Adipocytes and metabolism: Contributions to multiple myeloma.

Obesity contributes to many cancers, including breast cancer and multiple myeloma, two cancers that often colonize the bone marrow (BM). Obesity often causes metabolic disease, but at the cellular level, there is uncertainty regarding how these shifts affect cellular phenotypes. Evidence is building that different types of fuel affect tumor cell metabolism, mitochondrial function, and signaling pathways differently, but tumor cells are also flexible and adapt to less-than ideal metabolic conditions, suggesting that single-pronged attacks on tumor metabolism may not be efficacious enough to be effective clinically. In this review, we describe the newest research at the pre-clinical level on how tumor metabolic pathways and energy sources affect cancer cells, with a special focus on multiple myeloma (MM). We also describe the known forward-feedback loops between bone marrow adipocytes (BMAds) and local tumor cells that support tumor growth. We describe how metabolic targets and transcription factors related to fatty acid (FA) oxidation, FA biosynthesis, glycolysis, oxidative phosphorylation (OXPHOS), and other pathways hold great promise as new vulnerabilities in myeloma cells. Specifically, we describe the importance of the acetyl-CoA synthetase (ACSS) and the acyl-CoA synthetase long chain (ACSL) families, which are both involved in FA metabolism. We also describe new data on the importance of lactate metabolism and lactate transporters in supporting the growth of tumor cells in a hypoxic BM microenvironment. We highlight new data showing the dependency of myeloma cells on the mitochondrial pyruvate carrier (MPC), which transports pyruvate to the mitochondria to fuel the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), boosting OXPHOS. Inhibiting the MPC affects myeloma cell mitochondrial metabolism and growth, and synergizes with proteosome inhibitors in killing myeloma cells. We also describe how metabolic signaling pathways intersect established survival and proliferation pathways; for example, the fatty acid binding proteins (FABPs) affect MYC signaling and support growth, survival, and metabolism of myeloma cells. Our goal is to review the current the field so that novel, metabolic-focused therapeutic interventions and treatments can be imagined, developed and tested to decrease the burden of MM and related cancers.

Inhibition of Acyl-CoA Synthetase Long Chain Isozymes Decreases Multiple Myeloma Cell Proliferation and Causes Mitochondrial Dysfunction.

Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. The Cancer Dependency Map data suggested that ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show that inhibition of ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) causes apoptosis and decreases proliferation in a dose- and time-dependent manner. RNA-seq of MM.1S cells treated with TriC for 24 h showed a significant enrichment in apoptosis, ferroptosis, and ER stress. Proteomics of MM.1S cells treated with TriC for 48 h revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest, consistent with our observations of decreased mitochondrial membrane potential and increased mitochondrial superoxide levels. Interestingly, MM.1S cells treated with TriC for 24 h also showed decreased mitochondrial ATP production rates and overall lower cellular respiration.

Targeting the fatty acid binding proteins disrupts multiple myeloma cell cycle progression and MYC signaling.

Multiple myeloma is an incurable plasma cell malignancy with only a 53% 5-year survival rate. There is a critical need to find new multiple myeloma vulnerabilities and therapeutic avenues. Herein, we identified and explored a novel multiple myeloma target: the fatty acid binding protein (FABP) family. In our work, myeloma cells were treated with FABP inhibitors (BMS3094013 and SBFI-26) and examined in vivo and in vitro for cell cycle state, proliferation, apoptosis, mitochondrial membrane potential, cellular metabolism (oxygen consumption rates and fatty acid oxidation), and DNA methylation properties. Myeloma cell responses to BMS309403, SBFI-26, or both, were also assessed with RNA sequencing (RNA-Seq) and proteomic analysis, and confirmed with western blotting and qRT-PCR. Myeloma cell dependency on FABPs was assessed using the Cancer Dependency Map (DepMap). Finally, MM patient datasets (CoMMpass and GEO) were mined for FABP expression correlations with clinical outcomes. We found that myeloma cells treated with FABPi or with FABP5 knockout (generated via CRISPR/Cas9 editing) exhibited diminished proliferation, increased apoptosis, and metabolic changes in vitro. FABPi had mixed results in vivo, in two pre-clinical MM mouse models, suggesting optimization of in vivo delivery, dosing, or type of FABP inhibitors will be needed before clinical applicability. FABPi negatively impacted mitochondrial respiration and reduced expression of MYC and other key signaling pathways in MM cells in vitro. Clinical data demonstrated worse overall and progression-free survival in patients with high FABP5 expression in tumor cells. Overall, this study establishes the FABP family as a potentially new target in multiple myeloma. In MM cells, FABPs have a multitude of actions and cellular roles that result in the support of myeloma progression. Further research into the FABP family in MM is warrented, especially into the effective translation of targeting these in vivo.

Multiple myeloma is a type of blood cancer for which only a few treatments are available. Currently, only about half the patients with multiple myeloma survive for five years after diagnosis. Because obesity is a risk factor for multiple myeloma, researchers have been studying how fat cells or fatty acids affect multiple myeloma tumor cells to identify new treatment targets. Fatty acid binding proteins (FABPs) are one promising target. The FABPs shuttle fatty acids and help cells communicate. Previous studies linked FABPs to some types of cancer, including another blood cancer called leukemia, and cancers of the prostate and breast. A recent study showed that patients with multiple myeloma, who have high levels of FABP5 in their tumors, have worse outcomes than patients with lower levels. But, so far, no one has studied the effects of inhibiting FABPs in multiple myeloma tumor cells or animals with multiple myeloma. Farrell et al. show that blocking or eliminating FABPs kills myeloma tumor cells and slows their growth in a dish (in vitro) and in some laboratory mice. In the experiments, the researchers treated myeloma cells with drugs that inhibit FABPs or genetically engineered myeloma cells to lack FABPs. They also show that blocking FABPs reduces the activity of a protein called MYC, which promotes tumor cell survival in many types of cancer. It also changed the metabolism of the tumor cell. Finally, the team examined data collected from several sets of patients with multiple myeloma and found that patients with high FABP levels have more aggressive cancer. The experiments lay the groundwork for more studies to determine if drugs or other therapies targeting FABPs could treat multiple myeloma. More research is needed to determine why inhibiting FABPs worked in some mice with multiple myeloma but not others, and whether FABP inhibitors might work better if combined with other cancer therapies. There were no signs that the drugs were toxic in mice, but more studies must prove they are safe and effective before testing the drugs in humans with multiple myeloma. Designing better or more potent FABP-blocking drugs may also lead to better animal study results.

Obesity and myeloma: Clinical and mechanistic contributions to disease progression.

Obesity and obesogenic behaviors are positively associated with both monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma (MM). As the only known modifiable risk factor, this association has emerged as a new potential target for MM prevention, but little is known about the mechanistic relationship of body weight with MM progression. Here we summarize epidemiological correlations between weight, body composition, and the various stages of myeloma disease progression and treatments, as well as the current understanding of the molecular contributions of obesity-induced changes in myeloma cell phenotype and signaling. Finally, we outline groundwork for the future characterization of the relationship between body weight patterns, the bone marrow microenvironment, and MM pathogenesis in animal models, which have the potential to impact our understanding of disease pathogenesis and inform MM prevention messages.

Witch Nails (Krt90whnl): A spontaneous mouse mutation affecting nail growth and development.

Numerous single gene mutations identified in humans and mice result in nail deformities with many similarities between the species. A spontaneous, autosomal, recessive mutation called witch nails (whnl) is described here where the distal nail matrix and nail bed undergo degenerative changes resulting in formation of an abnormal nail plate causing mice to develop long, curved nails. This mutation arose spontaneously in a colony of MRL/MpJ-Faslpr/J at The Jackson Laboratory. Homozygous mutant mice are recognizable by 8 weeks of age by their long, curved nails. The whnl mutation, mapped on Chromosome 15, is due to a 7-bp insertion identified in the 3' region of exon 9 in the Krt90 gene (formerly Riken cDNA 4732456N10Rik), and is predicted to result in a frameshift that changes serine 476 to arginine and subsequently introduces 36 novel amino acids into the protein before a premature stop codon (p. Ser476ArgfsTer36). By immunohistochemistry the normal KRT90 protein is expressed in the nail matrix and nail bed in control mice where lesions are located in mutant mice. Immunoreactivity toward equine KRT124, the ortholog of mouse KRT90, is restricted to the hoof lamellae (equine hoof wall and lamellae are homologous to the mouse nail plate and nail bed) and the mouse nail bed. Equine laminitis lesions are similar to those observed in this mutant mouse suggesting that the latter may be a useful model for hoof and nail diseases. This first spontaneous mouse mutation affecting the novel Krt90 gene provides new insight into the normal regulation of the molecular pathways of nail development.

Development and characterization of three cell culture systems to investigate the relationship between primary bone marrow adipocytes and myeloma cells.

The unique properties of the bone marrow (BM) allow for migration and proliferation of multiple myeloma (MM) cells while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. BM adipocytes (BMAds) have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary BM cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and, reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes generated by differentiating BM-derived mesenchymal stromal cells (BMSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, then they likely will recapitulate in vivo biology better than BMSC-derived adipocytes, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd (pBMAds) may not be the same as the BMSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing pBMAds: two-dimensional (2D) coverslips, 2D transwells, and three-dimensional (3D) silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the BM microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human BM. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for up to 4 week in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In summary, we developed three in vitro cell culture systems to study pBMAds and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the BM, including other bone-homing tumor types.

Myeloma-bone marrow adipocyte axis in tumour survival and treatment response.

Multiple myeloma is an incurable cancer of the bone marrow that is dependent on its microenvironment, including bone marrow adipocytes (BMAds). Here, we discuss our findings that the reciprocal interaction of myeloma cells and BMAds, leads to myeloma cell survival and induces metabolic dysfunction and senescence-associated secretory phenotype in BMAds.

Sclerostin antibody increases trabecular bone and bone mechanical properties by increasing osteoblast activity damaged by whole-body irradiation in mice.

Irradiation therapy causes bone deterioration and increased risk for skeletal-related events. Irradiation interferes with trabecular architecture through increased osteoclastic activity, decreased osteoblastic activity, and increased adipocyte expansion in the bone marrow (BM), which further compounds bone-related disease. Neutralizing antibodies to sclerostin (Scl-Ab) increase bone mass and strength by increasing bone formation and reducing bone resorption. We hypothesized that treatment with Scl-Ab would attenuate the adverse effects of irradiation by increasing bone volume and decreasing BM adipose tissue (BMAT), resulting in better quality bone. In this study, 12-week-old female C57BL/6J mice were exposed to 6 Gy whole-body irradiation or were non-irradiated, then administered Scl-Ab (25 mg/kg) or vehicle weekly for 5 weeks. Femoral µCT analysis confirmed that the overall effect of IR significantly decreased trabecular bone volume/total volume (Tb.BV/TV) (2-way ANOVA, p < 0.0001) with a -43.8% loss in Tb.BV/TV in the IR control group. Scl-Ab independently increased Tb.BV/TV by 3.07-fold in non-irradiated and 3.6-fold in irradiated mice (2-way ANOVA, p < 0.0001). Irradiation did not affect cortical parameters, although Scl-Ab increased cortical thickness and area significantly in both irradiated and non-irradiated mice (2-way ANOVA, p < 0.0001). Femoral mechanical testing confirmed Scl-Ab significantly increased bending rigidity and ultimate moment independently of irradiation (2-way ANOVA, p < 0.0001). Static and dynamic histomorphometry of the femoral metaphysis revealed osteoblast vigor, not number, was significantly increased in the irradiated mice treated with Scl-Ab. Systemic alterations were assessed through serum lipidomic analysis, which showed that Scl-Ab normalized lipid profiles in the irradiated group. This data supports the theory of sclerostin as a novel contributor to the regulation of osteoblast activity after irradiation. Overall, our data support the hypothesis that Scl-Ab ameliorates the deleterious effects of whole-body irradiation on bone and adipose tissue in a mouse model. Our findings suggest that future research into localized and systemic therapies after irradiation exposure is warranted.

Bone Marrow Adipocytes: A Link between Obesity and Bone Cancer.

Cancers that grow in the bone marrow are for most patients scary, painful, and incurable. These cancers are especially hard to treat due to the supportive microenvironment provided by the bone marrow niche in which they reside. New therapies designed to target tumor cells have extended the life expectancy for these patients, but better therapies are needed and new ideas for how to target these cancers are crucial. This need has led researchers to interrogate whether bone marrow adipocytes (BMAds), which increase in number and size during aging and in obesity, contribute to cancer initiation or progression within the bone marrow. Across the globe, the consensus in the field is a unified "yes". However, how to target these adipocytes or the factors they produce and how BMAds interact with different tumor cells are open research questions. Herein, we review this research field, with the goal of accelerating research in the network of laboratories working in this area and attracting bright scientists with new perspectives and ideas to the field in order to bring about better therapies for patients with bone cancers.

Myeloma-Modified Adipocytes Exhibit Metabolic Dysfunction and a Senescence-Associated Secretory Phenotype.

Bone marrow adipocytes (BMAd) have recently been implicated in accelerating bone metastatic cancers, such as acute myelogenous leukemia and breast cancer. Importantly, bone marrow adipose tissue (BMAT) expands with aging and obesity, two key risk factors in multiple myeloma disease prevalence, suggesting that BMAds may influence and be influenced by myeloma cells in the marrow. Here, we provide evidence that reciprocal interactions and cross-regulation of myeloma cells and BMAds play a role in multiple myeloma pathogenesis and treatment response. Bone marrow biopsies from patients with multiple myeloma revealed significant loss of BMAT with myeloma cell infiltration of the marrow, whereas BMAT was restored after treatment for multiple myeloma. Myeloma cells reduced BMAT in different preclinical murine models of multiple myeloma and in vitro using myeloma cell-adipocyte cocultures. In addition, multiple myeloma cells altered adipocyte gene expression and cytokine secretory profiles, which were also associated with bioenergetic changes and induction of a senescent-like phenotype. In vivo, senescence markers were also increased in the bone marrow of tumor-burdened mice. BMAds, in turn, provided resistance to dexamethasone-induced cell-cycle arrest and apoptosis, illuminating a new possible driver of myeloma cell evolution in a drug-resistant clone. Our findings reveal that bidirectional interactions between BMAds and myeloma cells have significant implications for the pathogenesis and treatment of multiple myeloma. Targeting senescence in the BMAd or other bone marrow cells may represent a novel therapeutic approach for treatment of multiple myeloma. SIGNIFICANCE: This study changes the foundational understanding of how cancer cells hijack the bone marrow microenvironment and demonstrates that tumor cells induce senescence and metabolic changes in adipocytes, potentially driving new therapeutic directions.

Sclerostin-Neutralizing Antibody Treatment Rescues Negative Effects of Rosiglitazone on Mouse Bone Parameters.

Obesity, a growing pandemic, is a risk factor for many cancers and causes increased bone marrow adipose tissue (BMAT). in vitro studies and obese animal models suggest that BMAT contributes to cancer progression, but there is a lack of preclinical models to directly test BMAT's role in cancer. Overactivation of peroxisome-proliferator-activated receptor-γ (PPARγ) can skew bone formation and resorption rates, resulting in increased BMAT and trabecular bone loss. Thiazolidinediones (eg, rosiglitazone) are anti-diabetic therapies that promote adipogenesis through PPARγ activation. We investigated if rosiglitazone increases BMAT in an immunocompromised model, commonly used in cancer research, and if these effects could be reversed by co-administering a bone anabolic agent (sclerostin-neutralizing antibody [Scl-Ab]), which has been shown to inhibit adipogenesis, using DXA, µCT, OsO4 µCT, and dynamic histomorphometry. Four weeks of rosiglitazone in female SCID Beige mice (cohort 1) significantly decreased trabecular bone volume (BV/TV) by about one-half, through increased osteoclast and suppressed osteoblast activity, and significantly increased BMAT. In cohort 2, mice were administered rosiglitazone ± Scl-Ab for 4 weeks, and then rosiglitazone was discontinued and Scl-Ab or vehicle were continued for 6 weeks. Scl-Ab significantly increased bone parameters (eg, BV/TV, N.Ob/B.Pm, and MS/BS) in both groups. Scl-Ab also overcame many negative effects of rosiglitazone (eg, effects on trabecular bone parameters, increased mineralization lag time [MLT], and decreased bone formation rate [BFR]). Interestingly, Scl-Ab significantly decreased rosiglitazone-induced BMAT in the femur, mostly due to a reduction in adipocyte size, but had a much weaker effect on tibial BMAT. These data suggest targeting sclerostin can prevent rosiglitazone-induced bone loss and reduce BM adiposity, in some, but not all BMAT locations. Collectively, our data demonstrate that rosiglitazone increases BMAT in SCID Beige mice, but concomitant changes in bone may confound its use to specifically determine BMAT's role in tumor models. © 2020 American Society for Bone and Mineral Research (ASBMR).

Targeting Bone Cells During Sexual Maturation Reveals Sexually Dimorphic Regulation of Endochondral Ossification.

In endochondral ossification, chondroblasts become embedded in their matrix and become chondrocytes, which are mature cells that continue to proliferate, eventually becoming hypertrophic. Hypertrophic chondrocytes produce cartilage that is then resorbed by osteoclasts prior to bone matrix replacement via osteoblasts. Although sexually dimorphic bone phenotypes have long been characterized, specific modulation of the growth plate during a critical window in sexual maturation has not been evaluated. Here we report that specific depletion of osteocalcin- (OCN-) expressing cells in vivo during sexual maturation leads to dimorphic bone phenotypes in males and females. At 6 to 8 weeks of age, OCN-Cre;iDTR (inducible diphtheria toxin receptor-expressing) mice were treated with diphtheria toxin (DT) for 2 weeks to deplete OCN+ cells. At the end of the study, long bones were collected for µCT and histomorphometry, and serum was collected for proteomic and lipidomic analyses. Ablation of OCN+ cells in mice leads to consistent trends for weight loss after 2 weeks of treatment. Females exhibited decreased skeletal parameters in response to OCN+ cell ablation treatment, as expected. However, OCN+ cell ablation in males uniquely displayed an expansion of hypertrophic chondrocytes, a widening of the growth plate, and an abnormal "clubbing" anatomy of the distal femur. Following DT treatment, mice from both sexes also underwent metabolic cage analysis, in which both sexes exhibited decreased energy expenditure. We conclude that skewing endochondral bone formation during longitudinal growth has a profound effect on body weight and energy expenditure with sex-specific effects on developing bone. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The CD38low natural killer cell line KHYG1 transiently expressing CD16F158V in combination with daratumumab targets multiple myeloma cells with minimal effector NK cell fratricide.

Multiple myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of the CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (MoAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells that mediate in vivo tumour immunosurveillance. NK cells also play an important role during MoAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their FcγRIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring variant of CD16 harbouring a single-point polymorphism (F158V). However, the contribution of NK cells to the efficacy of daratumumab remains debatable as clinical data clearly indicate the rapid depletion of CD38high peripheral blood NK cells in patients upon daratumumab administration. In contrast, CD38low peripheral blood NK cells have been shown to survive daratumumab mediated fratricide in vivo, while still retaining their potent anti-MM cytolytic effector functions ex vivo. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach on CD38low NK cells in combination with daratumumab could represent a novel therapeutic option for treatment of MM. In the present study, we investigate a NK cell line (KHYG-1), derived from a patient with aggressive NK cell leukemia, as a platform for generating CD38low NK cells expressing CD16F158V which can be administered as an "off-the-shelf" therapy to target both CD38high and CD38low tumour clones in patients receiving daratumumab.

Multiple Myeloma Cells Alter Adipogenesis, Increase Senescence-Related and Inflammatory Gene Transcript Expression, and Alter Metabolism in Preadipocytes.

Within the bone marrow microenvironment, mesenchymal stromal cells (MSCs) are an essential precursor to bone marrow adipocytes and osteoblasts. The balance between this progenitor pool and mature cells (adipocytes and osteoblasts) is often skewed by disease and aging. In multiple myeloma (MM), a cancer of the plasma cell that predominantly grows within the bone marrow, as well as other cancers, MSCs, preadipocytes, and adipocytes have been shown to directly support tumor cell survival and proliferation. Increasing evidence supports the idea that MM-associated MSCs are distinct from healthy MSCs, and their gene expression profiles may be predictive of myeloma patient outcomes. Here we directly investigate how MM cells affect the differentiation capacity and gene expression profiles of preadipocytes and bone marrow MSCs. Our studies reveal that MM.1S cells cause a marked decrease in lipid accumulation in differentiating 3T3-L1 cells. Also, MM.1S cells or MM.1S-conditioned media altered gene expression profiles of both 3T3-L1 and mouse bone marrow MSCs. 3T3-L1 cells exposed to MM.1S cells before adipogenic differentiation displayed gene expression changes leading to significantly altered pathways involved in steroid biosynthesis, the cell cycle, and metabolism (oxidative phosphorylation and glycolysis) after adipogenesis. MM.1S cells induced a marked increase in 3T3-L1 expression of MM-supportive genes including Il-6 and Cxcl12 (SDF1), which was confirmed in mouse MSCs by qRT-PCR, suggesting a forward-feedback mechanism. In vitro experiments revealed that indirect MM exposure prior to differentiation drives a senescent-like phenotype in differentiating MSCs, and this trend was confirmed in MM-associated MSCs compared to MSCs from normal donors. In direct co-culture, human mesenchymal stem cells (hMSCs) exposed to MM.1S, RPMI-8226, and OPM-2 prior to and during differentiation, exhibited different levels of lipid accumulation as well as secreted cytokines. Combined, our results suggest that MM cells can inhibit adipogenic differentiation while stimulating expression of the senescence associated secretory phenotype (SASP) and other pro-myeloma molecules. This study provides insight into a novel way in which MM cells manipulate their microenvironment by altering the expression of supportive cytokines and skewing the cellular diversity of the marrow.

Identification of EOMES-expressing spermatogonial stem cells and their regulation by PLZF.

Long-term maintenance of spermatogenesis in mammals is supported by GDNF, an essential growth factor required for spermatogonial stem cell (SSC) self-renewal. Exploiting a transgenic GDNF overexpression model, which expands and normalizes the pool of undifferentiated spermatogonia between Plzf +/+ and Plzf lu/lu mice, we used RNAseq to identify a rare subpopulation of cells that express EOMES, a T-box transcription factor. Lineage tracing and busulfan challenge show that these are SSCs that contribute to steady state spermatogenesis as well as regeneration following chemical injury. EOMES+ SSCs have a lower proliferation index in wild-type than in Plzf lu/lu mice, suggesting that PLZF regulates their proliferative activity and that EOMES+ SSCs are lost through proliferative exhaustion in Plzf lu/lu mice. Single cell RNA sequencing of EOMES+ cells from Plzf +/+ and Plzf lu/lu mice support the conclusion that SSCs are hierarchical yet heterogeneous.

Inverse correlation between trabecular bone volume and bone marrow adipose tissue in rats treated with osteoanabolic agents.

There is currently an unmet clinical need for improved treatments for skeletal diseases such as osteoporosis and cancer-induced bone disease. This is due in part to a paucity of novel targets and an incomplete understanding of the mechanisms of action for established therapies. We defined the effects of anabolic treatments on bone and the bone marrow adipocyte (BMA). Sclerostin-neutralizing antibodies (Scl-Ab), romosozumab, human parathyroid hormone (hPTH, 1-34), and hPTH/hPTHrP analogues (e.g. teriparatide and abaloparatide) stimulate bone formation and have been studied in clinical trials for severe osteoporosis. In this study, eight-week-old male and female rats were administered vehicle, Scl-Ab (3 mg/kg or 50 mg/kg) weekly, or hPTH (1-34) (75 µg/kg) daily for 4 or 26 weeks. Histological analyses of distal femura were performed using a novel ImageJ method for trabecular bone and bone marrow adipose tissue (BMAT). Adipocyte number, circumference, and total adipose area were compared within the tissue area (T.Ar) or the marrow area (Ma.Ar), (defined as the T.Ar minus the trabecular bone area). After 26 weeks of treatment, a significant inverse correlation between bone and tissue adiposity (total adipocyte area divided by T.Ar) were observed in males and females (p < 0.0001). However, there were no significant correlations between bone and marrow adiposity (total adipocyte area divided by Ma.Ar) for either sex after 26 weeks of treatments. Scl-Ab treatments also resulted in no effect on adipocytes based on marrow adiposity for either sex after 26 weeks. However, chronic hPTH treatments significantly reduced adipocyte number and adiposity within the T.Ar and within the Ma.Ar in males. Overall, our data suggest that with long-term treatment, Scl-Abs decrease total tissue adiposity mainly by increasing trabecular bone, resulting in an overall reduction in the space in which adipocytes can reside. These findings were determined by developing and comparing two different methods of assessment of the marrow cavity, defined to either include or exclude trabecular bone. Thus, researchers should consider which adiposity measurement is more informative and relevant for their studies. Overall, our findings should help design improved therapies or combination treatments to target a potential new contributor to bone diseases: the bone marrow adipocyte.

Development of a 3D bone marrow adipose tissue model.

Over the past twenty years, evidence has accumulated that biochemically and spatially defined networks of extracellular matrix, cellular components, and interactions dictate cellular differentiation, proliferation, and function in a variety of tissue and diseases. Modeling in vivo systems in vitro has been undeniably necessary, but when simplified 2D conditions rather than 3D in vitro models are used, the reliability and usefulness of the data derived from these models decreases. Thus, there is a pressing need to develop and validate reliable in vitro models to reproduce specific tissue-like structures and mimic functions and responses of cells in a more realistic manner for both drug screening/disease modeling and tissue regeneration applications. In adipose biology and cancer research, these models serve as physiologically relevant 3D platforms to bridge the divide between 2D cultures and in vivo models, bringing about more reliable and translationally useful data to accelerate benchtop to bedside research. Currently, no model has been developed for bone marrow adipose tissue (BMAT), a novel adipose depot that has previously been overlooked as "filler tissue" but has more recently been recognized as endocrine-signaling and systemically relevant. Herein we describe the development of the first 3D, BMAT model derived from either human or mouse bone marrow (BM) mesenchymal stromal cells (MSCs). We found that BMAT models can be stably cultured for at least 3 months in vitro, and that myeloma cells (5TGM1, OPM2 and MM1S cells) can be cultured on these for at least 2 weeks. Upon tumor cell co-culture, delipidation occurred in BMAT adipocytes, suggesting a bidirectional relationship between these two important cell types in the malignant BM niche. Overall, our studies suggest that 3D BMAT represents a "healthier," more realistic tissue model that may be useful for elucidating the effects of MAT on tumor cells, and tumor cells on MAT, to identify novel therapeutic targets. In addition, proteomic characterization as well as microarray data (expression of >22,000 genes) coupled with KEGG pathway analysis and gene set expression analysis (GSEA) supported our development of less-inflammatory 3D BMAT compared to 2D culture. In sum, we developed the first 3D, tissue-engineered bone marrow adipose tissue model, which is a versatile, novel model that can be used to study numerous diseases and biological processes involved with the bone marrow.

3d Tissue Engineered In Vitro Models Of Cancer In Bone.

Biological models are necessary tools for gaining insight into underlying mechanisms governing complex pathologies such as cancer in the bone. Models range from in vitro tissue culture systems to in vivo models and can be used with corresponding epidemiological and clinical data to understand disease etiology, progression, driver mutations, and signaling pathways. In bone cancer, as with many other cancers, in vivo models are often too complex to study specific cell-cell interactions or protein roles, and 2D models are often too simple to accurately represent disease processes. Consequently, researchers have increasingly turned to 3D in vitro tissue engineered models as a useful compromise. In this review, tissue engineered 3D models of bone and cancer are described in depth and compared to 2D models. Biomaterials and cell types used are described, and future directions in the field of tissue engineered bone cancer models are proposed.

The skeletal cell-derived molecule sclerostin drives bone marrow adipogenesis.

The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.