Hydroxychloroquine and a low activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects.

Osteoporosis is incurable because there are no dual antiresorptive and anabolic therapeutic agents that can be administered long-term. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation and thus have limited effect in preventing osteoporotic fracture. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

RANKL inhibition reduces lesional cellularity and Gαs variant expression and enables osteogenic maturation in fibrous dysplasia.

Fibrous dysplasia (FD) is a rare, disabling skeletal disease for which there are no established treatments. Growing evidence supports inhibiting the osteoclastogenic factor receptor activator of nuclear kappa-B ligand (RANKL) as a potential treatment strategy. In this study, we investigated the mechanisms underlying RANKL inhibition in FD tissue and its likely indirect effects on osteoprogenitors by evaluating human FD tissue pre- and post-treatment in a phase 2 clinical trial of denosumab (NCT03571191) and in murine in vivo and ex vivo preclinical models. Histological analysis of human and mouse tissue demonstrated increased osteogenic maturation, reduced cellularity, and reduced expression of the pathogenic Gαs variant in FD lesions after RANKL inhibition. RNA sequencing of human and mouse tissue supported these findings. The interaction between osteoclasts and mutant osteoprogenitors was further assessed in an ex vivo lesion model, which indicated that the proliferation of abnormal FD osteoprogenitors was dependent on osteoclasts. The results from this study demonstrated that, in addition to its expected antiosteoclastic effect, denosumab reduces FD lesion activity by decreasing FD cell proliferation and increasing osteogenic maturation, leading to increased bone formation within lesions. These findings highlight the unappreciated role of cellular crosstalk between osteoclasts and preosteoblasts/osteoblasts as a driver of FD pathology and demonstrate a novel mechanism of action of denosumab in the treatment of bone disease.TRIAL REGISTRATION: ClinicalTrials.gov NCT03571191.

Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes.

Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction1. There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity1,2. Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments.

Nuclear Factor-Kappa B Regulation of Osteoclastogenesis and Osteoblastogenesis.

Maintenance of skeletal integrity requires the coordinated activity of multinucleated bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoclasts form resorption lacunae on bone surfaces in response to cytokines by fusion of precursor cells. Osteoblasts are derived from mesenchymal precursors and lay down new bone in resorption lacunae during bone remodeling. Nuclear factorkappa B (NF-κB) signaling regulates osteoclast and osteoblast formation and is activated in osteoclast precursors in response to the essential osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL), which can also control osteoblast formation through RANK-RANKL reverse signaling in osteoblast precursors. RANKL and some pro-inflammatory cytokines, including tumor necrosis factor (TNF), activate NF-κB signaling to positively regulate osteoclast formation and functions. However, these cytokines also limit osteoclast and osteoblast formation through NF-κB signaling molecules, including TNF receptor-associated factors (TRAFs). TRAF6 mediates RANKL-induced osteoclast formation through canonical NF-κB signaling. In contrast, TRAF3 limits RANKL- and TNF-induced osteoclast formation, and it restricts transforming growth factor ß (TGFß)-induced inhibition of osteoblast formation in young and adult mice. During aging, neutrophils expressing TGFß and C-C chemokine receptor type 5 (CCR5) increase in bone marrow of mice in response to increased NF-κB-induced CC motif chemokine ligand 5 (CCL5) expression by mesenchymal progenitor cells and injection of these neutrophils into young mice decreased bone mass. TGFß causes degradation of TRAF3, resulting in decreased glycogen synthase kinase-3ß/ß-catenin-mediated osteoblast formation and age-related osteoporosis in mice. The CCR5 inhibitor, maraviroc, prevented accumulation of TGFß+/CCR5+ neutrophils in bone marrow and increased bone mass by inhibiting bone resorption and increasing bone formation in aged mice. This paper updates current understanding of how NF-κB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast and osteoblast formation and activation with a focus on the role of TRAF3 signaling, which can be targeted therapeutically to enhance bone mass.

Molecular signatures distinguish senescent cells from inflammatory cells in aged mouse callus stromal cells.

Cellular senescence plays important roles in age-related diseases, including musculoskeletal disorders. Senescent cells (SCs) exert a senescence-associated secretory phenotype (SASP) by producing SASP factors, some of which overlap with factors produced by inflammatory cells (Inf-Cs). However, the differences between SCs and Inf-Cs and how they interact with each other during fracture repair have not been well studied. Here, we analyzed single cell RNA sequencing data of aged mouse fracture callus stromal cells. We defined Inf-Cs as cells that express NF-κB Rela/Relb, SCs as cells that express the senescence genes, Cdkn1a, Cdkn2a or Cdkn2c, and inflammatory SCs (Inf-SCs) as cells that express both NF-κB and senescence genes. Differentially expressed genes and pathway analyses revealed that Inf-SCs and SCs had a similar gene expression profile and upregulated pathways that are related to DNA damage/oxidation-reduction and cellular senescence, while Inf-Cs expressed different gene signatures and pathways from SCs and Inf-SCs, mainly related to inflammation. Cellchat software analysis indicated that SCs and Inf-SCs are potential ligand-producing cells that affect Inf-Cs as target cells. Cell culture experiments demonstrated that SC conditioned medium promoted inflammatory gene expression by callus-derived mesenchymal progenitor cells, and Inf-Cs had reduced osteoblast differentiation capacity. In summary, we have identified three cell subclusters associated with inflammation and senescence in callus stromal cells, predicted potential effects of Inf-SCs and SCs on Inf-Cs by production of active ligands, and demonstrated that when mesenchymal progenitors acquire inflammatory phenotypes their osteogenic potential is reduced.

TGFß1+CCR5+ neutrophil subset increases in bone marrow and causes age-related osteoporosis in male mice.

TGFß1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFß1+CCR5+) is the major source of TGFß1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFß1+CCR5+ neutrophil numbers in BM of young mice. During aging, TGFß1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFß1+CCR5+ neutrophil numbers in BM where they induce bone loss. TGFß1+CCR5+ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFß1+CCR5+ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFßRII specifically deleted in MPCs had lower numbers of TGFß1+CCR5+ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFß1+CCR5+ neutrophil numbers in BM could treat or prevent age-related osteoporosis.

Idiopathic Spontaneous Occurrence of Heterotrophic Occurrence During Pregnancy.

Heterotopic ossification (HO) typically presents in the hip, knee, and elbow joints in the setting of trauma or postsurgical intervention. Less commonly, it may occur secondary to neurologic dysfunction or underlying genetic conditions, but idiopathic HO is rare. Most cases of HO are managed nonoperatively with surgical resection remaining a controversy due to high recurrence rates. We describe a case of idiopathic HO of the shoulder that occurred in the absence of trauma, neurologic dysfunction, or underlying genetic disorder that was treated with surgical excision.

HSF1 is a driver of leukemia stem cell self-renewal in acute myeloid leukemia.

Acute myeloid leukemia (AML) is maintained by self-renewing leukemic stem cells (LSCs). A fundamental problem in treating AML is that conventional therapy fails to eliminate LSCs, which can reinitiate leukemia. Heat shock transcription factor 1 (HSF1), a central regulator of the stress response, has emerged as an important target in cancer therapy. Using genetic Hsf1 deletion and a direct HSF1 small molecule inhibitor, we show that HSF1 is specifically required for the maintenance of AML, while sparing steady-state and stressed hematopoiesis. Mechanistically, deletion of Hsf1 dysregulates multifaceted genes involved in LSC stemness and suppresses mitochondrial oxidative phosphorylation through downregulation of succinate dehydrogenase C (SDHC), a direct HSF1 target. Forced expression of SDHC largely restores the Hsf1 ablation-induced AML developmental defect. Importantly, the growth and engraftment of human AML cells are suppressed by HSF1 inhibition. Our data provide a rationale for developing efficacious small molecules to specifically target HSF1 in AML.

Intraligamentous synovial osteochondroma of the ligamentum teres: a series of 14 cases.

BACKGROUND: The ligamentum teres (LT) is covered by synovium. It acts as a stabilizer of the hip and as such it has been compared to the ACL of the knee joint. Pathologic changes occur in the LT with aging and osteoarthritis (OA), including degeneration, occasional chondroid metaplasia, and synovial chondromatosis are well-recognized in the literature. However, there are no reports of intraligamentous synovial osteochondroma occuring in the LT. METHODS: We reviewed the pathology reports of 542 osteoarthritic femoral arthroplasty specimens between January 2016 and December 2018. The LT was examined histologically in 55 cases because it was abnormal on gross examination. RESULTS: A single synovial osteochondroma, ranging in size from 0.4-1.7 cm in diameter, was present in the body of the LT in 14 cases (9 males; 5 females, aged 34 to 81 years), representing 2.6% of 542 arthroplasty cases. Ten of the osteochondromas had bone marrow fat without hematopoietic elements, 1 had hematopoietic elements, and 3 had no marrow among the bony trabeculae. Radiographically, all cases had moderate to severe osteoarthritis with no mention of an abnormality of LT. CONCLUSION: To our knowledge, this is the first report of intraligamentous synovial osteochondroma in the LT in osteoarthritis patients undergoing hip arthroplasty. It provides further support for microscopic examination of arthroplasty specimens for histologic abnormalities. Further prospective study is needed to determine if this lesion contributes adversely to the development or progression of osteoarthritis and if it is a reactive or neoplastic process.

Glomus Tumor of the Lower Extremity Previously Misdiagnosed as Complex Regional Pain Syndrome in Close Proximity to a Myxofibrosarcoma: A Case Report.

Complex regional pain syndrome (CRPS) is a potentially devastating condition that can result in severe psychological and social morbidity. It is a diagnosis of exclusion, and other pathologic entities must be ruled out first. Glomus tumors are exquisitely painful benign vascular tumors that are most common in the hand and are rarely found in the lower extremity. Here, we present a case of a patient who developed a focus of severe anterior knee pain and tenderness a few months after a car accident that had been misdiagnosed as CRPS for 15 years. She coincidentally developed a sarcoma of her ipsilateral leg distal to this site. Magnetic resonance imaging of the sarcoma included the area of knee pain where, interestingly, it identified a separate small soft-tissue mass. A glomus tumor was diagnosed histologically in a needle biopsy specimen from this mass, which was resected along with the sarcoma. For the first time in 15 years, despite the additional sarcoma surgery, she reported relief of her pain and complete resolution of her "CRPS."

Efficacy of Intraventricular Sponge Placement to Capture Debris in Aortic Valve Replacement.

Objective: Surgical aortic valve replacement (SAVR) carries the known risk of shedding debris into the left ventricle during valve leaflet excision and annulus debridement. Embolization of this debris may have devastating effects for the patient. Although surgeons have developed methods to mitigate this risk, no data exist as to their efficacy. Herein, we present the first study that evaluates the efficacy of a technique for capturing debris during SAVR. Methods: Our group conducted a prospective case series of 20 patients who underwent SAVR using the insertion of an intraventricular surgical sponge prior to valve leaflet excision and annulus debridement to capture debris. Surgical sponges were grossly, radiographically, and histologically examined for the presence of cellular and acellular debris to determine the efficacy of this technique. Results: Of the 20 surgical sponges analyzed, 15 specimens (75%) registered positivity for cellular and/or acellular debris. Seven sponges (35%) were grossly positive, 15 sponges (75%) were radiographically positive, and 4 sponges (20%) were histologically positive for calcified debris on examination. Conclusions: This is one of the few studies to objectively evaluate a method used to capture debris in SAVR procedures. Our results demonstrate a high frequency of debris captured within intraventricular surgical sponges and confirms the efficacy of this technique. While these data are promising, numerous additional approaches exist to capture debris, and a best practice standard should exist across the specialty.  In addition, this study does not address the clinical outcomes associated with this technique. To these ends, additional data and multicenter collaboration are required.

Age-associated callus senescent cells produce TGF-ß1 that inhibits fracture healing in aged mice.

Cellular senescence plays an important role in human diseases, including osteoporosis and osteoarthritis. Senescent cells (SCs) produce the senescence-associated secretory phenotype to affect the function of neighboring cells and SCs themselves. Delayed fracture healing is common in the elderly and is accompanied by reduced mesenchymal progenitor cells (MPCs). However, the contribution of cellular senescence to fracture healing in the aged has not to our knowledge been studied. Here, we used C57BL/6J 4-month-old young and 20-month-old aged mice and demonstrated a rapid increase in SCs in the fracture callus of aged mice. The senolytic drugs dasatinib plus quercetin enhanced fracture healing in aged mice. Aged callus SCs inhibited the growth and proliferation of callus-derived MPCs (CaMPCs) and expressed high levels of TGF-ß1. TGF-ß-neutralizing Ab prevented the inhibitory effects of aged callus SCs on CaMPCs and promoted fracture healing in aged mice, which was associated with increased CaMPCs and proliferating cells. Thus, fracture triggered a significant cellular senescence in the callus cells of aged mice, which inhibited MPCs by expressing TGF-ß1. Short-term administration of dasatinib plus quercetin depleted callus SCs and accelerated fracture healing in aged mice. Senolytic drugs represent a promising therapy, while TGF-ß1 signaling is a molecular mechanism for fractures in the elderly via SCs.

Proteasome inhibition-enhanced fracture repair is associated with increased mesenchymal progenitor cells in mice.

The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.

EULAR points to consider for minimal reporting requirements in synovial tissue research in rheumatology.

BACKGROUND: Synovial tissue research has become widely developed in several rheumatology centres, however, large discrepancies exist in the way synovial tissue is handled and, more specifically, how data pertaining to biopsy procedure, quality check and experimental results are reported in the literature. This heterogeneity hampers the progress of research in this rapidly expanding field. In that context, under the umbrella of European Alliance of Associations for Rheumatology, we aimed at proposing points to consider (PtC) for minimal reporting requirements in synovial tissue research. METHODS: Twenty-five members from 10 countries across Europe and USA met virtually to define the key areas needing evaluation and formulating the research questions to inform a systematic literature review (SLR). The results were presented during a second virtual meeting where PtC were formulated and agreed. RESULTS: Study design, biopsy procedures, tissue handling, tissue quality control and tissue outcomes (imaging, DNA/RNA analysis and disaggregation) were identified as important aspects for the quality of synovial tissue research. The SLR interrogated four databases, retrieved 7654 abstracts and included 26 manuscripts. Three OPs and nine PtC were formulated covering the following areas: description of biopsy procedure, overarching clinical design, patient characteristics, tissue handling and processing, quality control, histopathology, transcriptomic analyses and single-cell technologies. CONCLUSIONS: These PtC provide guidance on how research involving synovial tissue should be reported to ensure a better evaluation of results by readers, reviewers and the broader scientific community. We anticipate that these PtC will enable the field to progress in a robust and transparent manner over the coming years.

Bone-Targeted Bortezomib Inhibits Bortezomib-Resistant Multiple Myeloma in Mice by Providing Higher Levels of Bortezomib in Bone.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

TIFAB accelerates MLL-AF9-Induced acute myeloid leukemia through upregulation of HOXA9.

We previously showed stabilization of NIK-induced activation of NF-κB non-canonical signaling suppresses MLL-AF9-induced AML. In the current study, we demonstrate that deletion of NF-κB non-canonical RelB prevents the inhibitory effect of NIK stabilization in MLL-AF9 AML. Mechanistically, RelB suppresses its direct target, TIFAB, which is upregulated in human AML and correlates negatively with the survival of AML patients. Forced expression of TIFAB reverses NIK-induced impaired AML development through downregulation of RelB and upregulation of HOXA9. Consistent with upregulation of HOXA9, gene set enrichment analysis shows that forced expression of TIFAB blocks myeloid cell development, upregulates leukemia stem cell signature and induces similar gene expression patterns to those of HOXA9-MEIS1 and HOXA9-NUP98, and upregulates oxidative phosphorylation. Accordingly, forced expression of HOXA9 also largely releases the inhibitory impact of NIK stabilization via downregulation of RelB and upregulation of RelA. Our data suggest that NIK/RelB suppresses MLL-AF9-induced AML mainly through downregulation of TIFAB/HOXA9.

IKKß-NF-κB signaling in adult chondrocytes promotes the onset of age-related osteoarthritis in mice.

Canonical nuclear factor κB (NF-κB) signaling mediated by homo- and heterodimers of the NF-κB subunits p65 (RELA) and p50 (NFKB1) is associated with age-related pathologies and with disease progression in posttraumatic models of osteoarthritis (OA). Here, we established that NF-κB signaling in articular chondrocytes increased with age, concomitant with the onset of spontaneous OA in wild-type mice. Chondrocyte-specific expression of a constitutively active form of inhibitor of κB kinase ß (IKKß) in young adult mice accelerated the onset of the OA-like phenotype observed in aging wild-type mice, including degenerative changes in the articular cartilage, synovium, and menisci. Both in vitro and in vivo, chondrocytes expressing activated IKKß had a proinflammatory secretory phenotype characterized by markers typically associated with the senescence-associated secretory phenotype (SASP). Expression of these factors was differentially regulated by p65, which contains a transactivation domain, and p50, which does not. Whereas the loss of p65 blocked the induction of genes encoding SASP factors in chondrogenic cells treated with interleukin-1ß (IL-1ß) in vitro, the loss of p50 enhanced the IL-1ß­induced expression of some SASP factors. The loss of p50 further exacerbated cartilage degeneration in mice with chondrocyte-specific IKKß activation. Overall, our data reveal that IKKß-mediated activation of p65 can promote OA onset and that p50 may limit cartilage degeneration in settings of joint inflammation including advanced age.

Increased myocellular lipid and IGFBP-3 expression in a pre-clinical model of pancreatic cancer-related skeletal muscle wasting.

BACKGROUND: Skeletal muscle wasting (SMW) in cancer patients is associated with increased morbidity, mortality, treatment intolerance and discontinuation, and poor quality of life. This is particularly true for patients with pancreatic ductal adenocarcinoma (PDAC), as over 85% experience SMW, which is responsible for ~30% of patient deaths. While the established paradigm to explain SMW posits that muscle catabolism from systemic inflammation and nutritional deficiencies, the cause of death, and the cellular and molecular mechanisms responsible remain to be elucidated. To address this, we investigated the relationship between tumour burden and survival in the KCKO murine PDAC model. METHODS: Female C57BL/6J mice 6-8 weeks of age underwent orthotopic injection with KCKO-luc tumour cells. Solid tumour was verified on Day 5, post-tumour inoculation. In vivo, longitudinal lean mass and tumour burden were assessed via dual-energy X-ray absorptiometry and IVIS imaging, respectively, and total body weight was assessed, weekly. Animals were sacrificed at a designated end point of 'failure to thrive'. After sacrifice, lower limb hind muscles were harvested for histology and RNA extraction. RESULTS: We found a strong correlation between primary tumour size and survival (r2  = 0.83, P < 0.0001). A significant decrease in lower limb lean mass was first detected at Day 38 post-implantation vs. no tumour controls (NTCs) (P < 0.0001). SMW was confirmed by histology, which demonstrated a 38%, 32.7%, and 39.9% decrease in fibre size of extensor digitorum longus, soleus, and tibialis anterior muscles, respectively, in PDAC mice vs. NTC (P < 0.002). Histology also revealed a 67.6% increase in haematopoietic cells within the muscle of PDAC mice when compared with NTC. Bulk RNAseq on muscles from PDAC mice vs. NTC revealed significant increases in c/ebpß/Δ, il-1, il-6, and tnf gene expression. Pathway analyses to identify potential upstream factors revealed increased adipogenic gene expression, including a four-fold increase in igfbp-3. Histomorphometry of Oil Red-O staining for fat content in tibialis anterior muscles demonstrated a 95.5% increase in positively stained fibres from PDAC mice vs. NTC. CONCLUSIONS: Together, these findings support a novel model of PDAC-associated SMW and mortality in which systemic inflammation leads to inflammatory cell infiltration into skeletal muscle with up-regulated myocellular lipids.