Prospective evaluation of spatial heterogeneity at single cell resolution in multiple myeloma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 8524-8524
Author(s):  
Maximilian Merz ◽  
Almuth Maria Anni Merz ◽  
Jie Wang ◽  
Lei Wei ◽  
Qiang Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Single Cell ◽  
Bone Disease ◽  
Bone Destruction ◽  
The Body ◽  
Response To Treatment ◽  
Risk Scores ◽  
Specific Gene

8524 Background: Osteolytic lesions (OL) characterize symptomatic multiple myeloma (MM). It is still unclear why plasma cells (PC) cause OL in certain regions of the body while other areas show no signs of bone destruction despite significant bone marrow infiltration. We conducted the first study of single cell RNA sequencing (scRNA-seq) and whole-exome sequencing (WES) of PC obtained from random bone marrow samples (RS) and paired OL. Methods: As part of a prospective clinical trial, patients consented to an imaging-guided biopsy of new OL identified by PET/CT in addition to the RS from the iliac crest. Both samples were acquired in the same session. On the same day PC were isolated using a CD138 positive selection kit and single cell gene expression libraries were generated for scRNA-seq. Frozen PC were subjected to DNA extraction and WES. Results: We sequenced 93569 purified, viable PC from paired samples from 15 different locations in the first 7 consecutive patients (median PC from location: 7203; range 1121-10279). Quality assessment of scRNA-seq data revealed no differences between PC in OL and RS. Based on scRNA-seq, 9-24 different subpopulations of PC in individual patients were identified. Over 90% of clusters found in the RS were also present in corresponding OL suggesting a common ancestor. This was true for patients with overlapping as well as divergent mutational profiles in RS and OL as shown by WES. In each patient we found PC clusters that were predominantly present in OL. Respective clusters were characterized by expression of Wnt-signaling inhibitors like DKK-1, Frzb and sFRP-2 and other genes linked to MM bone disease (HGF, CXCL-12, CCL3). Lysosome-associated membrane protein-like molecule 5 (LAMP5) and J-chain were overexpressed in OL clusters. Analysis of genes (IKZF1 and IKZF3) associated with response to treatment and outcome revealed vast heterogeneity and differences in risk scores (UAMS70 and IFM15) on a single cell level from different locations in individual patients. Conclusions: Our study provides the first evidence that PC from OL have distinct transcriptomic profiles that link site-specific gene expression to development of bone disease and adverse outcome.

Bone disease as the first manifestation of systemic AL-amyloidosis

2020 ◽  
Vol 92 (7) ◽  
pp. 85-89
Author(s):  
L. P. Mendeleeva ◽  
I. G. Rekhtina ◽  
A. M. Kovrigina ◽  
I. E. Kostina ◽  
V. A. Khyshova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Plasma Cells ◽  
Interventricular Septum ◽  
Bone Destruction ◽  
Amyloid Deposition ◽  
Bone Lesions ◽  
Al Amyloidosis ◽  
Linear Type

Our case demonstrates severe bone disease in primary AL-amyloidosis without concomitant multiple myeloma. A 30-year-old man had spontaneous vertebral fracture Th8. A computed tomography scan suggested multiple foci of lesions in all the bones. In bone marrow and resected rib werent detected any tumor cells. After 15 years from the beginning of the disease, nephrotic syndrome developed. Based on the kidney biopsy, AL-amyloidosis was confirmed. Amyloid was also detected in the bowel and bone marrow. On the indirect signs (thickening of the interventricular septum 16 mm and increased NT-proBNP 2200 pg/ml), a cardial involvement was confirmed. In the bone marrow (from three sites) was found 2.85% clonal plasma cells with immunophenotype СD138+, СD38dim, СD19-, СD117+, СD81-, СD27-, СD56-. FISH method revealed polysomy 5,9,15 in 3% of the nuclei. Serum free light chain Kappa 575 mg/l (/44.9) was detected. Multiple foci of destruction with increased metabolic activity (SUVmax 3.6) were visualized on PET-CT, and an surgical intervention biopsy was performed from two foci. The number of plasma cells from the destruction foci was 2.5%, and massive amyloid deposition was detected. On CT scan foci of lesions differed from bone lesions at multiple myeloma. Bone fragments of point and linear type (button sequestration) were visualized in most of the destruction foci. The content of the lesion was low density. There was no extraossal spread from large zones of destruction. There was also spontaneous scarring of the some lesions (without therapy). Thus, the diagnosis of multiple myeloma was excluded on the basis based on x-ray signs, of the duration of osteodestructive syndrome (15 years), the absence of plasma infiltration in the bone marrow, including from foci of bone destruction by open biopsy. This observation proves the possibility of damage to the skeleton due to amyloid deposition and justifies the need to include AL-amyloidosis in the spectrum of differential diagnosis of diseases that occur with osteodestructive syndrome.

Prediction of Response to Treatment with Lenalidomide in Patients with Non-Del 5q Myelodysplastic Syndrome by Gene Expression Profiling Remains Difficult.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2781-2781
Author(s):  
Wolf-Karsten Hofmann ◽  
Florian Nolte ◽  
Ouidad Benlasfer ◽  
Eckhard Thiel ◽  
Gerhard Ehninger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Clinical Response ◽  
Expression Profiling ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Response To Treatment ◽  
Specific Gene ◽  
Prediction Of Response

Abstract Abstract 2781 Poster Board II-757 Lenalidomide belongs to a proprietary class of immunmodulatory drugs showing therapeutic activity in patients with myelodysplastic syndrome (MDS), in particular in those having the 5q-abnormality, but also in patients not showing this cytogenetical aberration. In 2008, Ebert et al. (PLos Med. 2, e35) could demonstrate that there is a specific gene expression profile in bone marrow cells collected from MDS-patients either with 5q- syndrome as well as MDS-patients having no 5q-abnormality which is strongly correlated with the clinical response to treatment with lenalidomide. Whereas this finding is not of clinical importance in patients with MDS del 5q (overall response 75 %) it may play a pivotal role for prediction of clinical response to lenalidomide in non-del 5q MDS-patients. Therefore, we have studied gene expression profile (HG-U133plus2.0, Affymetrix, Santa Clara, CA) of routinely isolated low-density mononuclear bone marrow cells from 8 patients with IPSS low/int-1 risk MDS having no deletion on chromosome 5 but were subsequently treated with lenalidomide 5 mg/day. All of the patients were transfusion dependent for red blood cells. The median duration of treatment with lenalidomide was 22 weeks. RNA was extracted by Trizol and quality controlled by using a Bioanalyzer 2100 system (Agilent, Waldborn, Germany) to exclude RNA degradation. Microarray hybridization was performed according to the standard Affymetrix protocol. Data were analyzed by Microarray Analysis Suites 5.0 (MAS 5.0; Affymetrix) and GeneSpring (Agilent Technologies, Santa Clara, CA). For clustering analysis we utilized the gene list of 68 discriminating genes as published by Ebert et al. the molecular analysis did clearly separate two groups of patients having specific gene expression profiles according to the responder/non-responder group as published previously. Furthermore, single sample prediction could discriminate three out of 8 patients to be possible responders to lenalidomide but this was not correlated to the clinical course of those patients while on treatment with lenalidomide. However, none of the MDS-patients receiving lenalidomide did show significant clinical response as defined by reduction of transfusion requirement by 50 % or transfusion independence. In conclusion, prediction of response to lenalidomide in non-del 5q patients by gene expression profiling so far remains critical. Prospective analysis of molecular changes including DNA analysis in larger clinical trials using lenalidomide in non-del 5q MDS-patients are required to establish reliable predictive markers in MDS. Disclosures: Hofmann: Celgene: Research Funding. Platzbecker:Celgene: Research Funding.

Bone Marrow Monocyte / Macrophage Derived Activin A Mediates the Osteoclastogenic Effects of IL-3 in Myeloma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3933-3933 ◽  
Author(s):  
Rebecca Silbermann ◽  
Marina Bolzoni ◽  
Paola Storti ◽  
Benedetta Dalla Palma ◽  
Sabrina Bonomini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Disease ◽  
Healthy Subjects ◽  
Research Funding ◽  
Bone Destruction ◽  
Activin A ◽  
Negative Regulator ◽  
Normal Donor ◽  
Myeloma Bone Disease

Abstract Abstract 3933 Multiple myeloma (MM) is characterized by bone destruction with suppressed new bone formation that is mediated by multiple factors including MIP-1α (CCL3), RANKL, IL-3, DKK1 and recently, Activin A (ActA). IL-3 is a bifunctional cytokine that indirectly increases osteoclastogenesis and suppresses osteoblastogenesis via CD14+ bone marrow monocytes (BMM). Additionally, IL-3 levels are elevated in the BM plasma of MM patients compared with normals, however the mediators of IL-3's effects on myeloma bone disease are unknown. To investigate this we performed gene expression profiling using Affymetrix GeneChip® analysis of IL-3 treated BMM from MM patients and found ActA gene expression was increased 180-fold and confirmed this finding at the protein level by ELISA. ActA is a negative regulator of bone mass that promotes osteoclastogenesis and is overproduced in MM patients. Interestingly, treatment of MM cells or bone marrow stromal cells (BMSC) with IL-3 did not induce secretion of ActA. We found that ActA was produced by MM and MGUS patient CD14+ cells treated with IL-3 to a significantly higher degree compared to healthy subjects. (Median ActA levels for MM and MGUS ActA levels were increased 66.57 and 51.6 fold respectively over untreated cells, while IL-3 treatment of normal cells increased ActA 8.5 fold.) ActA levels were also increased in freshly isolated marrow plasma of a cohort of patients with active MM as compared to patients with smoldering MM (SMM), MGUS, or healthy subjects (median ActA levels: active MM 453 pg/ml, SMM 328 pg/ml, MGUS 332 pg/ml, normal 286 pg/ml). ActA levels in MM patients with and without bone disease were not significantly different. (Median value with bone disease 463pg/ml vs. 407 pg/ml without bone disease.) ActA has also been reported to have a role in the differentiation and polarization of CD14+ tumor associated macrophages (TAMs), which are osteoclast precursors and can block MM cell apoptosis. Therefore, we examined the potential role of ActA in IL-3 mediated osteoclast (OCL) formation. Culture of normal BMM with ActA or IL-3 significantly enhanced osteoclastogenesis compared with control (mean number of OCL / 1×105 normal marrow non-adherent cells plated in IL-3 (100pg/ml) treated cultures 73; ActA (1ng/ml) treated cultures 123; cultures with vehicle alone, 8), and ActA enhanced RANKL-induced osteoclastogenesis. Osteoprotegerin treatment of normal donor BMM stimulated with ActA failed to block the osteoclastogenic effects of ActA, demonstrating that ActA's osteoclastogenic effects were RANKL independent. Importantly, the osteoclastogenic effect of IL-3 was dose-dependently inhibited by anti-ActA, and IL-3 induced ActA expression by BMM decreased during OCL differentiation. In support of early OCL precursors as the source of IL-3 induced ActA, we did not identify IL-3 receptors on mature OCL by flow cytometry. These results demonstrate that IL-3 induction of osteoclastogenesis is mediated by ActA produced by CD14+ BMM and is RANKL independent. Thus, we hypothesize that therapies targeting the ActA receptor, such as the recently developed ActA receptor antagonist, should block both IL-3 and ActA, and thereby significantly impact MM bone disease via their effects on TAMs. Disclosures: Bolzoni: Celgene Italy: Research Funding. Roodman:Millennium: Consultancy; Amgen: Consultancy. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

scholarly journals Deciphering spatial genomic heterogeneity at a single cell resolution in multiple myeloma

2021 ◽  
Author(s):  
Maximilian Merz ◽  
Almuth Merz ◽  
Jie Wang ◽  
Lei Wei ◽  
Qiang Hu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Single Cell ◽  
Bone Disease ◽  
Plasma Cells ◽  
Bone Destruction ◽  
Integrated Analysis ◽  
Myeloma Bone Disease ◽  
Additional Layer ◽  
Whole Exome ◽  
Transcriptional Changes

Abstract Osteolytic lesions (OL) characterize symptomatic multiple myeloma. The mechanisms of how malignant plasma cells (PC) cause OL in one region while others show no signs of bone destruction despite subtotal infiltration remain unknown. We report the first single-cell RNA sequencing (scRNA-seq) study of PC obtained prospectively from random bone marrow aspirates (BM) and paired imaging-guided biopsies of OL. We analyzed 148,630 PC from 24 different locations in 10 patients and observed vast inter- and intra-patient heterogeneity based on scRNA-seq analyses. Beyond the limited evidence for spatial heterogeneity from whole-exome sequencing, we found an additional layer of complexity by integrated analysis of anchored scRNA-seq datasets from the BM and OL. PC from OL were characterized by differentially expressed genes compared to PC from BM, including upregulation of genes associated with myeloma bone disease like DKK1, HGF and TIMP-1 as well as recurrent downregulation of JUN/FOS, DUSP1 and HBB. Assessment of PC from longitudinally collected samples revealed transcriptional changes after induction therapy. Our study, based on the largest number of PC analyzed by scRNA-seq, contributes to the understanding of destructive myeloma bone disease.

Osteoclast Gene Expression Profiling in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2735-2735
Author(s):  
Jerome Moreaux ◽  
Dirk Hose ◽  
Thierry Rème ◽  
Philippe Moine ◽  
Karène Mahtouk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Disease ◽  
Plasma Cells ◽  
Osteoclastic Bone Resorption ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

Abstract Multiple myeloma (MM) is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells (MMC) are found in close association with sites of active bone resorption, and the interactions between myeloma cells and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. In order to investigate the gene expression profile (GEP) of osteoclastic cells, we compare GEP of osteoclastic cells (7 samples) with normal B cells (7 samples), normal bone marrow plasma cells (7 samples), bone marrow stromal cells (5 samples), bone marrow CD3 cells (5 samples), CD14 cells (7 samples), CD15 cells (7 samples), CD34 cells (7 samples) and primary MMC (123 samples). Using SAM analysis, a set of 552 genes was overexpressed in osteoclasts compared to others cell subpopulations with a FDR ≤ 1% and a ratio ≥ 2. Osteoclasts specifically overexpressed genes coding for chemokines (CCL2, CCL7, CCL8, CCL13, CCL18, CXCL5 and CCL23) and MMC growth factors (IGF-1, APRIL and IL-10). Anti- IGF-1 receptor and TACI-Fc inhibit MMC growth induced by osteoclasts. Among the chemokines overexpressed by osteoclasts, the majority of them have a common receptor: CCR2 expressed by MMC. Anti-CCR2 MoAb inhibits migration of the CCR2+ HMCL in response to osteoclasts. Expression data of purified MMC were analyzed by supervised clustering of group with higher (CCR2high) versus lower (CCR2low) CCR2 expression level. Patients in the CCR2high group are characterized by a higher bone disease. A set of 176 genes was differentially expressed between CCR2high and CCR2low MMC. CCR2high displayed a gene signature linked to the dependency of MMC on the interactions with the BM osteoclastic subpopulation and the osteoclastic bone resorption. Taken together, our findings suggest addition of chemokine antagonists to current treatment regimens for MM should result in better therapeutic responses because of the loss of both the protective effect of the bone marrow environment on the MMC and the osteoclastic cells activity.

scholarly journals CST6 Is a Small Autocrine Molecule That Targets Myeloma Growth and Bone Destruction

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Dongzheng Gai ◽  
Stewart JP ◽  
Xuxing Shen ◽  
Jin-Ran Chen ◽  
Can Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Growth ◽  
Bone Resorption ◽  
Bone Formation ◽  
Bone Disease ◽  
Bone Destruction ◽  
Cathepsin K ◽  
Bone Marrow Biopsies ◽  
Lysosomal Protease

Bone destruction is a major complication of multiple myeloma (MM). Healthy bone is constantly remodeled through bone resorption by osteoclasts and bone formation by osteoblasts. New bone formation in MM is virtually non-existent, because differentiation of osteoblasts is inhibited by DKK1, a Wnt-β-catenin signaling inhibitor secreted by MM cells, reported by our group in NEJM, 2003. MM in its early stages is totally dependent on its microenvironment and for the hyperdiploid type MM this dependence is perpetual. Based on concordant gene expression signatures, predominantly driven by recurrent translocations and hyperdiploidy, we have classified MM into 7 distinct molecular entities. One subgroup, with significantly less bone disease and superior event-free and overall survival following high-dose therapy and stem cell transplantation than the other subgroups, defined as the Low Bone (LB) disease subgroup. Consistent with the LB phenotype, we have observed a strong inverse correlation between DKK1 and CST6 expression and by analyzing gene expression profiling (GEP) and RNA-sequencing data of more than 1,000 myeloma patients, we identified CST6 as the most upregulated gene in the LB subgroup. The aim of the present study was to determine the role of Cystatin E/M (CST6) in MM biology and to apply this knowledge to prevent both bone disease and MM cell growth. CST6, a 14-17 kD secretory protein, is a lysosomal protease inhibitor and suggested tumor suppressor gene. We showed that overexpression of CST6 in human MM cell lines prevents MM cell growth in vitro and in vivo. Also, purified CST6 protein from conditioned media of CST6-overexpressing MM cells significantly inhibits MM cell growth (p<0.01) and RANKL-induced osteoclast differentiation (p<0.01), decreases MM cell-induced bone destruction (p<0.05), and extends MM mouse survival (p<0.01). Mechanistic studies indicate that CST6 abrogates the alternative NF-kB signaling pathway as evidenced by a decrease in nuclear p52 protein in CST6-treated osteoclast precursors. Cathepsin K (CTSK), an osteoclast specific cysteine protease involved in bone resorption, was inhibited by CST6. GEP studies of whole bone marrow biopsies (WBMBx) across a spectrum of samples show higher expression of CTSK in WBMBx relative to purified plasma cells, while levels in MM remission WBMBx were higher than seen in healthy adult donors, MGUS/SMM, and newly diagnosed MM. Importantly, CTSK levels where not significantly different between remission and relapsed MM WBBx. These data show that Cathepsin K levels, and therefore osteoclasts, are elevated in the bone marrow of MM in remission and that these levels are similar to that seen in relapsed MM. Based on GEP data and experimental confirmation, we conclude that CST6, secreted by MM cells could be used clinically to target MM cells and prevent bone damage in MM. Inhibiting CTSK by CST6 in MM remission may aid in the prevention of MM relapse. Disclosures van Rhee: CDCN: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotech: Consultancy; EUSA: Consultancy.

scholarly journals Development of an in vivo model of human multiple myeloma bone disease

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
M Alsina ◽  
B Boyce ◽  
RD Devlin ◽  
JL Anderson ◽  
F Craig ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Disease ◽  
Bone Destruction ◽  
Long Bone ◽  
X Rays ◽  
Myeloma Bone Disease ◽  
Human Multiple Myeloma

Osteolytic bone destruction and its complications, bone pain, pathologic fractures, and hypercalcemia, are a major source of morbidity and mortality in patients with multiple myeloma. The bone destruction in multiple myeloma is due to increased osteoclast (OCL) activity and decreased bone formation in areas of bone adjacent to myeloma cells. The mechanisms underlying osteolysis in multiple myeloma in vivo are unclear. We used a human plasma cell leukemia cell line, ARH-77, that has disseminated growth in mice with severe combined immunodeficiency (SCID) and expresses IgG kappa, as a model for human multiple myeloma, SCID mice were irradiated with 400 rads and mice were injected either with 10(6) ARH-77 cells intravenously (ARH-77 mice) or vehicle 24 hours after irradiation. Development of bone disease was assessed by blood ionized calcium levels, x-rays, and histology. All ARH-77, but none of control mice that survived irradiation, developed hind limb paralysis 28 to 35 days after injection and developed hypercalcemia (1.35 to 1.46 mmol/L) a mean of 5 days after becoming paraplegic. Lytic bone lesions were detected using x-rays in all the hypercalcemic mice examined. No lytic lesions or hypercalcemia developed in the controls. Controls or ARH-77 mice, after developing hypercalcemia, were then killed and bone marrow plasma from the long bones were obtained, concentrated, and assayed for bone-resorbing activity. Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal rat long bone resorption assay when compared with controls (percentage of total 45Ca released = 35% +/- 4% v 11% +/- 1%). Histologic examination of tissues from the ARH-77 mice showed infiltration of myeloma cells in the liver and spleen and marked infiltration in vertebrae and long bones, with loss of bony trabeculae and increased OCL numbers. Interestingly, cultures of ARH-77 mouse bone marrow for early OCL precursors (colony-forming unit-granulocyte- macrophage [CFU-GM]) showed a threefold increase in CFU-GM from ARH-77 marrow versus controls (185 +/- 32 v 40 +/- 3 per 2 x 10(5) cell plated). Bone-resorbing human and murine cytokines such as interleukin- 6 (IL-6), IL-1 alpha or beta, TGF-alpha, lymphotoxin, and TNF alpha were not significantly increased in ARH-77 mouse sera or marrow plasma, compared with control mice, although ARH-77 cells produce IL-6 and lymphotoxin in vitro. Conditioned media from ARH-77 cells induced significant bone resorption in the fetal rat long bone resorption assay when compared with untreated media (percentage of total 45Ca released = 22% +/- 2% v 11% +/- 1%). This effect was not blocked by anti-IL-6 or antilymphotoxin (percentage of total 45Ca released = 19% +/- 1% and 22% +/- 1%, respectively). Thus, we have developed a model of human multiple myeloma bone disease that should be very useful to dissect the pathogenesis of the bone destruction in multiple myeloma.

Heparanase Expression Correlates with Bone Destruction in the Patients with Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1813-1813 ◽  
Author(s):  
Shi Wei ◽  
Racquel Innis-Shelton ◽  
Li Nan ◽  
Jian Ruan ◽  
Rebecca S Sollie ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Bone Marrow Cells ◽  
Bone Destruction ◽  
Bone Lesions ◽  
Bone Marrow Biopsies ◽  
Myeloma Cells ◽  
Lytic Bone Lesions ◽  
Marrow Cells

Abstract Abstract 1813 Multiple myeloma is an incurable malignancy, and excessive bone destruction is a major cause of morbidity in myeloma patients. However, the biologic mechanisms involved in the pathogenesis of myeloma-induced bone disease are poorly understood. Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in a variety of human tumors, including myeloma. In the present study, bone marrow biopsies from 40 myeloma patients were stained with antibodies raised against heparanase, RANKL (an osteoclastogenic cytokine), OPG (a decoy receptor for RANKL), TRAP (a marker of osteoclastogenesis) and osteocalcin (a marker of osteoblastogenesis). The radiologic studies for bone lesions of these patients were also recorded. We analyzed the correlations between heparanase expression in bone marrow myeloma cells with (1) the numbers of TRAP positive osteoclasts, (2) RANKL and OPG expression in myeloma cells and osteoblastic cells, (3) the numbers of osteocalcin positive osteoblasts in bone marrow, and (4) the presence/absence of lytic bone lesions. We found a positive correlation between heparanase expression and RANKL expression as well as the numbers of TRAP positive osteoclasts in myeloma and bone marrow cells, but no correlation was found between the expressions of heparanase and OPG in bone marrow cells (myeloma cells do not express OPG). In contrast, heparanase expression was negatively correlated with the numbers of osteocalcin positive osteoblasts. Taken together, these data suggest that heparanase expression by myeloma cells promotes osteoclastogenesis and at same time inhibits osteoblastogenesis. Clinical data show that 92% of patients with high level of heparanase had one or more lytic bone lesions, while only 63% of patients with median∼ low levels of heparanase had bone lesions (p<0.0001). In summary, enhanced heparanase expression in myeloma cells promotes bone resorption and inhibits bone formation; these events contribute to the uncontrolled bone destruction that is characteristic of myeloma. These data provide novel insight into the mechanisms driving myeloma bone disease and suggest that heparanase inhibitors are valid therapeutic targets for the treatment of multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

Brain-Derived Neurotrophic Factor Enhances Osteoclastogenesis in Multiple Myeloma via Upregulation of RANKL/OPG Ratio Both in Vitro and in a Murine Model of Myeloma Bone Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 568-568
Author(s):  
Li-Sha Ai ◽  
Chun-Yan Sun ◽  
Tao Guo ◽  
Ya-Dan Wang ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Neurotrophic Factor ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Myeloma Bone Disease ◽  
Trap Staining

Abstract Abstract 568 Osteolytic bone disease is a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption uncoupled with osteoblastic bone formation. Myeloma-induced osteoclastogenesis is largely depending on the increase of receptor activator of NF-κB ligand (RANKL) and decrease of osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as an MM-derived factor correlated with increased RANKL level and contributed to myeloma bone destruction. On the other hand, tyrosine receptor kinase B (TrkB), the receptor of BDNF, was found to be abundantly expressed by osteoblasts (OBs). Since OBs are the main source of RANKL and OPG in bone, here we sought to evaluate the involvement of BDNF/TrkB in the crosstalk between myeloma cells and OBs, as well as the effects of BDNF on RANKL/OPG ratio and myeloma bone disease. Co-cultures of OBs with pre-osteoclasts were performed in a non-contacted transwell system and treated with various concentration of BDNF. Osteoclast formation was detected with a tartrate-resistant acid phosphatase (TRAP) staining kit. Then, RANKL and OPG levels were measured when OBs cultures were exposed to BDNF or co-cultured with three human myeloma cell lines (RPMI8226, ARH-77 and U266). K252a (an inhibitor of TrkB) was present or absent in these systems to assess the effects of BDNF on RANKL/OPG expression in OBs. The involvement of downstream signaling molecules activated by BDNF in OBs was also investigated in this study, with the use of U0126 and a specific small interfering RNA (siRNA) for TrkB. For in vivo study, ARH-77 cells were stably transfected with an antisense short-hairpin RNA construct to BDNF (AS-ARH) or empty vector (EV-ARH). These cells were then intravenously injected to severe combined immunodeficiency (SCID) mice, to test their capacity to induce MM bone disease. Radiographs of mice tibiae and vertebrae were taken weekly by X ray. Changes in total body bone mineral density (BMD) of mice skeleton were recorded. At the end of the experiment, bone sections were stained with hematoxylin and eosin staining or TRAP staining. Secretion levels of RANKL and OPG in mice bone marrow were measured by ELISA. We showed that BDNF increased RANKL and decreased OPG production in OBs in a time- and dose-dependent manner, thus contributing to osteoclast formation in vitro. In addition, these effects were completely abolished by K252a and TrkB-siRNA (P < 0.05). BDNF regulates RANKL/OPG expression in OBs through the TrkB/ERK signaling pathway. Our in vivo results indicated that mice injected with AS-ARH cells, which expressed low levels of endogenous BDNF, were preserved and exhibited no radiologically identifiable osteolytic lesions. In addition, mice in AS-ARH group also had a lower incidence of vertebral compression deformities and paralysis in comparison with mice in EV-ARH group (P < 0.05). Further more, bones harboring AS-ARH cells showed marked reduction of RANKL/OPG ratio and osteoclast density when compared to the controls harboring EV-ARH cells (P < 0.05). Our results demonstrate that BDNF is an important contributor to osteoclastogenesis in MM. Antisense inhibition of BDNF in MM cells remarkably inhibited osteolytic bone destruction in SCID-ARH mice model. BDNF-induced bone destruction is partially mediated by MM-OB interactions via upregulation of RANKL/OPG ratio in the bone marrow milieu. These findings suggest targeting BDNF may become a new therapeutic strategy to improve patient outcome in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients

2021 ◽  
Vol 12 ◽  
Author(s):  
Duojiao Chen ◽  
Mohammad I. Abu Zaid ◽  
Jill L. Reiter ◽  
Magdalena Czader ◽  
Lin Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Single Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Type ◽  
Myeloma Cells ◽  
Freeze Thaw ◽  
Single Cell Rna Sequencing

Single-cell RNA sequencing reveals gene expression differences between individual cells and also identifies different cell populations that are present in the bulk starting material. To obtain an accurate assessment of patient samples, single-cell suspensions need to be generated as soon as possible once the tissue or sample has been collected. However, this requirement poses logistical challenges for experimental designs involving multiple samples from the same subject since these samples would ideally be processed at the same time to minimize technical variation in data analysis. Although cryopreservation has been shown to largely preserve the transcriptome, it is unclear whether the freeze-thaw process might alter gene expression profiles in a cell-type specific manner or whether changes in cell-type proportions might also occur. To address these questions in the context of multiple myeloma clinical studies, we performed single-cell RNA sequencing (scRNA-seq) to compare fresh and frozen cells isolated from bone marrow aspirates of six multiple myeloma patients, analyzing both myeloma cells (CD138+) and cells constituting the microenvironment (CD138−). We found that cryopreservation using 90% fetal calf serum and 10% dimethyl sulfoxide resulted in highly consistent gene expression profiles when comparing fresh and frozen samples from the same patient for both CD138+ myeloma cells (R ≥ 0.96) and for CD138– cells (R ≥ 0.9). We also demonstrate that CD138– cell-type proportions showed minimal alterations, which were mainly related to small differences in immune cell subtype sensitivity to the freeze-thaw procedures. Therefore, when processing fresh multiple myeloma samples is not feasible, cryopreservation is a useful option in single-cell profiling studies.

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