Multiple Myeloma Bone Research Articles

Bone mechanical properties were altered in a mouse model of multiple myeloma bone disease

Multiple myeloma bone disease (MMBD) is characterized by the growth of malignant plasma cells in bone marrow, leading to an imbalance in bone (re)modeling favoring excessive resorption. Loss of bone mass and altered microstructure characterize MMBD in humans and preclinical animal models, although, no study to date has examined bone composition or material properties. We hypothesized that MMBD alters bone composition, mineral crystal properties and mechanical properties in the MOPC315.BM.Luc model after intra-tibial injection of myeloma cells and three weeks of daily in vivo tibial loading. Decreased cortical bone elastic modulus and hardness measured by nanoindentation of tibiae were observed in MM-injected mice compared to PBS-injected mice, whereas cortical bone composition, mineral crystal properties measured by Fourier-transform infrared imaging or small angle X-ray scattering, respectively remained unchanged. However, MM-injected mice had thinner cancellous bone mineral particles compared to PBS-injected mice. Mechanical loading did not lead to altered cortical bone composition, mineral structure, or mechanical properties in the context of MM. Unexpectedly, we observed the intra-tibial injection itself altered the material composition of bone, manifested by increased matrix mineralization and crystal size of the hydroxyapatite crystals in the bone matrix. In conclusion, our data suggest that mechanical stimuli can be used as an adjuvant bone anabolic therapy in patients with MMBD to rebuild bone with unaltered composition and mineral structure to reduce subsequent fracture risk.

3D osteocyte lacunar morphometry of human bone biopsies with high resolution microCT: From monoclonal gammopathy to newly diagnosed multiple myeloma

Osteocytes are mechanosensitive, bone-embedded cells which are connected via dendrites in a lacuno-canalicular network and regulate bone resorption and formation balance. Alterations in osteocyte lacunar volume, shape and density have been identified in conditions of aging, osteoporosis and osteolytic bone metastasis, indicating patterns of impaired bone remodeling, osteolysis and disease progression. Osteolytic bone disease is a hallmark of the hematologic malignancy multiple myeloma (MM), in which monoclonal plasma cells in the bone marrow disrupt the bone homeostasis and induce excessive resorption at local and distant sites. Qualitative and quantitative changes in the 3D osteocyte lacunar morphometry have not yet been evaluated in MM, nor in the precursor conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). In this study, we characterized the osteocyte lacunar morphology in trabecular bone of the iliac crest at the ultrastructural level using high resolution microCT in human bone biopsy samples of three MGUS, two SMM and six newly diagnosed MM. In MGUS, SMM and MM we found a trend for lower lacunar density and a shift towards larger lacunae with disease progression (higher 50 % cutoff of the lacunar volume cumulative distribution) in the small osteocyte lacunae 20–900 μm3 range compared to control samples. In the larger lacunae 900–3000 μm3 range, we detected significantly higher lacunar density and microporosity in the MM group compared to the MGUS/SMM group. Regarding the shape distribution, the MGUS/SMM group showed a trend for flatter, more elongated and anisotropic osteocyte lacunae compared to the control group. Altogether, our findings suggest that osteocytes in human MM bone disease undergo changes in their lacunae density, volume and shape, which could be an indicator for osteolysis and disease progression. Future studies are needed to understand whether alterations of the lacunae architecture affect the mechanoresponsiveness of osteocytes, and ultimately bone adaptation and fracture resistance in MM and its precursors conditions.

Expression and Function of FAM72A Gene in Multiple myelomaFAM72A.

This study aims to comprehensively investigate the role of Family Member A with sequence similarity 72-A (FAM72A) in multiple myeloma. Multiple myeloma poses significant challenges. This study delves into FAM72A's impact on key cellular processes, shedding light on potential therapeutic targets and enhancing our understanding of multiple myeloma progression. Investigate the impact of FAM72A on the proliferation, apoptosis, and bortezomib sensitivity of multiple myeloma cell line U266. qRT-PCR analyzed FAM72A expression levels in bone marrow samples from 30 patients with multiple myeloma and 10 healthy donors at the Second Hospital of Shanxi Medical University. Cell lines overexpressing FAM72A were constructed, and Cell Counting Kit 8 (CCK-8) and flow cytometry were used to assess U266 cell proliferation, apoptosis, and sensitivity to bortezomib. Biological predictions for FAM72A were performed to find transcription factors binding to the FAM72A promoter region, verified using a luciferase assay. U266 cells were transfected with si-POU2F2 (POU class 2 homeobox 2), and the impact on cell proliferation was validated. Western blot analysis detected the expression of downstream proteins in the p53 signaling pathway. In vivo, experiments established a xenograft mouse model further to study the role of FAM72A in multiple myeloma. FAM72A was upregulated in multiple myeloma bone marrow tissues. Compared to the OE-NC group, the OE-FAM72A group showed increased Mouse Double Minute 2 homolog (MDM2) expression, decreased p53 expression, increased cell proliferation, and decreased apoptosis. POU2F2 was identified as the upstream transcription factor for FAM72A. Compared to the si-NC group, the si-POU2F2 group exhibited decreased MDM2 expression, increased p53 expression, slowed cell proliferation, and increased apoptosis. Silencing POU2F2 could reverse the pro-proliferative effect of over-expressing FAM72A in U266 cells. In vivo experiments in a xenograft mouse model further studied the role of FAM72A in multiple myeloma. Overexpression of FAM72A promotes U266 cell proliferation, inhibits apoptosis, and reduces sensitivity to bortezomib by regulating the POU2F2/FAM72A/p53 signaling pathway.

First-Line Combination with Proteasome Inhibitor-Based Treatment and Zoledronic Acid Is Effective in Reducing Later Fractures in Multiple Myeloma Irrespective of Multiple Myeloma Bone Disease at Diagnosis.

The present study provides real-world evidence on the treatment of multiple myeloma (MM) bone disease with various bisphosphonates combined for different myeloma-specific treatments as no validated data regarding the best combination treatment for bone disease associated with MM are available. We examined retrospectively 345 MM patients treated with autologous stem cell transplantation in Finland during 1996-2020. The median age of the patients was 60 years with a median follow-up time of 50 months (1-339). At diagnosis, 72.1% of the patients had myeloma-associated bone disease and 45.8% had fractures. Most patients (58.8%) received proteasome inhibitor (PI)-containing treatment at first line. MM bone disease was treated in 91.6% of the patients; 49.9% received zoledronic acid (ZA) and 29.9% pamidronate. Inferior overall survival was associated with MM bone disease at diagnosis (p = 0.005) or a fracture at diagnosis (p = 0.003). A later fracture was identified in 29% of the patients, and in those patients without MM bone disease at diagnosis later fractures were less common after ZA treatment (p = 0.049). PI-based treatment plus ZA (p = 0.019) seemed to be the best combination to prevent later fractures, even though the same patient subgroup was more likely to experience relapse (p = 0.018), and also when excluding patients with previous induction therapy without novel agents (p = 0.008). To conclude, this study suggests that the best therapy to prevent later fractures in MM might be PI-based treatment combined with ZA.

Coefficient of variation and texture analysis of 18F-FDG PET/CT images for the prediction of outcome in patients with multiple myeloma

In multiple myeloma (MM) bone marrow infiltration by monoclonal plasma cells can occur in both focal and diffuse manner, making staging and prognosis rather difficult. The aim of our study was to test whether texture analysis of 18 F-2-deoxy-d-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) images can predict survival in MM patients. Forty-six patients underwent 18 F-FDG-PET/CT before treatment. We used an automated contouring program for segmenting the hottest focal lesion (FL) and a lumbar vertebra for assessing diffuse bone marrow involvement (DI). Maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean) and texture features such as Coefficient of variation (CoV), were obtained from 46 FL and 46 DI. After a mean follow-up of 51 months, 24 patients died of myeloma and were compared to the 22 survivors. At univariate analysis, FL SUVmax (p = 0.0453), FL SUVmean (p = 0.0463), FL CoV (p = 0.0211) and DI SUVmax (p = 0.0538) predicted overall survival (OS). At multivariate analysis only FL CoV and DI SUVmax were retained in the model (p = 0.0154). By Kaplan-Meier method and log-rank testing, patients with FL CoV below the cut-off had significantly better OS than those with FL CoV above the cut-off (p = 0.0003), as well as patients with DI SUVmax below the threshold versus those with DI SUVmax above the threshold (p = 0.0006). Combining FL CoV and DI SUVmax by using their respective cut-off values, a statistically significant difference was found between the resulting four survival curves (p = 0.0001). Indeed, patients with both FL CoV and DI SUVmax below their respective cut-off values showed the best prognosis. Conventional and texture parameters derived from 18F-FDG PET/CT analysis can predict survival in MM patients by assessing the heterogeneity and aggressiveness of both focal and diffuse infiltration.

Single‐cell sequencing reveals the correlation of aggrephagy signaling and multiple myeloma immune microenvironment composition

AbstractAggrephagy, a type of autophagy, degrades the aggregation of misfolded protein in cells. However, the role of aggrephagy in multiple myeloma (MM) has not been fully demonstrated. In this study, we first investigated the correlation between aggrephagy signaling, MM immune microenvironment composition and disease prognosis. Single‐cell RNA‐seq data, including the expression profiles of 12,187 single cells from seven MM bone marrow (BM) and seven healthy BM samples, were analyzed by non‐negative matrix factorization for 44 aggrephagy‐related genes. Bulk RNA‐seq cohorts from the Gene Expression Omnibus database were used to evaluate the prognostic value of aggrephagy‐related immune cell subtypes and predict immune checkpoint blockade immunotherapeutic response in MM. Compared with healthy BM, MM BM exhibited different patterns of aggrephagy‐related gene expression. In MM BM, macrophages, CD8+ T cells, B cells and natural killer cells could be grouped into four to nine aggrephagy‐related subclusters. The signature of aggrephagy signaling molecule expression in the immune cells correlates with the patient's prognosis. Our investigation provides a novel view of aggrephagy signaling in MM tumor microenvironment cells, which might be a prognostic indicator and potential target for MM treatment.

Bone Metabolism of Multiple Myeloma Bone Disease Patients with Different Blood Separation Results

To investigate the clinical significance of bone metabolic indexes for disease assessment and curative effect monitoring in multiple myeloma (MM) bone disease (MBD) patients with different blood separation results. A total of 134 newly diagnosed MM patients treated in Cangzhou Hospital of Integrated TCM-WM-Hebei were enrolled and divided into control group [119 cases, serum, colloid and red blood cell (RBC) from top to bottom of sample] and abnormal group (15 cases, serum, mixed layer of RBC and serum, colloid and RBC from top to bottom of sample) according to the results of blood separation. According to the imaging findings, MBD was classified into grade 0-4, grade 0-2 was mild, and grade 3-4 was severe. The MBD grade of patients in the two groups was analyzed. The curative effect of MBD patients after chemotherapy and the changes of blood separation results and bone metabolic indexes before and after treatment were evaluated. The correlation between β2-microglobulin (MG) and bone metabolic indexes was analyzed by Pearson correlation analysis. In the control group, there were 69 cases of grade 0-2 and 50 cases of grade 3-4, while in the abnormal group, there were 5 cases of grade 0-2 and 10 cases of grade 3-4, the difference was statistically significant (P < 0.05). The serum β2-MG, β-CTX levels in abnormal group were both significantly higher than those in control group, while the levels of P1NP and osteocalcin (OC) were significantly lower (all P < 0.001). In the control group, there were 95 patients with ≥ partial response (PR) and the blood separation results were not changed, while 24 patients with <PR and 5 of them had abnormal blood separation results. In the abnormal group, 9 patients with efficacy ≥PR showed normal blood separation results, while 6 patients with efficacy < PR and 5 of them still remained abnormal blood separation results. Compared with before treatment, β-CTX and β2-MG of patients with efficacy ≥PR were significantly decreased but P1NP and OC increased in the control group (all P < 0.001), which was the same as abnoraml group (both P < 0.001, P < 0.01). There were no significant changes in the levels of all indexes in the two groups of patients with efficacy < PR (P >0.05). Compared with before treatment, the levels of β-CTX and β2-MG in the control group with unchanged blood separation results were significantly decreased (both P < 0.001), while the levels of P1NP and OC were significantly increased (P < 0.01, P < 0.001), and the level of each index in the patients transformed to abnormal blood separation result after treatment did not significantly change (P >0.05); the levels of β-CTX and β2-MG in the abnormal group transformed to normal blood separation result were significantly decreased (both P < 0.01), while the levels of P1NP and OC were significantly increased (P < 0.001, P < 0.01), and the level of each index in patients with unchanged blood separation results did not significantly change (P>0.05). Pearson correlation analysis showed that serum β2-MG was positively correlated with β-CTX (r =0.709, P < 0.001), and negatively correlated with P1NP and OC (r =-0.410,r =-0.412, both P < 0.001). MBD patients with abnormal blood separation results have higher bone disease grade and poor prognosis, which is closely related to the significant increase of bone resorption index β-CTX level and decrease of bone formation index P1NP and OC levels, leading to more serious bone metabolic homeostasis disorder. The results of blood separation combined with the changes of bone metabolic indexes can be used as one of the comprehensive predictors of disease condition, efficacy monitoring and prognosis evaluation of MBD patients.

Value of third-generation of VNCa dual-energy CT for differentiating diffuse marrow infiltration of multiple myeloma from red bone marrow.

This study aims to investigate the ability of bone marrow imaging using third-generation dual-energy computed tomography (CT) virtual noncalcium (VNCa) to differentiate between multiple myeloma (MM) with diffuse bone marrow infiltration and red bone marrow (RBM). Bone marrow aspiration or follow-up results were used as reference. We retrospectively reviewed 188 regions of interests (ROIs) from 21 patients with confirmed MM and diffuse bone marrow infiltrations who underwent VNCa bone marrow imaging between May 2019 and September 2022. At the same time, we obtained 98 ROIs from 11 subjects with RBM for comparative study, and 189 ROIs from 20 subjects with normal yellow bone marrow for the control group. The ROIs were delineated by 2 radiologists independently, the interobservers reproducibility was evaluated by interclass correlation coefficients. The correlation with MRI grade results was analyzed by Spearman correlation coefficient. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal threshold for differentiating between these groups and to assess diagnostic performance. There were statistically significant differences in VNCa CT values of bone marrow among the MM, RBM, and control groups (all P < .001), with values decreasing sequentially. A strong positive rank correlation was observed between normal bone marrow, subgroup MM with moderately and severe bone marrow infiltration divided by MRI and their corresponding CT values (ρ = 0.897, 95%CI: 0.822 to 0.942, P < .001). When the CT value of VNCa bone marrow was 7.15 HU, the area under the curve (AUC) value for differentiating RBM and MM was 0.723, with a sensitivity of 50.5% and a specificity of 89.8%. When distinguishing severe bone marrow infiltration of MM from RBM, the AUC value was 0.80 with a sensitivity 70.9% and a specificity 78.9%. The AUC values for MM, RBM, and the combined group compared to the control group were all >0.99, with all diagnostic sensitivity and specificity exceeding 95%. VNCa bone marrow imaging using third-generation dual-energy CT accurately differentiates MM lesions from normal bone marrow or RBM. It demonstrates superior diagnostic performance in distinguishing RBM from MM with diffuse bone marrow infiltration.

Abstract 2305: Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2305.

In Vitro Low-Bortezomib Doses Induce Apoptosis and Independently Decrease the Activities of Glutathione S-Transferase and Glutathione Peroxidase in Multiple Myeloma, Taking into Account the GSTT1 and GSTM1 Gene Variants.

Multiple myeloma (MM) is a malignancy derived from plasma cells. Bortezomib affects the concentration of reduced glutathione (GSH) and the activity of glutathione enzymes. The aim of our study was to analyze deletion (null/present) variants of GSTT1 and GSTM1 genes and their association with the levels of glutathione and its enzymes in bortezomib-treated cell cultures derived from MM patients. This study included 180 individuals (80 MM patients and 100 healthy blood donors) who were genotyped via multiplex PCR (for the GSTT1/GSTM1 genes). Under in vitro conditions, MM bone marrow cells were treated with bortezomib (1-4 nM) to determine apoptosis (via fluorescence microscopy), GSH concentration, and activity of glutathione enzymes (via ELISA). Bortezomib increased the number of apoptotic cells and decreased the activity of S-glutathione transferase (GST) and glutathione peroxidase (GPx). We found significant differences in GST activity between 1 nM (GSTT1-null vs. GSTT1-present), 2 nM (GSTT1-null vs. GSTT1-present), and 4 nM (GSTM1-null vs. GSTM1-present) bortezomib: 0.07 vs. 0.12, p = 0.02; 0.06 vs. 0.10, p = 0.02; and 0.03 vs. 0.08, p = 0.01, respectively. Bortezomib affects the activities of GST and GPx. GST activity was associated with GSTT1 and GSTM1 variants but only at some bortezomib doses.

A Prospective Study of the IlluminOss Photodynamic Nail System for Pelvic Stabilization: Treatment of Impending and Actual Fractures from Metastatic Bone Disease, Multiple Myeloma, and Primary Bone Lymphoma.

The stabilization of metastatic lesions in the periacetabular region can be successfully performed using percutaneous techniques. Photodynamic nails (PDNs) are among the available tools for stabilization. Data on postoperative complications and functional outcomes are, however, scarce. Patients undergoing percutaneous stabilization using PDNs (IlluminOss Medical) for impending or actual minimally displaced pathological fractures of the pelvis from metastatic bone disease, multiple myeloma, or primary bone lymphoma were enrolled prospectively. Outcomes were assessed preoperatively and postoperatively at the 2-day, 2-week, 6-week, 3-month, 6-month, and 1-year time points. Functional outcomes assessed included the Patient-Reported Outcomes Measurement Information System (PROMIS) Physical Function, PROMIS Pain Interference, Combined Pain and Ambulatory Function (CPAF), EuroQol-Visual Analogue Scale (EQ-VAS), and Musculoskeletal Tumor Society (MSTS) scores. Pain was assessed using a VAS. A total of 30 patients treated with PDNs were included. The median VAS pain score dropped from 60 points preoperatively to 30 at 6 weeks postoperatively (p = 0.004). The median CPAF score improved from 6 preoperatively to 7 postoperatively at the 6-week mark. The median EQ-VAS score showed significant improvement at 6 weeks (70 versus 50; p = 0.006). The median 2-week PROMIS Pain Interference score was significantly lower than preoperatively (64.1 versus 66.9; p = 0.03). An improvement in the median PROMIS Physical Function score was seen at 6 weeks following surgery compared with preoperatively (37 versus 30.1; p = 0.001). A significant improvement in the MSTS score was seen as soon as 2 days after surgery (77% versus 40%; p < 0.0001). Among patients with pelvic bone metastases, multiple myeloma, or primary bone lymphoma, we found that treatment using PDNs resulted in immediate return to ambulation and rapid functional outcome improvement, with low complication rates. In this population, this technique represents a safe alternative to open surgery. Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions.

We have previously demonstrated that cystatin E/M (CST6), which is elevated in a subset of patients with multiple myeloma (MM) lacking osteolytic lesions (OLs), suppresses MM bone disease by blocking osteoclast differentiation and function. CST6 is a secreted type 2 cystatin, a cysteine protease inhibitor that regulates lysosomal cysteine proteases and the asparaginyl endopeptidase legumain. Here, we developed B cell maturation antigen (BCMA) CST6 chimeric antigen receptor T cells (CAR-T cells), which lysed MM cells and released CST6 proteins. Our in vitro studies show that these CAR-T cells suppressed the differentiation and formation of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts. Using xenografted MM mice, bioluminescence images showed that both BCMA-CAR-T and BCMA-CST6-CAR-T cells inhibited MM growth to a similar extent. Reconstructed micro-computed tomography images revealed that BCMA-CST6-CAR-T cells, but not BCMA-CAR-T cells, prevented MM-induced bone damage and decreased osteoclast numbers. Our results provide a CAR-T strategy that targets tumor cells directly and delivers an inhibitor of bone resorption.

Recent advances in imaging and artificial intelligence (AI) for quantitative assessment of multiple myeloma.

Multiple myeloma (MM) is a malignant blood disease, but there have been significant improvements in the prognosis due to advancements in quantitative assessment and targeted therapy in recent years. The quantitative assessment of MM bone marrow infiltration and prognosis prediction is influenced by imaging and artificial intelligence (AI) quantitative parameters. At present, the primary imaging methods include computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET). These methods are now crucial for diagnosing MM and evaluating myeloma cell infiltration, extramedullary disease, treatment effectiveness, and prognosis. Furthermore, the utilization of AI, specifically incorporating machine learning and radiomics, shows great potential in the field of diagnosing MM and distinguishing between MM and lytic metastases. This review discusses the advancements in imaging methods, including CT, MRI, and PET/CT, as well as AI for quantitatively assessing MM. We have summarized the key concepts, advantages, limitations, and diagnostic performance of each technology. Finally, we discussed the challenges related to clinical implementation and presented our views on advancing this field, with the aim of providing guidance for future research.

Cells of the Osteoblast Lineage Cross-Talk with Myeloid-Derived Suppressor Cells to Promote Multiple Myeloma Growth

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment. Osteolytic bone disease is a hallmark of MM and is partly due to osteoblast (OB) inhibition. MM cells suppress osteoblast maturation and function via inhibition of the critical Runt-related transcription factor 2 (Runx2) in immature OBs. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present and even increased in the MM bone microenvironment and their contribution to the progression of MM has not been well understood. We previously developed a syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice), and demonstrated that OB-Runx2 deficiency creates a highly inflammatory BM microenvironment that is responsible for MM cell bone-homing to new bone sites. We therefore hypothesize that pre-osteoblasts play a role as promoters of MM cell survival and proliferation. To determine the direct effect of pre-osteoblasts on MM cells, we established co-cultures of pre-osteoblasts with MM cells. 5TGM1 murine MM cells were cocultured with MC3T3 murine pre-osteoblasts and newborn mouse calvarial pre-osteoblasts respectively. Within 48hrs of co-culture, each of pre-osteoblast populations slightly stimulated proliferation of 5TGM1 MM cells. These data suggest that pre-osteoblasts do not directly affect myeloma growth in vitro. Therefore, we next focus our studies on the role of pre-osteoblasts in MM progression in vivo. Firstly, we used OB-Runx2 deficient mouse model to assess the impact of pre-osteoblast accumulation on MM progression in vivo. 2x10 5 5TGM1-Luciferase MM cells were injected into the right tibia of OB-Runx2 -/- mice and control mice. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA demonstrated that, compared with controls, OB-Runx2 -/- mice had a much larger tumor burden. Following, to more thoroughly evaluate the role of pre-osteoblasts in early stage of MM cell engraftment in bones, we used MC3T3 murine pre-osteoblasts to modulate pre-osteoblast populations at the sites of MM engraftment. 2x10 5 5TGM1-Luciferase MM cells were injected directly into the right tibia of NSG mice with or without equal numbers of MC3T3 pre-osteoblasts. Tumors grew significantly larger when co-injected with 5TGM1 MM cells and MC3T3 pre-osteoblasts than injected with 5TGM1 MM cells alone, which was confirmed by luciferin imaging and serum IgG2bκ ELISA. These data suggest that pre-osteoblasts provide critical support for MM progression in vivo. Given that pre-osteoblasts have no direct effect on the growth of MM cells in vitro, we hypothesized that the pre-osteoblastic niche may regulate MM cells via other intermediary cells in BM. We previously showed that OB-Runx2 deficiency induces an immunosuppressive BM microenvironment that is marked by an increase of myeloid-derived suppressor cells (MDSCs). To investigate whether pre-osteoblasts support MM cell survival and proliferation through the functional crosstalk with MDSCs, 5TGM1 MM cells were co-cultured with MC3T3 pre-osteoblasts and mouse calvarial pre-osteoblasts respectively in the presence or absence of MDSCs. Interestingly, pre-osteoblasts significantly promoted the proliferation of MM cells under the existence of MDSCs. To extend these findings in vivo, we next determined whether MDSC depletion can alleviate the promoting effect of pre-osteoblasts on MM progression. Gemcitabine (GEM) is an FDA-approved anti-cancer agent and MDSC inhibitor used in the treatment of solid tumors but not commonly used in MM therapy. We again injected 5TGM1-Luciferase MM cells into the right tibia of NSG mice with or without co-injection of MC3T3 pre-osteoblasts. We treated tumor-bearing mice with PBS or GEM (i.p. 30 mg/kg/week) for 4 weeks. Bioluminescence imaging and serum IgG2bκ ELISA demonstrated that GEM could overcome the proliferative effect of pre-osteoblasts on MM cells in mouse model, and this effect was independent of NK, B and T cells. Taken together, using in vivo and in vitro models, we show that pre-osteoblasts have a positive regulatory impact on MM cells through communication with MDSCs. Ongoing mechanistic studies aim to delineate how pre-osteoblasts modulate the functions of MDSCs to promote MM progression.

E3 Ubiquitin Ligase TRIM21 Enhances Macrophage-Mediated Bortezomib Resistance By Inducing M2 Polarization in Multiple Myeloma

Background: Multiple myeloma (MM) is a malignant plasma cell hematologic tumor with an increasing incidence and has become the second most common hematologic malignancy after non-Hodgkin lymphoma. Macrophage is an abundant component of the MM bone marrow microenvironment and contributes to the enhancement of MM drug resistance. Our previous study found that the expression of the E3 ubiquitin ligase TRIM21 was reduced in MM cells, and MM cells with low expression of TRIM21 showed reduced sensitivity to Bortezomib (Bort). This study aims to investigate the role and mechanism of TRIM21 in macrophage-mediated MM Bort resistance. Objective ：Explore the effect and mechanism of TRIM21 in mediating multiple myeloma Bortezomib resistance. Methods ：1. Immunofluorescence to analyze the expression of TRIM21 on macrophages in bone marrow biopsy specimens from patients with MM; 2. QPCR, western blot (WB) and flow cytometry (FCM) to detect the level of macrophage polarization markers after constructing overexpression or knockdown of TRIM21; 3. WB and FCM to detect the sensitivity of MM cells to Bort after co-culture of MM cells and macrophage; 4. Transcriptome sequencing to identify TRIM21-mediated pathway changes in macrophage; 5. QPCR and WB to verify the effect of TRIM21 on macrophage polarization after treatment with pathway inhibitors. Results:1. Immunofluorescence analysis showed increased expression of TRIM21 in macrophage from relapsed and refractory MM (RRMM) patients compared to newly diagnosed multiple myeloma (NDMM) patients;2. QPCR, WB and FCM results showed that overexpression of TRIM21 in macrophage led to decreased polarization markers of M1 phenotype and increased the polarization markers of M2 phenotype. In contrast, knockdown of TRIM21 increased M1 polarization and decreased M2 polarization; 3. WB and FCM results demonstrated that co-culture of MM cells with TRIM21-overexpressing macrophages significantly enhanced MM cell tolerance to Bortezomib and decreased MM cell apoptosis; 4. Transcriptome sequencing revealed activation of the JAK-STAT3 pathway in macrophages after TRIM21 overexpression; 5. QPCR and WB results showed that the macrophage polarization effect induced by TRIM21 overexpression was reversed after treatment with the STAT3 inhibitor C188-9. Conclusions: Our study shows that TRIM21 is overexpressed in macrophages in patients with relapsed and refractory MM. In addition, TRIM21 induces M2 polarization by activating the JAK-STAT3 pathway, thereby enhancing the protective effect of macrophage on MM cells. These findings provide a new insight into the role of TRIM21 in MM drug resistance and lay an important theoretical foundation for targeting macrophages in MM. Key words: TRIM21, Macrophage, Bortezomib, JAK-STAT3

Label-Free High Dimensional Single Cell Morphological Profiling of Different Hematological Malignancies By Ghost Cytometry

Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern 'omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6) (Figure 1). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information (Figure 2). For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified unique cell populations across blood cell populations that were distinct from the healthy BM-MNC controls. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Keywords: flow cytometry, acute leukemia, multiple myeloma, label-free cell analysis, biomarker, liquid biopsy, ghost cytometry, artificial intelligence, machine learning Figure 1. Research design overview. Bone marrow samples from patients and non-diseased controls will be analyzed by Ghost Cytometry to assess disease by conventional markers and identify novel morphometric subpopulations. Morphological information is acquired as label-free GMI signals without any labeling. The label-free GMI information is transformed into two-dimensional space by UMAP dimensionality reduction method to identify subpopulations of interest. Both samples are stained with known cell surface markers for validation of unsupervised classification. Figure 2. Identification of disease-specific cell subpopulations by unsupervised approach. Using an unsupervised machine learning technique called UMAP, label-free morphologically distinct cellular subpopulations can be identified in B-ALL, AML, MM when compared to healthy donor controls (left panels, inside the dotted circles). For validation of the subpopulations, known cell surface markers (CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM) and cell trace marker (CellTracker) were overlaid. The intensity increases monotonically through the colormaps. The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population.

Checkpoint Inhibitor PD-1H/VISTA Affects Myeloma Bone Disease By Osteoclast Cytoskeleton Remodeling

Introduction We have recently described the critical role of checkpoint inhibitor PD-1H/VISTA in osteoclast activation and multiple myeloma bone disease. Highly expressed in osteoclast precursors and mature osteoclasts, PD-1H functions as the receptor for osteoclastogenic factor MMP-13 and mediates MMP-13 induced osteoclast activation and myeloma bone disease ( Nat Commun14, 4271 (2023)). Despite the extensive functional study of PD-1H in immune disease models, the PD-1H interacting cellular complex is yet to be defined. Here we report how PD-1H interacts with cytoskeleton proteins contributing to osteoclast remodeling in multiple myeloma bone disease. Methods and Results To identify interacting proteins of PD-1H, PD-1H-His 6 recombinant protein was overexpressed in mouse mononuclear BMCs, and PD-1H-His 6 binding protein complex was pulled down by Ni-NTA agarose beads followed by mass spectrum analysis (Figure A). LIM and SH3 domain protein 1 (LASP1) was one of the most abundant cytoskeleton proteins identified in the PD-1H complex. We therefore first confirmed its binding to PD-1H by two-way co-IP assays (Figure B and C). Co-localization of LASP1 and PD-1H were further confirmed by immunofluorescence assay when co-expressed in HEK293 cells (Figure D). LASP1 is an F-actin-binding protein that stabilizes F-actin bundles and is involved in podosome regulation in macrophages (PLoS ONE 7(4): e35340). It has four domains: LIM, two nebulin-like repeats (N1 and N2) and SH3 domains. While nebulin-like repeats mediate binding to F-actin, SH3 domain is the major protein-protein binding site (PLoS ONE 7(4): e35340). In co-IP assay using PD-1H and domain deleted LASP1, deletion of LIM (△LIM) or either one or both nebulin-like repeats (△N1, △N2 and △N1/2) did not affect the binding to PD-1H. Instead, deletion of SH3 (△SH3) in LASP1 lead to loss of PD-1H binding, demonstrating that PD-1H binds to LASP1 at the SH3 domain (Figure E). Next, the association of LASP1 and PD-1H in the osteoclast cytoskeleton was demonstrated by confocal immunofluorescence microscopy. LASP1 co-localized with F-actin (Figure F) and PD-1H (Figure G) at the sealing ring in mature osteoclasts. Interestingly, although MMP-13 stimulation increased the size of osteoclasts and the sealing ring, MMP-13 did not change the co-localization pattern of LASP1 with PD-1H or F-actin. Conclusions Taken together, this study reveals the novel mechanism of PD-1H regulation of osteoclasts cytoskeleton reorganization, which is critical for osteoclast bone resorption activity. PD-1H directly associates with LASP1 at its C-terminal SH3 domain. Although LASP1 was reported to regulates podosomes function in macrophages, its role in osteoclast cytoskeletons remain largely obscure. The future study will be focused on delineating the role of LASP1 in osteoclast function, and its role in MMP-13/PD-1H signaling regulated myeloma bone disease.

Novel_circ_003686 regulates the osteogenic differentiation of MSCs in patients with myeloma bone disease through miR-142-5p/IGF1 axis

ObjectivesCirc_003686 is a novel_circRNA with abnormally low expression found in the samples of multiple myeloma bone disease (MBD) patients. The current research intended to investigate the effects of novel_circ_003686 in osteogenesis-induced differentiation of bone marrow mesenchymal stem cells (BMSCs) in MBD. MethodsBMSCs were extracted from MBD patients and normal participants, the pcDNA3.1 encoding the circ_003686 (ov-circ_003686), miR-142-5p-mimic/inhibitor and siRNA oligonucleotides targeting insulin like growth factor 1 (IGF1, si-IGF1) were applied to intervene circ_003686, miR-142-5p and IGF1 levels, respectively. Results: Results showed that ov-circ_003686 could mediate the osteogenesis-induced differentiation of MBD-BMSC, and luciferase assay and RIP experiments confirmed that circ_003686 could bind to miR-142-5p. MiR-142-5p-inhibitor helped osteogenesis-induced differentiation, while miR-142-5p-mimic inhibited osteogenesis-induced differentiation and reversed the promoting effect of ov-circ_003686, suggesting that circ_003686/miR-142-5p axis participated in osteogenesis-induced differentiation of MBD-BMSC. In addition, miR-142-5p binds to the target gene IGF1 and negatively adjust its expression. Si-IGF1 significantly inhibited the osteogenesis-induced differentiation and reversed the promotion effects of miR-142-5p-inhibitor and ov-circ_003686. Moreover, circ_003686/miR-142-5p/IGF1 axis meaningfully regulates protein expressions in the PI3K/AKT pathway. ConclusionIn conclusion, this research confirmed that circ_003686 regulated the osteogenesis-induced differentiation of MBD-BMSC by sponging miR-142-5p and mediating IGF1, and the PI3K/AKT pathway may also be involved.

Current Status and Future of Artificial Intelligence in MM Imaging: A Systematic Review.

Artificial intelligence (AI) has attracted increasing attention as a tool for the detection and management of several medical conditions. Multiple myeloma (MM), a malignancy characterized by uncontrolled proliferation of plasma cells, is one of the most common hematologic malignancies, which relies on imaging for diagnosis and management. We aimed to review the current literature and trends in AI research of MM imaging. This study was performed according to the PRISMA guidelines. Three main concepts were used in the search algorithm, including "artificial intelligence" in "radiologic examinations" of patients with "multiple myeloma". The algorithm was used to search the PubMed, Embase, and Web of Science databases. Articles were screened based on the inclusion and exclusion criteria. In the end, we used the checklist for Artificial Intelligence in Medical Imaging (CLAIM) criteria to evaluate the manuscripts. We provided the percentage of studies that were compliant with each criterion as a measure of the quality of AI research on MM. The initial search yielded 977 results. After reviewing them, 14 final studies were selected. The studies used a wide array of imaging modalities. Radiomics analysis and segmentation tasks were the most popular studies (10/14 studies). The common purposes of radiomics studies included the differentiation of MM bone lesions from other lesions and the prediction of relapse. The goal of the segmentation studies was to develop algorithms for the automatic segmentation of important structures in MM. Dice score was the most common assessment tool in segmentation studies, which ranged from 0.80 to 0.97. These studies show that imaging is a valuable data source for medical AI models and plays an even greater role in the management of MM.

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.