protease inhibitors, have been used in clinics to treat MM in both initial therapy or maintenance therapy post autologous hematopoietic stem cell transplantation (ASCT). We previously discovered that caffeic acid phenethyl ester (CAPE), a water-insoluble natural compound, inhibited the growth of MM cells by inducing oxidative stress. As part of our continuous effort to pursue a less toxic yet more effective therapeutic approach for MM, the objective of this study is to investigate the potential of CAPE for in vivo applications by using magnetic resonance imaging (MRI)-capable superparamagnetic iron oxide nanoparticles (IONP) as carriers. Cyclo (Arg-Gly-Asp-D-Phe-Cys) (RGD) is conjugated to IONP (RGD-IONP/CAPE) to target the overexpressed αvβ3 integrin on MMcells for receptor-mediated internalization and intracellular delivery of CAPE. A stable loading of CAPE on IONP can be achieved with a loading efficiency of 48.7% ± 3.3% (wt%). The drug-release studies indicate RGD-IONP/CAPE is stable at physiological (pH 7.4) and basic pH (pH 9.5) and subject to release of CAPE at acidic pH (pH 5.5) mimicking the tumor and lysosomal condition. RGD-IONP/CAPE causes cytotoxicity specific to human MM RPMI8226, U266, and NCI-H929 cells, but not to normal peripheral blood mononuclear cells (PBMCs), with IC50s of 7.97 ± 1.39, 16.75 ± 1.62, and 24.38 ± 1.71μM after 72-h treatment, respectively. Apoptosis assays indicate RGD-IONP/CAPE induces apoptosis of RPMI8226 cells through a caspase-9 mediated intrinsic pathway, the same as applying CAPE alone. The apoptogenic effect of RGD-IONP/CAPE was also confirmed on the RPMI8226 cells co-cultured with human bone marrow stromal cells HS-5 in a Transwell model to mimic the MM microenvironment in the bone marrow. In conclusion, we demonstrate that water-insoluble CAPE can be loaded to RGD-IONP to greatly improve the biocompatibility and significantly inhibit the growth of MMcells in vitro through the induction of apoptosis. This study paves the way for investigating the MRI-trackable delivery of CAPE for MM treatment in animal models in the future.

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

Identification of an angiogenesis-related risk score model for survival prediction and immunosubtype screening in multiple myeloma.

Multiple myeloma (MM) is an incurable B-cell malignancy, but with the emergence of immunotherapy, a potential cure is hopeful. The individualized interaction between the tumor and bone marrow (BM) microenvironment determines the response to immunotherapy. Angiogenesis is a constant hallmark of the BM microenvironment in MM. However, little is known about the potency ability of angiogenesis-associated genes (AAGs) to regulate the immune microenvironment of MM patients. We comprehensively dissected the associations between angiogenesis and genomic landscapes, prognosis, and the immune microenvironment by integrating 36 AAGs. Immunohistochemistry was performed to verify the correlation between angiogenic factor expression and patient prognosis. Single-sample gene set enrichment analysis was applied to quantify the relative abundance of 28 infiltrating cells. The AAG score was constructed using the least absolute shrinkage and selection operator Cox regression model. Angiogenesis was closely correlated with MM patient prognosis, and the mutation intensity of the AAGs was low. Immunohistochemistry confirmed that high microvessel density predicted poor prognosis. Three AAG clusters and two gene clusters with distinct clinical outcomes and immune characteristics were identified. The established AAG_score model performed well in predicting patient prognosis and active immunotherapy response. The high-AAG_score subgroup was characterized by reduced immune cell infiltration, poor prognosis, and inactive immunotherapy response. Multivariate analyses indicated that the AAG_score was strongly robust and independent among the prognostic variables. This study revealed that angiogenesis is significantly related to MM patient prognosis and immune phenotype. Evaluating the AAG signature was conducive to predicting patient response to immunotherapy and guiding more efficacious immunotherapy strategies.

A neutrophil extracellular trap-related risk score predicts prognosis and characterizes the tumor microenvironment in multiple myeloma

Multiple myeloma (MM) is a distinguished hematologic malignancy, with existing studies elucidating its interaction with neutrophil extracellular traps (NETs), which may potentially facilitate tumor growth. However, systematic investigations into the role of NETs in MM remain limited. Utilizing the single-cell dataset GSE223060, we discerned active NET cell subgroups, namely neutrophils, monocytes, and macrophages. A transcriptional trajectory was subsequently constructed to comprehend the progression of MM. Following this, an analysis of cellular communication in MM was conducted with a particular emphasis on neutrophils, revealing an augmentation in interactions albeit with diminished strength, alongside abnormal communication links between neutrophils and NK cells within MM samples. Through the intersection of differentially expressed genes (DEGs) between NET active/inactive cells and MM versus healthy samples, a total of 316 genes were identified. This led to the development of a 13-gene risk model for prognostic prediction based on overall survival, utilizing transcriptomics dataset GSE136337. The high-risk group manifested altered immune infiltration and heightened sensitivity to chemotherapy. A constructed nomogram for predicting survival probabilities demonstrated encouraging AUCs for 1, 3, and 5-year survival predictions. Collectively, our findings unveil a novel NET-related prognostic signature for MM, thereby providing a potential avenue for therapeutic exploration.

Novel Insights into the Initiation, Evolution, and Progression of Multiple Myeloma by Multi-Omics Investigation

The evolutionary history of multiple myeloma (MM) includes malignant transformation, followed by progression to pre-malignant stages and overt malignancy, ultimately leading to more aggressive and resistant forms. Over the past decade, large effort has been made to identify the potential therapeutic targets in MM. However, MM remains largely incurable. Most patients experience multiple relapses and inevitably become refractory to treatment. Tumor-initiating cell populations are the postulated population, leading to the recurrent relapses in many hematological malignancies. Clonal evolution of tumor cells in MM has been identified along with the disease progression. As a consequence of different responses to the treatment of heterogeneous MM cell clones, the more aggressive populations survive and evolve. In addition, the tumor microenvironment is a complex ecosystem which plays multifaceted roles in supporting tumor cell evolution. Emerging multi-omics research at single-cell resolution permits an integrative and comprehensive profiling of the tumor cells and microenvironment, deepening the understanding of biological features of MM. In this review, we intend to discuss the novel insights into tumor cell initiation, clonal evolution, drug resistance, and tumor microenvironment in MM, as revealed by emerging multi-omics investigations. These data suggest a promising strategy to unravel the pivotal mechanisms of MM progression and enable the improvement in treatment, both holistically and precisely.

BCMA-targeted bortezomib nanotherapy improves therapeutic efficacy, overcomes resistance, and modulates the immune microenvironment in multiple myeloma

Bortezomib (BTZ) is a standard-of-care treatment in multiple myeloma (MM); however, adverse side effects and development of resistance limit its long term benefit. To improve target specificity, therapeutic efficacy, and overcome resistance, we designed nanoparticles that encapsulate BTZ and are surface-functionalized with BCMA antibodies (BCMA-BTZ-NPs). We confirmed efficient cellular internalization of the BCMA-BTZ-NPs only in BCMA-expressing MM cells, but not in BCMA-knockout (KO) cells. In addition, BCMA-BTZ-NPs showed target-specific cytotoxicity against MM cell lines and primary tumor cells from MM patients. The BCMA-BTZ-NPs entered the cell through receptor-mediated uptake, which escapes a mechanism of BTZ resistance based on upregulating P-glycoprotein. Furthermore, BCMA-BTZ-NPs induced cell death more efficiently than non-targeted nanoparticles or free BTZ, triggering potent mitochondrial depolarization followed by apoptosis. In BTZ-resistant cells, BCMA-BTZ-NPs inhibited proteasome activity more effectively than free BTZ or non-targeted nanoparticles. Additionally, BCMA-BTZ-NPs enhanced immunogenic cell death and activated the autophagic pathway more than free BTZ. Finally, we found that BCMA-BTZ-NPs selectively accumulated at the tumor site in a murine xenograft model, enhanced tumor reduction, and prolonged host survival. These results suggest BCMA-BTZ-NPs provide a promising therapeutic strategy for enhancing the efficacy of BTZ and establish a framework for their evaluation in a clinical setting.

Elucidating the Role of Tolerogenic Dendritic Cells in Immune Tolerance and Disease Progression in Multiple Myeloma

Introduction: Previous research has demonstrated that programmed death-ligand 1 (PD-L1) in multiple myeloma (MM) cells not only suppresses anti-tumor immune responses but also enhances malignant potential through the PD-L1-PD-1 interaction [Ishibashi M, et al. Cancer Immunol Res. 2016]. However, it has not yet been shown that monotherapy and combination therapies utilizing PD-1/PD-L1 antibodies are sufficiently effective for MM. One reason for this is the absence of myeloma-specific cytotoxic T lymphocytes induction in the MM tumor microenvironment. In certain cancers, tumor-infiltrating dendritic cells (DCs) undergo conversion into tolerogenic DCs, which are crucial for inducing and maintaining T-cell tolerance, although this remains unclear in MM. Thus, this study aimed to investigate the function and induction mechanisms of tolerogenic DCs in the MM microenvironment. Materials and Methods: (1) Flow cytometry (FCM) was employed to assess the two conventional DC (cDC; lineage negative, HLA-DR +CD11c high) subsets, cDC1 (CD141 + cDC) and cDC2 (CD1c + cDC), in bone marrow (BM) samples obtained from MM patients (n=17) and healthy controls (n=13). (2) CD14 + monocytes were cultured with IL-4 and GM-CSF to generate monocyte-derived DCs (moDCs). Simultaneously, moDCs were co-cultured with MM cell lines using a trans-well system. The characteristics and functions of moDCs were analyzed using real-time PCR, RNA-sequencing (RNA-seq), western blotting, ELISA, and FCM after lipopolysaccharide stimulation. Results: The frequency of cDC1 subsets was significantly lower in MM patients compared to controls, while the major cDC2 subset did not differ. Similarly, when moDCs were co-cultured with MM cells, moDC numbers were markedly reduced. These co-cultured moDCs exhibited downregulated expression of activation markers CD80, CD86, and CD83, along with decreased phosphorylation of ERK and S6K. Conversely, these moDCs upregulated the expression of immunosuppressive factors IL-10, ARG1, and NOS2 through increased transcription factor C/EBPβ. Moreover, these moDCs inhibited T-cell proliferation, activation, and interferon-γ production in comparison to control moDCs, indicating the presence of immature and tolerogenic phenotypes. However, these phenotypes were not observed in moDCs co-cultured with soluble factors secreted from MM and stromal cells. Treatment with AZD3965 improved the phenotype of moDCs co-cultured with MM cells, reversing their suppressive effect on T-cell proliferation. AZD3965 targets monocarboxylate transporter 1 (MCT1) in MM cells, thereby suppressing extracellular lactate accumulation and acidic pH in the MM microenvironment. Furthermore, RNA-seq analysis comparing moDCs co-cultured with and without MM cells revealed downregulation of gene sets associated with activated DCs in moDCs co-cultured with MM. Notably, moDCs co-cultured with MM showed high enrichment of gene sets related to lysosome biogenesis and NOD-like receptor signaling. This suggests that inflammasome activation in these moDCs leads to caspase-1 activation, resulting in increased levels of mature IL-1β and IL-18 in their cell culture supernatants. Additionally, co-culturing CD38-positive MM cells with CD203a/CD73-positive HS-5 stromal cells and NAD + resulted in the generation of adenosine in the cell culture supernatant. Adenosine promotes the immaturation of mature DCs; however, inflammasome activation does not occur in these DCs. Conclusions: Patients with MM exhibit a reduced frequency of cDC1 subsets, which are crucial for cross-presentation to CD8 + T cells. moDCs in acid pH conditions derived from MM cells display an immature phenotype and impaired function due to decreased DC activation signals. Furthermore, inflammasome activation promotes the secretion of IL-1β and IL-18, contributing to pro-tumorigenic inflammation in the MM microenvironment. Moreover, adenosine generated via the CD38/CD203a/CD73 adenosinergic pathway promotes the immaturation and dysfunction of moDCs. Thus, the characteristics of tolerogenic DCs may vary depending on the MM microenvironment. These findings highlight the induction of tolerogenic DCs by the metabolic microenvironment in MM, leading to immune tolerance and disease progression.

Tuning the Innate Immune Multiple Myeloma Microenvironment By Modulating IRF4

The tumor microenvironment (TME) has emerged as a key contributor to cancer progression in numerous solid tumors as well as blood cancers that evolve in the bone marrow (BM). In the context of multiple myeloma (MM), survival of malignant plasma cells is supported by interactions with the BM microenvironment, which is comprised of various components including cells, extracellular matrix, and soluble factors. Therefore, the development of strategies that specifically target the MM BM microenvironment is crucial for improving current disease prognosis. Within the BM niche, M2-like myeloma-associated macrophages (MAMs) play a significant role in cancer cell survival and progression and have been implicated in producing an immune-suppressive TME by generating inflammatory mediators, growth factors, cytokines, chemokines, etc. Macrophages exhibit plasticity with regard to their polarization state, which enable them to dynamically respond and adapt to different environmental stimuli in order to fulfill important immune functions. Thus, the reprogramming of M2-like, pro-tumoral MAMs toward a M1-like, anti-tumoral phenotype represents a promising therapeutic strategy. In MM, inflammatory and anti-viral pathways promote disease progression. Interferon regulatory factor 4 (IRF4) is a transcription factor that has previously been shown to promote MM progression through enhanced survival of malignant plasma cells. Under physiological conditions, IRF4 also regulates macrophage polarization, where its expression drives alternative activation of macrophages primarily to M2-like (anti-inflammatory) phenotypes. However, the extent to which IRF4 activation or direct inhibition governs the polarization status and pro-tumoral activity of macrophages in the MM microenvironment has not been fully elucidated. In this study, we sought to further explore the role of IRF4 in macrophage polarization and investigated the potential of modulating IRF4 expression to reprogram myeloma-associated macrophages (MAMs) as a novel therapeutic strategy. First, we developed myeloma-tailored macrophage polarization models using primary human peripheral blood-derived monocytes and the THP1 monocyte cell line. Our M1 and M2 polarization models utilize a customized cocktail of myeloid growth factors and cytokines, supplemented with interleukin (IL)-6 to mimic the IL-6-enriched MM microenvironment. Characterization of M1 (CD80, TNFα, and CXCL10) and M2 (MRC1, FN1, and CCL22) markers confirmed the presence of distinct macrophage subpopulations, as well as upregulated expression of IRF4 in M2-like progeny and IRF5 in M1-like macrophages. To investigate the extent to which IRF4 activation or direct inhibition governs macrophage polarization and function, overexpression and knockdown experiments on macrophages were performed in vitro, along with whole transcriptome sequencing of IRF4-overexpressing THP1 monocytes compared to vector-transduced controls. Gene expression analyses revealed that genetic down-modulation of IRF4 in M2-like macrophages shifts the polarization status toward a hybrid phenotype that displays enhanced M1-like properties, while still retaining M2-like characteristics. This shift in polarization state was further amplified when monocytes were transduced to overexpress IRF4 prior to differentiation into M2-like macrophages and subsequent IRF4 knockdown, suggesting that IRF4 plays a key regulatory role in tuning macrophage polarization. Taken together, our preclinical findings provide groundwork for future studies on MAMs and suggest that an IRF4-mediated macrophage reprogramming strategy is a promising therapeutic approach for patients with MM and a variety of other malignancies typified by an anti-inflammatory TME.

Spatial Dissection of the Bone Marrow Microenvironment in Multiple Myeloma By High Dimensional Multiplex Tissue Imaging

Introduction Multiple myeloma (MM) is shaped by interactions between immune, stromal and tumor cells in the bone marrow (BM) microenvironment (BMME). Our understanding of these processes is based on high parametric flow cytometry and single cell transcriptomics, which lack spatial resolution. Thus, crucial aspects of the in situ tumor ecosystem, including cell-cell interactions, can only be predicted. Novel high dimensional, multiplex tissue imaging methods are able to capture similar granularity while also preserving spatial organization. By optimizing antibody selection, as well as staining, imaging and data processing protocols we have established a BMME-targeted CODEX workflow based on a 60 marker panel. Here, we conducted the first comprehensive multiplex protein imaging survey of the BMME across the spectrum of MM and precursor stages and leveraged paired FISH and whole genome sequencing (WGS) to elucidate the interplay of tumor intrinsic features and the BMME. Methods We imaged 489 BM trephine biopsies in tissue microarray format on the CODEX (Akoya Biosciences) platform. After quality control (≥750 cells per core, <25% unclassifiable cells) we retained data from 12 non-malignant controls, 13 MGUS, 20 smoldering MM, 349 uniformly treated newly diagnosed MM (NDMM) patients from a phase III clinical trial, as well as 22 relapsed MM samples with a median of 3 (range 1-6) tissue cores per sample, comprising a total of 3.1 million single cells. CODEX data was processed and analyzed using custom scripts. Cellular neighborhoods were computed by clustering the cell type composition of sliding windows (n=10 nearest neighboring cells) across the tissue. WGS (n=212) and FISH (n=309) were performed on CD138-purified BM mononuclear cells and analyzed using in-house pipelines. Results We identified 40 cell types, encompassing the relevant lineages (myeloid, lymphoid, stromal, endothelial, tumor), subtypes (e.g. T, B, NK cells) and functional states (e.g GRZB, Ki67, and PD1) of the BMME. The degree of plasma cell infiltration determined by CODEX correlated with the clinical pathologist's quantification based on classical immunohistology (r=0.77, p<0.0001), and the kappa/lambda status of MM cells matched the reported light chain restriction, supporting the validity of our data. At the compositional level, we observed shifts in relative cell type abundance from precursor to MM stages. Major changes in the BMME included enriched stromal, endothelial and CD8+ T cells as well as depleted polymorphonuclear (PMN), erythropoietic and CD4+ T cells in advanced stages. The highest proportion of monocytoid dendritic cells, monocytes, mast cells and cytotoxic CD8+ T cells was seen in relapsed MM. Leveraging the spatial resolution of the CODEX data, we identified multiple distinct cellular neighborhoods (CNs) defined by recurrent local cell type composition. CNs enriched for plasma cells in combination with different immune cell subsets including exhausted T cells increased towards the MM stage, while CNs dominated by PMNs, erythropoietic cells and CD206 negative macrophages decreased. To investigate links between MM subtypes and BMME patterns, we correlated CODEX data with MM-initiating and driver events. While patients with t(4;14) showed a significant increase in eosinophils, patients with gain1q had higher levels of endothelial and stromal subsets colocalizing in the same CN. Distinct types of stromal cells were also enriched in patients with double hits (≥2 FISH high risk features). RAS mutations and biallelic inactivation of tumor suppressor genes were associated with altered T cell subset composition. Further corroborating the importance of BMME architecture, a CN with colocalization of plasma and exhausted CD8 T cells was associated with unfavorable prognosis in NDMM patients. Conclusions High dimensional, multiplex tissue imaging of the BMME enables interrogation of cellular interactions and tissue architecture in situ in clinical samples at single cell resolution. To our knowledge this is the first highly multiplexed spatial study of the human BMME in a large clinical cohort and the first such study in multiple myeloma. We track characteristic shifts in tissue composition associated with disease progression, reveal genotype-phenotype associations in the BMME as a correlate of tumor-microenvironment co-evolution and detect prognostically relevant tissue architectural features.

Integrative Multi-Omics Analysis of Tumor-Immune Microenvironment in Multiple Myeloma: Novel Insights and Therapeutic Implications

Despite notable therapeutic advances that have improved the survival of multiple myeloma (MM) patients, development of drug resistance remains a major problem. Transcriptomic analysis provides an opportunity to dissect the complexity of tumors, including the surrounding microenvironment, which has a significant impact on MM tumor progression and patients' response to treatment, as demonstrated by the effectiveness of immunomodulatory therapies. To improve the tailoring of targeted and immune based therapeutic strategies, it is crucial to decipher the tumor-immune microenvironment profile in MM patients. We used a multi-omics data integration approach, including RNAseq-based gene expression for MM tumor cells (MMCs) (n=196) and for the corresponding purified tumor microenvironment (TME) (n=124), single nucleotide variant data from whole exome sequencing of MMCs (n=100), deconvolution of TME immune subtypes (n=124), and relevant clinical metadata. This allowed us to thoroughly characterize both the tumor and its TME. Applying Multiomics Factor Analysis (MOFA) identified 13 principal factors capturing the diversity of the disease, encompassing both MM malignant and microenvironment components. Factors 1 and 2 specifically represented the heterogeneity in the TME, incorporating gene expression profiles and deconvolution-based immune cell subtypes. On the other hand, Factors 6, 9, 11, and 12 were specific to variations in the gene expression profile of the malignant component. Importantly, the analysis of the variables linked to these main factors uncovers not only genes with established functions in myeloma but also novel candidates with potential importance. Based on the top-weight in the gene expression and deconvolution data, Factor 1 showed a positive association with MMP8 expression by TME cells and a negative correlation with the presence of CD8 positive T cells in the TME. Factor 2, on the other hand, exhibited positive alignments with the expression of BHLHA15, TNFRSF17 (BCMA), KLF15, and IGF1 by TME cells. Regarding Factor 6 and Factor 11, which capture tumor fraction heterogeneity, Factor 6 showed alignment with the expression of cancer testis antigens ( MAGEA6, MAGEA3), CXCL12, and KLF4. Meanwhile, Factor 11 was associated with the expression of genes such as SULF2, as well as CRP levels, percentage of CD138+ cells, and the percentage of cells in cell cycle phase S. Next, we investigated the predictive potential of the latent factors inferred by MOFA in models of clinical outcomes. Interestingly, 6 out of the 13 factors identified by MOFA showed significant associations with patients' overall survival (cox regression, p <= 0.05). These factors include Factor 1 and 2, which are related to the composition of the TME, and Factor 6 and 9, which are associated with myeloma progression status, osteolysis, t(11;14), t(4;14), and MM molecular classification. Finally, performing unsupervised clustering using the latent MOFA factors, we identified two main groups with distinct TME subtypes. These groups can be further divided into nine subgroups, each exhibiting unique TME characteristics. Notably, the TME subtypes showed correlations with essential gene expression signatures related to immune response regulation, T cell activation, and natural killer mediated immunity. Additionally, the significant presence of deconvolution-based immune cell types, including T cell CD8+, T cell regulatory, T cell CD4+ (non-regulatory), NK cells and Monocytes was observed. Moreover, the TME subtypes were associated with patients' overall survival and response to daratumumab. Patients presenting TME enriched in immune cells were associated with a significant better outcome ( p = 0.0026). These findings highlight the importance of TME analysis in predicting treatment response and MM patients' outcome in the context of immune-based therapies. Our integrative multi-omics analysis revealed comprehensive MM heterogeneity and distinct immune subtypes that could be of therapeutic interest for personalized therapeutic approaches.

Targeted Inhibition of Myeloperoxidase (MPO): A New Therapeutic Strategy for the Treatment of Multiple Myeloma

Despite major improvements in therapeutic strategies for patients with multiple myeloma (MM), effective treatment still remains a persistent challenge, as patients ultimately relapse and succumb to the disease. In the last decade, studies have highlighted the reciprocal interaction between MM plasma cells (PC) and the bone marrow (BM) microenvironment in regulating immune evasion, disease progression and persistence. As MM PC rely on BM stromal cells and their secreted factors for their survival and growth, the therapeutic targeting of the BM microenvironment may prove to be a novel and successful strategy for myeloma care in the future. Myeloid-derived suppressor cells (MDSC), a heterogenous population of myeloid cells are described to promote MM progression through immunosuppression and induction of angiogenesis. Myeloperoxidase (MPO), a key inflammatory enzyme important in host defence, is reported to be the most highly upregulated gene in MDSCs in murine cancer models. Recently, the accumulation of MPO within the tumour microenvironment has attracted much attention with a number of studies describing a role for MPO in regulating cancer development due to its potent pro-oxidative and proinflammatory properties. Our most recent findings have revealed new functional roles for myeloid-derived MPO in the BM microenvironment of MM. Specifically, we demonstrate that myeloid cell populations are increased within the BM of 5TGM1 tumour-bearing mice and that MM PC may directly influence Mpo gene expression in BM-derived myeloid cells. Mechanistically, we report that MPO has the capacity to induce the expression of key MM growth factors, and exerts potent immune suppression by inhibiting anti-tumour T-cell responses. Remarkably, in the syngeneic KaLwRij/5TGM1 mouse model of MM, targeted inhibition of MPO with the suicide substrate 4-Aminobenzoic acid hydrazide (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Here, we investigate for the first time the efficacy of an orally bioavailable irreversible small molecule inhibitor of MPO (MPOi) in the preclinical Vk*MYC murine model of myeloma. Twelve-week-old C57BL/6J mice were intravenously inoculated with Vk*MYC (Vk14451-GFP) cells and tumour progression was monitored by serum paraprotein electrophoresis (SPEP), whilst endpoint GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. To characterise myeloid cell populations and associated Mpo expression in the Vk*MYC tumour landscape, we utilised flow cytometry to quantitate CD11b+ cells and used magnetic activated cell separation to isolate these populations and characterise the expression of Mpo by qPCR. Our studies confirm that CD11b+ myeloid cells are significantly increased in the BM of Vk*MYC tumour-bearing mice (p<0.01), accompanied by an upregulation of MPO mRNA expression (p<0.001). To assess the efficacy of MPOi in MM, mice were treated with MPOi or vehicle alone twice daily by oral gavage, with treatment initiated at the time of Vk*MYC inoculation. Notably, mice receiving MPOi presented with significantly reduced endpoint tumour burden (9 weeks post tumour cell inoculation). We observed a 15.2% and 31.4% reduction in MM tumour as determined by hind limb GFP% (p<0.05) and SPEP (p<0.01) respectively, compared to vehicle control. Additionally, mRNA analysis of complete BM revealed an upregulation of the critical cytokine IFN gamma (p<0.05) and the downregulation of the potent proangiogenic factor VEGF (p<0.05) in mice treated with MPOi, suggesting MPO inhibition in MM may be an advantageous means to regulate the BM microenvironment and impede disease progression. However, when MPOi treatment was initiated at first signs of detectable disease, (as identified by the presence of a monoclonal spike by SPEP; 5 weeks post tumour cell inoculation), no difference in endpoint tumour burden was observed. This suggests that targeted inhibition of MPO using MPOi may be more effective in at early stages of MM development. In conclusion, the findings presented in this study indicate that inhibiting MPO activity with MPOi, as a single agent therapy, attenuates MM tumour growth in the Vk*Myc mouse model. With limited therapies used in the clinic that target the stromal microenvironment, these findings suggest MPOi could be investigated as a potential novel treatment option that may be included in combination with current frontline therapeutic agents.

Levels of Individual Mid and Long Chain Polyunsaturated Triacylglycerols (TAGs) Are Different between the Bone Marrow Microenvironment of Patients with Multiple Myeloma (MM) and Monoclonal Gammopathy of Unknown Significance (MGUS)

Background: It has been recently demonstrated that altered lipid metabolism involving increased free fatty acids (FFAs) uptake supports the growth and proliferation of MM cells (Panaroni et al. Blood 2022; Farell et al. Elife 2023). Whether the availability of FFAs in the bone marrow (BM) microenvironment is associated with the pathogenesis of MM from MGUS has not been previously evaluated. Thus, we evaluated if individual species of FAs or FA-containing lipids such as triacylglycerols (TAGs), diacylglycerols (DAGs), and monoacylglycerols (MAGs) are differentially present between the BM microenvironment of MGUS and MM patients. Methods: Lipid profiles of BM plasma samples (reflective of the BM microenvironment) from patients with a spectrum of plasma cell disorders (22 MGUS and 24 MM (15 newly diagnosed and 9 relapsed/refractory)) were acquired by liquid chromatography (LC)/tandem mass spectrometry (MS), and individual lipid species were quantified based on single-point quantification using 55 isotopically labeled internal standards. Concentration-based lipid species data was analyzed by the web-based tool MetaboAnalyst 5.0. Data was normalized by quantile normalization, log transformed and scaled by Pareto scaling. p values <0.05 were considered significant. Results: A total of 993 distinct lipid species were quantified by the MS-based targeted lipidomics assessment. Of these lipid species, the levels of individual mid-chain (≤50 carbon chain length) and long-chain (≥52 carbon chain length) polyunsaturated TAGs were the most differentially present in the BM plasma of MM compared to MGUS patients (Figure 1A). Even though a principal component analysis (PCA) involving all lipid species showed moderate separation of MM from MGUS patients, five MGUS patients (categorized as MGUS-other (MGO)) who clustered more towards MM patients compared to the remaining MGUS patients had a distinct pattern of individual mid and long-chain polyunsaturated TAG levels that were comparable to MM patients (Figure 1B). Furthermore, the proliferation rate of clonal plasma cells in the BM of these MM and MGUS as measured by the S-phase% was found to be directly correlated with the level of long-chain polyunsaturated TAGs. Conclusions: Mid and long-chain polyunsaturated TAGs are present in lower and higher levels, respectively, in the BM microenvironment of MM compared to MGUS patients. These findings continue to support the importance of FA-containing TAGs and other lipids in the pathogenesis of MM. Future confirmatory studies evaluating the biological significance of these differences are required to determine if they can be exploited for diagnostic and therapeutic purposes.

Phenotype Molding of Monocytes in the Multiple Myeloma Microenvironment

Human monocytes are mononuclear phagocytes that comprise a major population of innate immune cells. Various monocyte-derived cells, including osteoclasts, dendritic cells, and macrophages, have been implicated in immunosuppression within the multiple myeloma (MM) tumor microenvironment (TME). Despite recent developments in understanding the link between monocyte-derived cells and immunosuppression in myeloma, there are still critical knowledge gaps in the association between monocytes themselves and immunosuppression within the context of MM. Specifically, the functional and developmental alterations of monocytes in the MM TME have not been explored based on the single-cell studies. In the present study, we generated a comprehensive single-cell map of peripheral blood (PB) and bone marrow (BM) monocytes from both healthy donors (HDs) and MM patients. Specifically, Heparin-anticoagulated PB samples or BM aspirates were collected from seven newly diagnosed MM (NDMM) patients and healthy donors (HDs). Paired BM aspirates were obtained from 10 MM patients at the time of diagnosis and after induction therapy. Differential expression analysis showed that type I interferon (IFN) response was upregulated in MM monocytes. And BM monocytes derived from MM patients exhibited impaired T cell attraction abilities and demonstrated an elevated capacity for suppressing T cell function. Moreover, immune checkpoint molecules, including CD274 (encoding PD-L1) and PDCD1LG2 (encoding PD-L2), were significantly upregulated in MM monocytes. Additionally, a positive correlation was observed between type I IFN response and T cell suppression capacity of MM monocytes. Trajectory analyses revealed an IFN-focused disruption of developmental pathways of both BM and PB monocytes in MM. Lastly, we included 10 MM patients at the time of diagnosis and after induction therapy to monitor transcriptional changes over time. Our results revealed that anti-tumor therapy successfully mitigated the excessive type-I interferon response of BM monocytes in MM. We document that MM monocytes exhibited an augmented type-I IFN response compared to monocytes from HDs, while their overall heterogeneity remained unchanged. Furthermore, we extended the knowledge on immunosuppression of the MM TME that the excessive type-I IFN response in MM had a significant impact on the differentiation of both BM and PB monocytes, leading to T-cell immunosuppression, specifically in BM monocytes. In the cohort that we longitudinally followed, we observed a decrease in the type-I IFN response across nearly all monocyte subsets in MM BM monocytes after induction therapy, accompanied by a reduction in the expression of the T-cell suppression gene set. In sum, our findings indicates that monocytes in the BM promotes the immunosuppression of the MM TME and represents potential therapeutic targets for modulating the MM TME.

Single Cell Analysis of Bone Marrow Stromal Cells Shows That a Pro-Inflammatory and Stress Response Together with Polarized Differentiation of Stromal Elements Characterize the Bone Marrow Microenvironment during Multiple Myeloma Development

Current knowledge of the biological role of the non-hemopoietic components of the bone marrow (BM) niche to the pathogenesis of multiple myeloma (MM) is poorly understood. We utilized a murine model to interrogate niche forming cells early in the natural history of MM, at a precursor stage (MGUS/smoldering-like) in more detail. Studying interactions between the MM clone and the stromal components of the MM niche are challenging as they represent only approximately 0.2% of the total marrow content and are difficult to isolate. However, by isolating the stromal compartment of the BM by depleting hemopoietic cells via flow sorting combined with the use of single cell RNA sequencing (scRNA-seq) it is possible to perform an in-vivo analysis of the individual cellular components of the stromal microenvironment and to characterize their relative abundance, cellular differentiation state, and transcriptomic profile. To comprehensively assess changes in the stromal cells during the early stage of MM, 5TGM1 cells were intravenously injected into aged (6 month old) KaLwRij mice. KaLwRij mice without MM were used as controls. Stromal cells were isolated following tumor engraftment in the marrow at a precursor stage and enriched as previously described (Baryawno et al, Cell 2019). scRNA-seq was performed on 5 libraries of cells using the 10X Genomics Chromium platform. All data analysis was performed in R. After filtering out low quality cells, 45,030 cells remained for analysis. The 5 libraries were integrated with Seurat and run through a standard clustering workflow involving log-normalization, finding variable features, dimensional reduction, and clustering. Cell type annotation for each cluster was performed manually using established markers for both the stromal and immune compartments. After excluding immune cell populations, 14,219 cells remained. Select subpopulations were re-clustered by isolating the cell types of interest and running them through separate standard clustering workflows. For each population of interest, differentially expressed genes were determined using the Wilcox rank sum test in Seurat, trajectory analysis and pseudotime were computed using Monocle3, and enriched gene sets were computed using GSEAPreranked. We identified 7 distinct populations of stromal cells including mesenchymal stromal cells (MSCs) ( Lepr, Adipoq, and Cxcl12), osteo-lineage cells (OLCs) ( Bglap, Spp1, and Sp7), fibroblasts ( S100a4, Fn1, and Dcn), chondrocytes ( Col2a1, Sox9, and Acan), pericytes ( Acta2, Myh11, and Mcam), and two endothelial cell (ECs) populations, arterial (AEC) and sinusoidal (SEC) ( Cdh5, Cd34, and Pecam1). Compared to normal BM, MM engraftment was associated with numerical differences in stromal cell populations between normal and MM. Gene set enrichment analysis showed an inflammatory and oxidative stress signal associated with the MM microenvironment. Sub-clustering analysis showed MSC differentiation was polarized away from osteocyte formation towards adipocytes with the identification of a novel population only seen in MM. Bone marrow endothelial cell populations were also substantially altered at this early disease stage, with differentiation polarized towards sinusoidal endothelial cells generating a pro-angiogenic /pro-inflammatory phenotype. An increase in cells undergoing endothelial to mesenchymal transition (EndMT) was also present. Taken together, we show, for the first time, existence of remodeling of the stromal populations induced by the MM clone characterized by a pro-inflammatory phenotype together with polarized differentiation. These changes result in the expansion of a number of key populations that increase the MM niche contributing to growth and survival signals and shaping of the content of the immune microenvironment. These changes result in a self-perpetuating signaling loop between cells, which needs to be broken therapeutically in order to stop progression, induce remission and long-term disease outcomes. Conclusion - In early stages of MM pathogenesis, MM cells remodel the stromal microenvironment by altering the amount and function of MSCs and endothelial cells. Through favoring an adipocytic fate of MSCs, endothelial mesenchymal transition and altering the balance between arterial and sinusoidal endothelial cells, MM cells promote an inflammatory environment that contributes to MM development and progression.

Mitochondrial Fitness and Immune Function of Natural Killer Cells Are Impaired By the Multiple Myeloma (MM) Tumor Cells: The Role of MM Microenvironment Interactions

The immune system including Natural Killer (NK) activity was shown to be impaired in active MM. We and others have previously demonstrated the important role of the bone marrow (BM) microenvironment in MM pathophysiology, however, data on its effects on NK activity in MM is limited. We first assessed the phenotype of the BM-derived NK cells. BM-NK from MM pts (n=10) showed an exhausted phenotypic signature with significantly reduced expression of CD16 (p<0.01), and increased expression of exhaustion markers CD39 (p<0.001) and CD57 (p<0.001) compared to BM-NK cells from healthy controls (n=8). Furthermore, MM-BM NK cells demonstrated reduced mitochondrial membrane potential, suggesting impaired mitochondrial fitness. The dysfunction of the mitochondria led to deficiency of ATP synthesis; mitochondrial ATP levels were reduced in NK cells from MM BM samples in comparison to the normal BMs (p<0.001). Next, the functional response of BM NK cells to the stimulation with IL-2 (2000 IU/ml for 7 and 14 days) was evaluated in vitro. NK cells from MM BM demonstrated reduced proliferation capacity, increased CD39, reduced expression of activating receptors NKp44 and NKp30, and reduced mitochondrial membrane potential upon the activation with IL-2. The impaired mitochondrial fitness correlated with reduced levels of NKp44, indicating a potential link between the decreased mitochondrial activity and the reduced functionality of NK cells. Indeed, in vitro, the cytotoxic activity of IL-2-stimulated MM-BM NK cells against MM cell lines was significantly diminished compared to healthy IL-2-activated BM-NK cells. Glucose and fatty acids (FA) are key substrates of the mitochondrial bioenergetic pathways and essential for NK cell activation. Of note, glucose uptake was increased comparably upon IL-2 stimulation in NK cells from both MM patients and healthy donors. In contrast, MM-BM NK cells demonstrated reduced FA uptake in response to IL-2 stimulation compared to BM NK cells from healthy donors. These data indicate impaired mitochondrial and NK cell properties in the BM MM niche. Next, we established an in vitro model to interrogate the relationship between NK cell mitochondrial and immune dysfunction within the MM BM microenvironment. Specifically, we cultured MM cell lines (n=5) with the NK92 cell line or primary IL-2-activated or not activated blood-derived NK cells, and assessed NK cell mitochondrial fitness, metabolic activity, and immune functional properties. Co-culture with MM cells significantly impaired mitochondrial mass and mitochondrial membrane potential in NK cells, decreased mitochondrial ATP levels, while upregulating MM mitochondrial ROS in NK cells, indicating that MM cells induce mitochondrial damage and impair ATP synthesis in NK cells. Mitochondrial fitness and energy production are pivotal for NK cell persistence and cytotoxicity. In accordance, direct interaction with MM cells suppressed NK cell proliferation in vitro, inducing G0/G1 cell cycle arrest. Moreover, MM-induced mitochondrial impairment in NK cells correlated with reduced responsiveness of MM cells to NK-induced killing, while short-term exposure to MM cells significantly diminished the anti-myeloma cytotoxic activity of NK cells. Accordingly, CD39 expression was upregulated, while NKp44 levels significantly decreased in NK cells upon interaction with MM cells. To further characterize the molecular mechanisms associated with MM-mediated NK cell mitochondrial impairment, we assessed the gene expression profile of MM-exposed sorted NK92 cells versus unexposed cells. A distinct metabolic signature characterized by reduced expression of genes involved in the mitochondrial activity was detected in MM-exposed NK cells. Thus, genes involved in respiratory activity and mitochondrial dynamics were downregulated post-MM exposure. Furthermore, a profound increase in fructose-1,6-biphosphatase 1 (FBP1) expression was detected in MM-exposed NK cells. FBP1 is the rate-limiting enzyme for gluconeogenesis that simultaneously inhibits glycolysis, possibly interfering with NK cell metabolic activity and immune function. Altogether, our data suggest that MM BM microenvironment and direct interactions with malignant plasma cells impair NK mitochondrial fitness, diminishing energy production, increasing oxidative stress, and driving exhaustion with reduced anti-tumor capacity in NK cells.

The Clinical Relevance of Selected Cytokines in Newly Diagnosed Multiple Myeloma Patients.

Multiple myeloma (MM) is the second most common hematological neoplasm. Cytokines, chemokines, and their receptors, induced by the microenvironment of MM, participate in tumor growth, the attraction of leukocytes, cell homing, and bone destruction. This study aimed to assess the correlation between the pretreatment serum concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), angiogenic chemokine monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) and the clinical outcomes and survival of patients newly diagnosed with MM. The study group consisted of 82 individuals. The IL-8 concentration was significantly positively correlated with the age of onset (p = 0.007), the International Staging System (ISS) stage (p = 0.03), the Eastern Cooperative Oncology Group (ECOG) performance status (p < 0.001), the degree of anemia before treatment (p < 0.0001), the degree of kidney disease (p < 0.001), and VEGF (p = 0.0364). Chemotherapy responders had significantly lower concentrations of IL-8 (p < 0.001), IL-6 (p < 0.001), and VEGF (p = 0.04) compared with non-responders. Patients with treatment-induced polyneuropathy had significantly higher levels of IL-8 (p = 0.033). Patients with a high level of IL-6 had a 2-fold higher risk of progression-free survival (PFS) reduction (17 vs. 35 months; HR = 1.89; p = 0.0078), and a more than 2.5-fold higher risk of overall survival (OS) reduction (28 vs. 78 months; HR = 2.62; p < 0.001). High levels of IL-6, IL-8, and VEGF demonstrated significant predictive values for some clinical conditions or outcomes of newly diagnosed MM patients. Patients with an early response to chemotherapy had a significantly lower concentration of these cytokines. A high pretreatment IL-6 concentration was an independent negative prognostic marker for newly diagnosed MM patients.

Bleeding and Thrombosis in Multiple Myeloma: Platelets as Key Players during Cell Interactions and Potential Use as Drug Delivery Systems

Multiple myeloma (MM) is a hematological malignancy originated in the bone marrow and characterized by unhindered plasma cell proliferation that results in several clinical manifestations. Although the main role of blood platelets lies in hemostasis and thrombosis, platelets also play a pivotal role in a number of other pathological conditions. Platelets are the less-explored components from the tumor microenvironment in MM. Although some studies have recently revealed that MM cells have the ability to activate platelets even in the premalignant stage, this phenomenon has not been widely investigated in MM. Moreover, thrombocytopenia, along with bleeding, is commonly observed in those patients. In this review, we discuss the hemostatic disturbances observed in MM patients and the dynamic interaction between platelets and myeloma cells, along with present and future potential avenues for the use of platelets for diagnostic and therapeutic purposes.

Interleukin-10 increases macrophage-mediated chemotherapy resistance via FABP5 signaling in multiple myeloma

Macrophages (MΦs) protect multiple myeloma (MM) cells from chemotherapy-induced apoptosis, and interleukin-10 (IL-10) is frequently elevated in the MM microenvironment. However, the role of IL-10 in MΦ-induced tumor chemotherapy resistance has not yet been clarified. In the present study, bone marrow-derived MΦs were treated with IL-10 (IL10-MΦs), and IL10-MΦ-induced MM chemotherapy resistance was evaluated. IL-10 promoted MΦ-mediated resistance to MM chemotherapy. In addition, IL-10 treatment increased lipid accumulation and fatty acid β-oxidation in MΦs. Mechanistically, IL-10 increased fatty acid binding protein 5 (FABP5) expression in MΦs, and targeting FABP5 decreased MM chemotherapy resistance induced by IL10-MΦs in vitro and enhanced chemotherapeutic efficacy in vivo. Inhibition of FABP5 decreased the expression of Carnitine Palmitoyltransferase 1A (CPT1A) in IL10-MΦs. In addition, inhibition of CPT1A in IL10-MΦs decreased IL10-MΦ-mediated MM chemotherapy resistance. Peroxisome proliferator-activated receptor γ (PPARγ) is upstream of FABP5 signaling. Inhibition of PPARγ in IL10-MΦs decreased IL10-MΦ-mediated MM chemotherapy resistance in vitro. Collectively, our work indicates that IL-10 enhances MΦ-mediated MM chemotherapy resistance via FABP5 signaling and targeting FABP5 has potentially important clinical implications.

Intratumor microbiota: Occult participants in the microenvironment of multiple myeloma.

More recently, microbiota was detected in several tumorous tissues including multiple myeloma (MM), but the roles of which is still under-studied as paucity of research on tumor biology. Moreover, we also detected the presence of microbiota in the bone marrow of patients with MM by 2bRAD-M sequencing technology, which is an incurable hematological malignancy characterized by accumulation of abnormal plasma cells in the bone marrow. However, the roles of intratumor microbiota in tumor disease remains poorly understood. In this review, we critically reviewed recent literature about microbiota in the tumorigenesis and progression of MM. Importantly, we proposed that the emergence of microbiota in the microenvironment of multiple myeloma may be attributed to microbial dysbiosis and impaired intestinal barrier, due to the increased prevalence of MM in patients with obesity and diabetes, of which the characteristic phenotype is gut microbial dysbiosis and impaired intestinal barrier. When the intestinal barrier is damaged, dysbiotic microbiota and their metabolites, as well as dysregulated immune cells, may participate in the reshaping of the local immune microenvironment, and play pivotal roles in the tumorigenesis and development of multiple myeloma, probably by migrating to the bone marrow microenvironment from intestine. We also discuss the emerging microbiological manipulation strategies to improve long-term outcomes of MM, as well as the prospective of the state-of-the-art techniques to advance our knowledge about the biological implication in the microbiome in MM.