Marrow Stroma Research Articles

PDGF-BB Deficiency in the Blood Serum from Aplastic Anemia Patients Affects Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells

Aplastic anemia (AA) is characterized by bone marrow (BM) aplasia and pancytopenia. BM stromal microenvironment is closely intertwined with hematopoietic cells by reciprocal regulation. It is still unclear how hematopoietic deficiency affects the bone marrow stroma of the AA patients. Multipotent mesenchymal stromal cells (MMSCs) are the progenitors of stromal cells. In vitro, proliferation rate of MMSCs of AA patients is decreased compared to those of healthy donors. This may be explained by the influence of pathological environmental condition in the patients’ BM. The aim of the study was to compare the effect of AA patients’ sera on healthy donor MMSCs to healthy donors’ sera and to elucidate the nature of their difference. Proliferation test showed 3-fold decrease in number of MMSCs after incubation in medium supplemented with AA patients’ sera compared to donors’ serum samples. The degree of this effect correlated with the severity of thrombocytopenia in patients. The decrease in cell number was not associated with cell death, as the number of apoptotic cells defined by flow cytometry did not differ between the groups. ELISA revealed a decreased level of PDGF-BB in the patients’ sera compared to donors’ serum samples (69 ± 5 pg/mL vs 112 ± 21 pg/mL, respectively). The addition of recombinant PDGF-BB or healthy donor’s platelet lysate to the culture medium supplemented with AA patients’ serum restored its ability to support MMSCs growth. Thus, PDGF-BB deficiency is one of the environmental factors causing MMSCs damage in AA patients.

Skeletal stem and progenitor cells in bone physiology, ageing and disease.

Skeletal stem cells (SSCs) and related progenitors with osteogenic potential, collectively termed skeletal stem and/or progenitor cells (SSPCs), are crucial for providing osteoblasts for bone formation during homeostatic tissue turnover and fracturerepair. Besides mediating normal bone physiology, they also have important roles in various metabolic bone diseases, including osteoporosis. SSPCs are of tremendous interest because they represent prime future targets for osteoanabolic therapies and bone regenerative medicine. Remarkable progress has been made in characterizing various SSC and SSPC populations in postnatal bone. SSPCs exist in the periosteum and within the bone marrow stroma, including subsets localizing around arteriolar and sinusoidal blood vessels; they can display osteogenic, chondrogenic, adipogenic and/or fibroblastic potential, and exert critical haematopoiesis-supportive functions. However, much remains to be clarified. By the current markers, bona fide SSCs are commonly contained within broader SSPC populations characterized by considerable heterogeneity and overlap, whose common versus specific functions in health and disease have not been fully unravelled. Here, we review the present knowledge of the identity, fates and relationships of SSPC populations in the postnatal bone environment, their contributions to bone maintenance, the changes observed upon ageing, and the effect of metabolic diseases such as osteoporosis and diabetes mellitus.

Cell-based therapy in the treatment of musculoskeletal diseases.

A limited number of tissues can spontaneously regenerate following injury, and even fewer can regenerate to a state comparable to mature, healthy adult tissue. Mesenchymal stem cells (MSCs) were first described in the 1960s-1970s by Friedenstein et al as a small population of bone marrow cells with osteogenic potential and abilities to differentiate into chondrocytes. In 1991, Arnold Caplan coined the term "mesenchymal cells" after identifying these cells as a theoretical precursor to bone, cartilage, tendon, ligament, marrow stroma, adipocyte, dermis, muscle, and connective tissues. MSCs are derived from periosteum, fat, and muscle. Another attractive property of MSCs is their immunoregulatory and regenerative properties, which result from crosstalk with their microenvironment and components of the innate immune system. Collectively, these properties make MSCs potentially attractive for various therapeutic purposes. MSCs offer potential in sports medicine, aiding in muscle recovery, meniscal tears, and tendon and ligament injuries. In joint disease, MSCs have the potential for chondrogenesis and reversing the effects of osteoarthritis. MSCs have also demonstrated potential application to the treatment of degenerative disc disease of the cervical, thoracic, and lumbar spine.

Dual therapeutic targeting of MYC and JUNB transcriptional programs for enhanced anti-myeloma activity

Deregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes.

IL-33-dependent NF-κB activation inhibits apoptosis and drives chemoresistance in acute myeloid leukemia

BackgroundDespite recent advances in therapeutic regimens, the prognosis of acute myeloid leukemia (AML) remains poor. Following our previous finding that interleukin-33 (IL-33) promotes cell survival along with activated NF-κB in AML, we further investigated the role of NF-κB during leukemia development. MethodsFlow cytometry was performed to value the apoptosis and proliferation. qRT-PCR and western blot were performed to detect the expression of IL-6, active caspase 3, BIRC2, Bcl-2, and Bax, as well as activated NF-κB p65 and AKT. Finally, xenograft mouse models and AML patient samples were used to verify the findings observed in AML cell lines. ResultsIL-33-mediated NF-κB activation in AML cell lines contributes to a reduction in apoptosis, an increase in proliferation rate as well as a decrease in drug sensitivity, which were reversed by NF-κB inhibitor, Bay-117085. Moreover, IL-33 decreased the expression of active caspase-3 while increasing the levels of BIRC2, Bcl-2, and Bax, and these effects were blocked by Bay-117085. Additionally, NF-κB activation induced by IL-33 increases the production of IL-6 and autocrine activation of AKT. Co-culture of bone marrow stroma with AML cells resulted in increased IL-33 expression by leukemia cells, along with decreased apoptosis level and reduced drug sensitivity. Finally, we confirmed the in vivo pro-tumor effect mediated by IL-33/ NF-κB axis using a xenograft model of AML. ConclusionOur data indicate that IL-33/IL1RL1-dependent signaling contributes to AML cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pAKT, supporting IL-33/NF-κB/pAKT as a potential target for AML therapy.

ISOLATION OF BONE MARROW MESENCHYMAL STEM CELLS EMBEDDED IN NATIVE TISSUE STROMA YIELDS ENRICHED HARVEST, IMPROVED ADHERENCE AND PROLIFERATION, AND UNIQUE SECRETOME

Background: The field of orthobiologics traditionally utilizes cellular products, including bone-marrow aspirate concentrate (BMAC), micronized adipose tissue, and platelet preparations to address pain from degenerative processes, orthopedic injuries and medical conditions characterized by chronic inflammation and tissue degradation. For BMAC, maximizing the concentration of mesenchymal stem cells (MSCs) in a reduced volume is thought to allow for the therapeutic delivery of the cellular concentrate, secretome, and extracellular vesicles to a site of orthopedic injury or surgical repair. The extracellular matrix (ECM) within the bone marrow stroma contains collagens and proteoglycans known to regulate cell proliferation, migration, differentiation, and cell-cell communication among resident bone marrow cells. This study aimed to evaluate the cellular effects on MSC health and function when harvested to retain their native tissue stroma. Methods: We evaluated a novel and unique processing method and device (BMAX™) to mechanically generate a purified MSC product derived from bone marrow in a nonenzymatic manner. BMAX™ products, including cells and stroma, were plated in MSC culture media and incubated for 3–14 days (P0-P1) before evaluation with flow cytometry for cell phenotyping and immunoassays for secretome profiling. Results: The orthobiologic product containing three-dimensional stromal components can be produced in minutes using an automated bedside device requiring minimal benchtop space. We found increased MSC adherence, improved proliferative density in culture, and significantly elevated enrichment of stromal-derived MSCs versus traditional BMAC centrifugation-based preparations. Further, we demonstrate a unique secretome profile in BMAX™ versus traditional BMAC centrifugation-based preparations. Conclusions: These qualities provide a novel and unique platform for autologous and allogeneic bone-marrow-derived therapy to better address inflammatory and destructive processes that may improve bone-marrow-derived cell therapies’ efficacy.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions

BackgroundDespite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets.MethodsWe collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis.ResultsThe bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption.ConclusionsThese results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

Role of the mesenchymal stromal cells in bone marrow failure of Fanconi Anemia patients.

Fanconi anemia (FA) is an inherited disorder characterized by bone marrow failure, congenital malformations, and predisposition to malignancies. Alterations in hematopoietic stem cells (HSC) have been reported, but little is known regarding the bone marrow (BM) stroma. Thus, the characterization of Mesenchymal Stromal Cells (MSC) would help to elucidate their involvement in the BM failure. We characterized MSCs of 28 FA patients (FA-MSC) before and after treatment (hematopoietic stem cell transplantation, HSCT; or gene therapy, GT). Phenotypic and functional properties were analyzed and compared with MSCs expanded from 26 healthy donors (HD-MSCs). FA-MSCs were genetically characterized through, mitomycin C-test and chimerism analysis. Furthermore, RNA-seq profiling was used to identify dysregulated metabolic pathways. Overall, FA-MSC had the same phenotypic and functional characteristics as HD-MSC. Of note, MSC-GT had a lower clonogenic efficiency. These findings were not confirmed in the whole FA patients' cohort. Transcriptomic profiling identified dysregulation in HSC self-maintenance pathways in FA-MSC (HOX), and was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR). Our study provides a comprehensive characterization of FA-MSCs, including for the first time MSC-GT and constitutes the largest series published to date. Interestingly, transcript profiling revealed dysregulation of metabolic pathways related to HSC self-maintenance. Taken together, our results or findings provide new insights into the pathophysiology of the disease, although whether these niche defects are involved in the hematopoietic defects seen of FA deserves further investigation.

A gravity-driven tissue chip to study the efficacy and toxicity of cancer therapeutics.

Tissue chip and organs-on-chip technologies have emerged as promising tools in preclinical studies. In oncology, this is driven by the high failure rates of candidate drugs in clinical trials mainly due to inadequate efficacy or intolerable toxicity and the need for better predictive preclinical models than those traditionally used. However, the intricate design, fabrication, operation, and limited compatibility with automation limit the utility of tissue chips. To tackle these issues, we designed a novel 32-unit tissue chip in the format of standard 96-well plates to streamline automation, fabricated it using 3D printing, and leveraged gravity-driven flow to bypass the need for external flow devices. Each unit includes three interconnected tissue compartments that model liver, tumor, and bone marrow stroma. The focus on liver and bone marrow stroma was due to their respective roles in drug metabolism and disturbances to the bone marrow niche from off-target toxicity of chemotherapies. We analyzed flow patterns, mixing, and oxygen transport among and within the compartments through finite element simulations and demonstrated the utility of the tissue chip to study the efficacy of commonly-used cytotoxic cancer drugs against tumor cells and their toxicity toward liver and bone marrow cells. The ability to simultaneously study drug efficacy and toxicity in high throughput can help select promising therapeutics in early stages of drug discovery in preclinical studies.

Bone Marrow Stroma Co-cultivation Model of Breast Cancer Dormancy.

Breast cancer cells metastasize to the bone marrow before a primary tumor is detected. Most micrometastases die in this hostile microenvironment, but some survive and enter a state of dormancy and chemoresistance due to their close interaction with cells in the bone marrow hematopoietic niche. Over many years, some of the cells reawaken and result in metastatic disease that cannot be cured. Analyzing the cellular and molecular interactions between cancer and bone marrow niche cells requires relevant models that can be manipulated and studied. Generation of bone marrow stromacultures in vitro permits the formation of cellular monolayers upon which breast cancer cells can be co-cultivated and their behavior interrogated using a variety of techniques. This manuscript describes the basic techniques for generating bone marrow stromal monolayers, co-incubating cancer cells and determining the effects on cancer cell proliferation and molecular signaling.

Targeting CDK9 in KMT2A-Rearranged Infant Leukemia: Evidence for Activity and Drug Synergy with Enitociclib

Introduction: Translocations of the lysine methyltransferase 2A (KMT2A) gene on 11q23 are seen in most infant leukemias. The outcomes in cases with KMT2A rearrangements (KMT2Ar) are significantly worse due to higher disease relapse rates and the emergence of chemo-resistance. Studies have shown that P-TEFb (CDK9/Cyclin T1) is an important component of the KMT2Ar complex and drives the transcriptional elongation step, subsequently contributing to the development of leukemia (Mueller, Dorothee et al.,2009). Based on this rationale, we investigated the potential of the selective P-TEFb/CDK9 inhibitor enitociclib in infant leukemia. Enitociclib potently inhibits MYC and MCL-1 in various tumor models (Sher, Steven et al.,2023). In addition, menin has been shown to be an important protein in the activation of target genes by KMT2Ar fusions, and menin inhibitors are considered promising therapeutic agents against infant leukemias, with at least four menin inhibitors currently in clinical development. We show preclinical data to support the activity of enitociclib with respect to cellular cytotoxicity, apoptosis induction and drug synergy in infant leukemia cells. Methods: A panel of pediatric leukemia cell lines representing high-risk and treatment resistant KMT2Ar cells were used. Leukemia cells with wild-type KMT2A as well as normal CD34+ cells and PBMCs from healthy donors were used as controls. Leukemic cells were treated with enitociclib (0.5µM - 0.03µM) and cell viability was measured using alamar blue assay. Apoptotic cell death following enitociclib treatment was measured by Annexin V/Propidium Iodide flow cytometry. Target modulation analyses were carried out by western blotting. Bone marrow (BM) stroma and leukemia co-culture experiments were performed to identify the effect of enitociclib on BM-derived growth support provided to leukemic cells. Drug combination assays were carried out with the menin inhibitor MI-463 and combination indices were calculated using the Chao-Talalay method. Rat xenograft models bearing infant leukemia cells were used to study the efficacy and tolerability of enitociclib therapy. Results: Data from in vitro cytotoxicity assays showed high sensitivity of KMT2Ar infant leukemia cells to enitociclib with IC 50 values of 30nM for KMT2Ar cells and 406nM for KMT2A-WT cells. Annexin V/PI staining showed a dose-dependent increase in cells in the late apoptotic stage for KMT2Ar cells after treatment. Target validation assays in KMT2Ar cells showed inhibition of transcriptional elongation by decreased serine phosphorylation by approximately 75% of RNA POL II CTD starting as early as 6 hours after treatment with 0.1µM enitociclib. Additionally, in KMT2Ar cells, an 80%-85% reduction in the amount of MCL-1 and MYC protein levels was observed after treatment with 0.1µM enitociclib for 6 hours. Furthermore, our data show that enitociclib may help to overcome the BM derived stromal mediated survival advantage in KMT2Ar leukemic cells. Enitociclib also potentiated the anti-leukemic effect of chemotherapies for childhood leukemia with the most effective combination observed with doxorubicin (0.01µM IC 50 for doxorubicin alone, 0.002µM IC 50 when combined with enitociclib) and prednisolone (25µM IC 50 for prednisolone only, 0.01µM IC 50 in combination). Drug combination assays showed effective synergy between enitociclib and the KMT2A-menin inhibitor MI-463 in KMT2Ar infant leukemia cells with a combination index of less than 1. As a detectable biomarker of activity, HOXA9 protein levels were found to be robustly diminished with 60% decrease after 24-hour treatment with the drug combination. In vivo, enitociclib given once weekly for 3 weeks led to complete remission in 10 out of 12 rats bearing KMT2Ar xenografts. Discussion: Our data demonstrate the ability of enitociclib to induce effective cellular cytotoxicity in high-risk KMT2Ar leukemia cells. We further show that enitociclib potentiated the anti-leukemic effect of chemotherapies that constitute the backbone of childhood leukemia protocols. Importantly, we show synergy with menin inhibition that is currently in consideration to be included in new generation clinical trials, suggesting the effective use of this novel combination in future treatment protocols. We provide the first in vitro and animal data supporting enitociclib in future treatment approaches for infants with KMT2Ar leukemia.

Hematopoietic Stem Cell Numbers Are Regulated at Both Systemic and Local Levels

Since Schofield proposed the niche model in the 1970s, it has been widely believed that the number of hematopoietic stem cells (HSCs) is determined by the niche size. We and others have identified several components of the HSC niche, including Nestin-GFP + mesenchymal stem cells (MSCs). However, the number of defined niche cells vastly exceeds that of HSCs, raising the question about the actual regulatory mechanisms governing HSC numbers. To address this question, we initially established a mathematical model of niche availability and occupancy. The model predicted that an increase in available niches initially leads to a corresponding rise in the number of occupying HSCs, which aligns with the concept of local, niche availability-driven restriction on HSC numbers. Interestingly, our model also suggested that this increase in HSC numbers saturates when the number of available niches reaches a critical level. One potential mechanism for this saturation is that HSC numbers may also be regulated at the systemic level, with total HSC numbers in the body being restricted. To experimentally test this model and hypothesis, we developed a bone transplantation technique that allows us to increase the number of available HSC niches. When a single femur (referred to as a graft) was transplanted per mouse (called a host), we observed the progressive regeneration of bone marrow (BM) stroma and hematopoietic recovery in the grafts. While the number of HSCs (Lin -Sca-1 +c-Kit +CD150 +CD48 -CD34 -) in the grafts was still 41.7 ± 7.60% of that in the host femurs at 5 months after transplantation (n = 8, P = 0.0002), they were comparable at 3 months after transplantation when granulocyte colony-stimulating factor (G-CSF) was administered to hosts at 1 month after transplantation (n = 8, P = 0.660). By utilizing Nestin-GFP transgenic mice and a CD45 congenic system, we found that MSCs in grafts are derived from the graft, while hematopoietic cells are derived from the host. Given that a single femur harbors only 8.3 ± 0.440% of HSCs in the total body (n = 11), 6 femurs were transplanted per mouse, followed by G-CSF administration, to assess the impact of increased HSC niches on total HSC numbers. Strikingly, HSC numbers per host femur and graft femur were lower than those per femur from sham-operated mice (n = 7-48, P = 0.012). Moreover, we found that the sum of HSC numbers in the bone transplantation host and graft bones was equivalent to that in the sham-operated group (n = 7-8, P = 0.364). Total HSC numbers were also maintained when 6 femurs from wild-type (WT) mice were transplanted into Cdh2 (N-Cadherin)-CreER; Cxcl12fl/fl mice, which have an impaired ability to retain HSCs in the BM (n = 8, P = 0.711). These results indicate that the addition of normal niches does not alter total HSC numbers in the body, regardless of whether the endogenous niche is functional, supporting the conclusion that HSC numbers are indeed restricted at the systemic level (Figure 1A). To determine whether HSC numbers are also limited at the local level, as suggested by our mathematical model, we transplanted a single WT femur to Cxcl12fl/fl or Cdh2-CreER; Cxcl12fl/fl mice. We hypothesized that implanting only one femur would have a minimal impact on the total BM, and therefore HSC numbers in the grafts transplanted to Cxcl12fl/fl mice would reach the physiological level. We reasoned that if there is no local restriction on HSC numbers, there would be more HSCs in the WT grafts transplanted to Cdh2-CreER; Cxcl12fl/fl mice compared with those implanted to Cxcl12fl/fl mice. As expected, HSC numbers per WT graft femur in Cxcl12fl/fl mice were comparable to those per femur of sham-operated Cxcl12fl/fl mice (corresponding to the physiological level) (n = 8, P = 0.433). However, CXCL12 deficiency in the host BM did not affect HSC numbers per WT graft femur (n = 8, P = 0.986). Importantly, the sum of HSC numbers in the host body and the graft femur of Cdh2-CreER; Cxcl12fl/fl recipient mice was lower than that of sham-operated Cxcl12fl/fl mice (n = 8, P = 0.003), indicating that total HSC numbers in the former mice are below the systemic limit. Collectively, these data suggest that the failure to increase HSC numbers per WT graft in Cdh2-CreER; Cxcl12fl/fl hosts is, at least in part, due to a local restriction on HSC numbers (Figure 1B). Our study demonstrates that HSC numbers are restricted at both systemic and local levels, thereby rewriting the long-standing model for the regulation of HSC numbers.

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

P-001 The bone marrow stroma influences extrinsic apoptotic signaling and results in resistance to BCMA CAR-T cell induced cell death

P-001 The bone marrow stroma influences extrinsic apoptotic signaling and results in resistance to BCMA CAR-T cell induced cell death

Germ line variants in patients with acute myeloid leukemia without a suspicion of hereditary hematologic malignancy syndrome

AbstractGerm line predisposition in acute myeloid leukemia (AML) has gained attention in recent years because of a nonnegligible frequency and an impact on management of patients and their relatives. Risk alleles for AML development may be present in patients without a clinical suspicion of hereditary hematologic malignancy syndrome. In this study we investigated the presence of germ line variants (GVs) in 288 genes related to cancer predisposition in 47 patients with available paired, tumor-normal material, namely bone marrow stroma cells (n = 29), postremission bone marrow (n = 17), and saliva (n = 1). These patients correspond to 2 broad AML categories with heterogeneous genetic background (AML myelodysplasia related and AML defined by differentiation) and none of them had phenotypic abnormalities, previous history of cytopenia, or strong cancer aggregation. We found 11 pathogenic or likely pathogenic variants, 6 affecting genes related to autosomal dominant cancer predisposition syndromes (ATM, DDX41, and CHEK2) and 5 related to autosomal recessive bone marrow failure syndromes (FANCA, FANCM, SBDS, DNAJC21, and CSF3R). We did not find differences in clinical characteristics nor outcome between carriers of GVs vs noncarriers. Further studies in unselected AML cohorts are needed to determine GV incidence and penetrance and, in particular, to clarify the role of ATM nonsense mutations in AML predisposition.

The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression

AbstractMyelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.

The Effect of Lanthanum (III) Nitrate on the Osteogenic Differentiation of Mice Bone Marrow Stromal Cells.

To study the species of lanthanum (III) nitrate (La[NO3]3) dispersed in cell media and the effect on the osteoblast differentiation of bone marrow stroma cells (BMSCs). Different La-containing precipitations were obtained by adding various concentrations of La(NO3)3 solutions to Dulbecco's modified Eagle medium (DMEM) or DMEM with fetal bovine serum (FBS). A series of characterisation methods, including dynamic light scattering, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and protein quantification were employed to clarify the species of the different La-containing precipitations. The primary BMSCs were isolated, and the cell viability, alkaline phosphatase activity, and the formation of a mineralised nodule of BMSCs were tested when treated with different La-containing precipitations. The La(NO3)3 solutions in DMEM could form LaPO4, which exits in the particle formation, while the La(NO3)3 solutions in DMEM with FBS could form a La-PO4-protein compound. When treated with La(NO3)3 solutions in DMEM, the cell viability of the BMSCs was inhibited at the concentrations of 1, 10, and 100μM at 1day and 3days. Meanwhile, the supernatant derived from the La(NO3)3 solutions in DMEM did not affect the cell viability of the BMSCs. In addition, the precipitate derived from the La(NO3)3 solutions in DMEM added to the complete medium inhibited the cell viability of the BMSCs at concentrations of 10μM and 100μM. When treated with La(NO3)3 solutions in DMEM with FBS, the derived precipitate and supernatant did not affect the cell viability of the BMSCs, except for the concentration of 100μM La(NO3)3. The La-PO4-protein formed from the La(NO3)3 solutions in DMEM with FBS inhibited the osteoblast differentiation of BMSCs at the concentration of 1μM La(NO3)3 (P < 0.05) but had no effect on either the osteoblast differentiation at the concentrations of 0.001 and 0.1μM or on the formation of a mineralised nodule at all tested concentrations of La(NO3)3. Overall, La(NO3)3 solutions in different cell culture media could form different La-containing compounds: La-PO4 particles (in DMEM) and a La-PO4-protein compound (in DMEM with FBS). The different La-containing compounds caused different effects on the cell viability, osteoblast differentiation, and the formation of a mineralised nodule of the BMSCs. The La-containing precipitation inhibited the osteoblast differentiation by inhibiting the expression of osteoblast-related genes and proteins, providing a theoretical basis for clinical doctors to apply phosphorus-lowering drugs such as lanthanum carbon.

Matrix stiffness controls megakaryocyte adhesion, fibronectin fibrillogenesis, and proplatelet formation through Itgβ3

AbstractMegakaryocytes (MKs) are the precursor cells of platelets, located in the bone marrow (BM). Once mature, they extend elongated projections named proplatelets through sinusoid vessels, emerging from the marrow stroma into the circulating blood. Not all signals from the microenvironment that regulate proplatelet formation are understood, particularly those from the BM biomechanics. We sought to investigate how MKs perceive and adapt to modifications of the stiffness of their environment. Although the BM is one of the softest tissue of the body, its rigidification results from excess fibronectin (FN), and other matrix protein deposition occur upon myelofibrosis. Here, we have shown that mouse MKs are able to detect the stiffness of a FN-coated substrate and adapt their morphology accordingly. Using a polydimethylsiloxane substrate with stiffness varying from physiological to pathological marrow, we found that a stiff matrix favors spreading, intracellular contractility, and FN fibrils assembly at the expense of proplatelet formation. Itgb3, but not Itgb1, is required for stiffness sensing, whereas both integrins are involved in fibrils assembly. In contrast, soft substrates promote proplatelet formation in an Itgb3-dependent manner, consistent with the ex vivo decrease in proplatelet formation and the in vivo decrease in platelet number in Itgb3-deficient mice. Our findings demonstrate the importance of environmental stiffness for MK functions with potential pathophysiological implications during pathologies that deregulate FN deposition and modulate stiffness in the marrow.

Abstract 631: Single-cell analysis of immune and stroma cell remodeling in renal cell carcinoma primary tumors and bone metastatic lesions

Abstract Background: Renal cell carcinoma (RCC) is the most common renal tumor in adults, and among these clear cell RCC (ccRCC) is most common subtype and accounts for most renal cancer-related deaths. One-third of patients with advanced ccRCC present with bone metastases at the time of diagnosis. Despite therapeutic advances, once cancer has metastasized to the bone, it represents a highly morbid and ultimately lethal disease. Comprehensive genomic studies in ccRCC have provided important insights into the somatic alterations and intra-tumor heterogeneity. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined. Methods: We collected fresh patient samples directly from the operating room and performed single-cell transcriptomic profiling of human ccRCC tumors (ccRCC primary), solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow stroma cells from patients undergoing hip replacement surgery (Benign) as control. We provide a comparative single cell transcriptomic analysis between ccRCC primary tumors and bone metastatic lesions. Results: Comparison of malignant cells from primary and metastatic sites reveal a distinct transcriptional signature that is predictive of metastatic potential and patient survival. The bone marrow in metastatic patients has sustained immune-suppressive microenvironment, featuring increased, exhausted CD8+ cytotoxic T-cells, T-regulatory cells and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival, markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. Conclusions: These results provide insights into mechanisms underlying of ccRCC tumors progression leading bone metastasis. We also highlight potential communication channels and therefore therapeutic targets within both the immune and the tumor cell populations. Citation Format: Shenglin Mei, Adele M. Alchahin, Ioanna Tsea, Youmna Kfoury, Taghreed Taghreed, Hirak Sarkar, Shulin Wu, Alexander O. Subtelny, Yida Zhang, Keyan Salari, Chin-Lee Wu, Mark A. Randolph, David T. Scadden, Douglas Dahl, John Shin, Peter Kharchenko, Philip Saylor, David Sykes, Ninib Baryawno. Single-cell analysis of immune and stroma cell remodeling in renal cell carcinoma primary tumors and bone metastatic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 631.

Inhibition of lysyl oxidases synergizes with 5-azacytidine to restore erythropoiesis in myelodysplastic and myeloid malignancies

Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN.