Combination breast cancer chemotherapy with doxorubicin and cyclophosphamide damages bone and bone marrow in a female rat model.

PURPOSE: Anthracyclines (including doxorubicin) are still the backbone of commonly used breast cancer chemotherapy regimens. Despite increasing use of doxorubicin and cyclophosphamide (AC) combinations for treating breast cancer, their potential to cause adverse skeletal effects remains unclear. METHODS: This study examined the effects of treatments with the AC regimen on bone and bone marrow in adult female rats. RESULTS: AC treatment for four cycles (weekly intravenous injection of 2 mg/kg doxorubicin and 20 mg/kg cyclophosphamide) resulted in a reduced volume of trabecular bone at the metaphysis, which was associated with reduced serum levels of 25-hydroxy vitamin D3 and alkaline phosphatase. Reductions in densities of osteocytes and bone lining cells were also observed. In addition, bone marrow was severely damaged, including a severe reduction in bone marrow cellularity and an increase in marrow adipocyte content. Accompanying these changes, there were increases in mRNA expression of adipogenesis regulatory genes (PPARγ and FABP4) and an inflammatory cytokine (TNFα) in metaphysis bone and bone marrow. CONCLUSIONS: This study indicates that AC chemotherapy may induce some bone loss, due to reduced bone formation, and bone marrow damage, due to increased marrow adiposity. Preventive strategies for preserving the bone and bone marrow microenvironment during anthracycline chemotherapy warrant further investigation.

Combination chemotherapy with cyclophosphamide, epirubicin and 5-fluorouracil causes trabecular bone loss, bone marrow cell depletion and marrow adiposity in female rats.

The introduction of anthracyclines to adjuvant chemotherapy has increased survival rates among breast cancer patients. Cyclophosphamide, epirubicin and 5-fluorouracil (CEF) combination therapy is now one of the preferred regimens for treating node-positive breast cancer due to better survival with less toxicity involved. Despite the increasing use of CEF, its potential in causing adverse skeletal effects remains unclear. Using a mature female rat model mimicking the clinical setting, this study examined the effects of CEF treatment on bone and bone marrow in long bones. Following six cycles of CEF treatment (weekly intravenous injections of cyclophosphamide at 10 mg/kg, epirubicin at 2.5 mg/kg and 5-flurouracil at 10 mg/kg), a significant reduction in trabecular bone volume was observed at the metaphysis, which was associated with a reduced serum level of bone formation marker alkaline phosphatase (ALP), increased trends of osteoclast density and osteoclast area at the metaphysis, as well as an increased size of osteoclasts being formed from the bone marrow cells ex vivo. Moreover, a severe reduction of bone marrow cellularity was observed following CEF treatment, which was accompanied by an increase in marrow adipose tissue volume. This increase in marrow adiposity was associated with an expansion in adipocyte size but not in marrow adipocyte density. Overall, this study indicates that six cycles of CEF chemotherapy may induce some bone loss and severe bone marrow damage. Mechanisms for CEF-induced bone/bone marrow pathologies and potential preventive strategies warrant further investigation.

Methotrexate-induced bone marrow adiposity is mitigated by folinic acid supplementation through the regulation of Wnt/ß-catenin signalling.

Antimetabolite Methotrexate (MTX) is commonly used in childhood oncology. As a dihydrofolate reductase inhibitor it exerts its action through the reduction of cellular folate, thus its intensive use is associated with damage to soft tissues, bone marrow, and bone. In the clinic, MTX is administered with folinic acid (FA) supplementation to alleviate some of this soft tissue damage. However, whether and how FA alleviates damage to the bone and bone marrow requires further investigation. As the Wnt/ß-catenin signalling pathway is critical for commitment and differentiation of mesenchymal stem cells down the osteogenic or adipogenic lineage, its deregulation has been found associated with increased marrow adiposity following MTX treatment. In order to elucidate whether FA supplementation prevents MTX-induced bone marrow adiposity by regulating Wnt/ß-catenin signalling, young rats were given saline or 0.75 mg/kg MTX once daily for 5 days, receiving saline or 0.75 mg/kg FA 6 h after MTX. FA rescue alleviated the MTX-induced bone marrow adiposity, as well as inducing up-regulation of Wnt10b mRNA and ß-catenin protein expression in the bone. Furthermore, FA blocked up-regulation of the secreted Wnt antagonist sFRP-1 mRNA expression. Moreover, secreted sFRP-1 protein in the bone marrow and its expression by osteoblasts and adipocytes was found increased following MTX treatment. This potentially indicates that sFRP-1 is a major regulator of defective Wnt/ß-catenin signalling following MTX treatment. This study provides evidence that folate depletion caused by MTX chemotherapy results in increased bone marrow adiposity, and that FA rescue alleviates these defects by up-regulating Wnt/ß-catenin signalling in the bone.

Amyloid beta1₋42 (Aß42) up-regulates the expression of sortilin via the p75(NTR)/RhoA signaling pathway.

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co-receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6-month-old swedish-amyloid precursor protein/PS1dE9 transgenic mice. Aß42 enhanced the protein and mRNA expression levels of sortilin in a dose- and time-dependent manner in SH-SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75(NTR) (P75ECD-FC), or the antibody against the extracellular domain of p75(NTR), blocked the up-regulation of sortilin induced by Amyloid-ß protein (Aß), suggesting that Aß42 increased the expression level of sortilin and mRNA in SH-SY5Y via the p75(NTR) receptor. Inhibition of ROCK, but not Jun N-terminal kinase, suppressed constitutive and Aß42-induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aß42 oligomer increases sortilin gene and protein expression through p75(NTR) and RhoA signaling pathways, suggesting a potential physiological interaction of Aß42 and sortilin in Alzheimer's disease.

Methotrexate chemotherapy promotes osteoclast formation in the long bone of rats via increased pro-inflammatory cytokines and enhanced NF-κB activation.

Cancer chemotherapy with methotrexate (MTX) is known to cause bone loss. However, the underlying mechanisms remain unclear. This study investigated the potential role of MTX-induced pro-inflammatory cytokines and activation of NF-κB in the associated osteoclastogenesis in rats. MTX (0.75 mg/kg per day) was administered for 5 days, and bone and bone marrow specimens were collected on days 6, 9, and 14. Compared with a normal control, MTX increased the density of osteoclasts within the metaphyseal bone and the osteoclast formation potential of marrow cells on day 9. RT-PCR analysis of mRNA expression for pro-osteoclastogenic cytokines in the metaphysis indicated that, although the receptor activator of NF-κB ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-α, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-α on day 6 and of IL-6 on day 14. Plasma from treated rats induced osteoclast formation from normal bone marrow cells, which was attenuated by a TNF-α-neutralizing antibody. Indicative of a role for NF-κB signaling, plasma on day 6 increased NF-κB activation in RAW(264.7) cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-κB inhibitor, parthenolide. Our results demonstrate mechanisms for MTX-induced osteoclastogenesis and show that MTX induces osteoclast differentiation by generating a pro-osteoclastogenic environment in both bone and the circulation, specifically with increased TNF-α levels and activation of NF-κB.

Methotrexate chemotherapy reduces osteogenesis but increases adipogenic potential in the bone marrow.

Intensive use of cancer chemotherapy is increasingly linked with long-term skeletal side effects such as osteopenia, osteoporosis and fractures. However, cellular mechanisms by which chemotherapy affects bone integrity remain unclear. Methotrexate (MTX), used commonly as an anti-metabolite, is known to cause bone defects. To study the pathophysiology of MTX-induced bone loss, we examined effects on bone and marrow fat volume, population size and differentiation potential of bone marrow stromal cells (BMSC) in adult rats following chemotherapy for a short-term (five once-daily doses at 0.75 mg/kg) or a 6-week term (5 doses at 0.65 mg/kg + 9 days rest + 1.3 mg/kg twice weekly for 4 weeks). Histological analyses revealed that both acute and chronic MTX treatments caused a significant decrease in metaphyseal trabecular bone volume and an increase in marrow adipose mass. In the acute model, proliferation of BMSCs significantly decreased on days 3-9, and consistently the stromal progenitor cell population as assessed by CFU-F formation was significantly reduced on day 9. Ex vivo differentiation assays showed that while the osteogenic potential of isolated BMSCs was significantly reduced, their adipogenic capacity was markedly increased on day 9. Consistently, RT-PCR gene expression analyses showed osteogenic transcription factors Runx2 and Osterix (Osx) to be decreased but adipogenic genes PPARγ and FABP4 up-regulated on days 6 and 9 in the stromal population. These findings indicate that MTX chemotherapy reduces the bone marrow stromal progenitor cell population and induces a switch in differentiation potential towards adipogenesis at the expense of osteogenesis, resulting in osteopenia and marrow adiposity.

Deregulation of the CXCL12/CXCR4 axis in methotrexate chemotherapy-induced damage and recovery of the bone marrow microenvironment.

Cancer chemotherapy disrupts the bone marrow (BM) microenvironment affecting steady-state proliferation, differentiation and maintenance of haematopoietic (HSC) and stromal stem and progenitor cells; yet the underlying mechanisms and recovery potential of chemotherapy-induced myelosuppression and bone loss remain unclear. While the CXCL12/CXCR4 chemotactic axis has been demonstrated to be critical in maintaining interactions between cells of the two lineages and progenitor cell homing to regions of need upon injury, whether it is involved in chemotherapy-induced BM damage and repair is not clear. Here, a rat model of chemotherapy treatment with the commonly used antimetabolite methotrexate (MTX) (five once-daily injections at 0.75 mg/kg/day) was used to investigate potential roles of CXCL12/CXCR4 axis in damage and recovery of the BM cell pool. Methotrexate treatment reduced marrow cellularity, which was accompanied by altered CXCL12 protein levels (increased in blood plasma but decreased in BM) and reduced CXCR4 mRNA expression in BM HSC cells. Accompanying the lower marrow CXCL12 protein levels (despite its increased mRNA expression in stromal cells) was increased gene and protein levels of metalloproteinase MMP-9 in bone and BM. Furthermore, recombinant MMP-9 was able to degrade CXCL12 in vitro. These findings suggest that MTX chemotherapy transiently alters BM cellularity and composition and that the reduced cellularity may be associated with increased MMP-9 expression and deregulated CXCL12/CXCR4 chemotactic signalling.

Methotrexate toxicity in growing long bones of young rats: a model for studying cancer chemotherapy-induced bone growth defects in children.

The advancement and intensive use of chemotherapy in treating childhood cancers has led to a growing population of young cancer survivors who face increased bone health risks. However, the underlying mechanisms for chemotherapy-induced skeletal defects remain largely unclear. Methotrexate (MTX), the most commonly used antimetabolite in paediatric cancer treatment, is known to cause bone growth defects in children undergoing chemotherapy. Animal studies not only have confirmed the clinical observations but also have increased our understanding of the mechanisms underlying chemotherapy-induced skeletal damage. These models revealed that high-dose MTX can cause growth plate dysfunction, damage osteoprogenitor cells, suppress bone formation, and increase bone resorption and marrow adipogenesis, resulting in overall bone loss. While recent rat studies have shown that antidote folinic acid can reduce MTX damage in the growth plate and bone, future studies should investigate potential adjuvant treatments to reduce chemotherapy-induced skeletal toxicities.

Circulating tumour cell RNA characterisation from colorectal cancer patient blood after inertial microfluidic enrichment.

The detection and molecular analysis of circulating tumour cells (CTCs) potentially provides a significant insight to the characterisation of disease, stage of progression and therapeutic options for cancer patients. Following on from the protocol by Warkiani et al. 2016, which describes a method of enriching CTCs from cancer patient blood with inertial microfluidics, we describe a method to measure the CTC RNA expression in the enriched fraction using droplet digital PCR and compare transcript detection with and without RNA pre-amplification. •Inertial microfluidics combined with droplet digital PCR is advantageous as it allows for CTC enrichment and subsequent RNA analysis from patient blood. This allows for patient tumour analysis with increased sensitivity and precision compared to quantitative Real Time PCR and enables the direct quantification of nucleic acids without the need for tumour biopsy.•This method demonstrates an efficient approach providing important insights into the analysis of colorectal cancer patients' CTCs using a specific gene subset or biomarkers, an approach that may be tailored to tumour type or expanded to larger panels.

Surgical site infection in overweight and obese Total Knee Arthroplasty patients.

PURPOSE: This aim of this study was to evaluate the rate of surgical site infection (SSI) in patients undergoing Total Knee Arthroplasty (TKA), to improve our understanding of the associations between infection rate and obesity. METHODS: Data was reviewed for 839 primary TKA procedures performed at a National Arthroplasty Centre over one year (April 2007-March 2008). SSI data was collected at 30 days and one year post-operatively. Patients were grouped guided by the WHO classifications of obesity; normal (BMI < 25.0), overweight (BMI 25.00-29.99), obese class I (BMI 30.00-34.99), obese class II (BMI 35.00-39.99), obese class III (BMI ≥ 40.00). Statistical significance was assessed by Fisher's Exact Test. RESULTS: When grouped by BMI, 30.9% of patients were obese class I, 19.0% obese class II and 8.7% obese class III. Of the total cohort, 22 patients (2.6%) had superficial SSI and 13 (1.5%) had deep SSI. When comparing the obese class III cohort to all other cohorts (non-obese class III), the odds ratios for superficial SSI was 4.20 (95% CI [1.59, 11.09]; p = 0.009) and deep SSI was 6.97 (95% CI [2.22, 21.89]; p = 0.003). In the obese class III cohort, superficial SSI rate was higher in females (8.9%) than males (5.9%), yet deep SSI demonstrated the opposite, with a higher occurrence in males (11.8%) compared to females (5.4%). CONCLUSION: This study suggests that obese class III TKA patients are at increased odds of superficial and deep SSI compared to other BMI cohorts. Interestingly, male obese class III patients demonstrated a higher rate of deep infection compared to their female counterparts. However, it must be noted that study findings are limited as confounders were unable to be accounted for in this retrospective study design.

Regulatory pathways associated with bone loss and bone marrow adiposity caused by aging, chemotherapy, glucocorticoid therapy and radiotherapy.

The bone marrow is a complex environment that houses haematopoietic and mesenchymal cell populations and regulates bone turnover throughout life. The high proliferative capacity of these cell populations however, makes them susceptible to damage and injury, altering the steady-state of the bone marrow environment. Following cancer chemotherapy, irradiation and long-term glucocorticoid use, reduced bone and increased fat formation of marrow stromal progenitor cells results in a fatty marrow cavity, reduced bone mass and increased fracture risk. These bone and marrow defects are also observed in age-related complications such as estrogen deficiency and increased oxidative stress. Although the underlying mechanisms are yet to be clarified, recent investigations have suggested a switch in lineage commitment of bone marrow mesenchymal stem cells down the adipogenic lineage at the expense of osteogenic differentiation following such stress or injury. The Wnt/ß-catenin signalling pathway is however has been recognized the key mechanism regulating stromal commitment, and its involvement in the osteogenic and adipogenic lineage commitment switch under the damaging conditions has been of great interest. This article reviews the effects of various types of stress or injury on the commitment to the adipogenic and osteogenic lineages of bone marrow stromal progenitor cells, and summarizes the roles of the Wnt/ß-catenin and associated signalling pathways in the lineage commitment, switch, and recovery after damage, and as a therapeutic target.

Microarray expression analysis of genes and pathways involved in growth plate cartilage injury responses and bony repair.

The injured growth plate cartilage is often repaired by a bone bridge which causes bone growth deformities. Whilst previous studies have identified sequential inflammatory, fibrogenic, osteogenic and bone remodelling responses involved in the repair process, the molecular pathways which regulated these cellular events remain unknown. In a rat growth plate injury model, tissue from the injury site was collected across the time-course of bone bridge formation using laser capture microdissection and was subjected to Affymetrix microarray gene expression analysis. Real Time PCR and immunohistochemical analyses were used to confirm changes in levels of expression of some genes identified in microarray. Four major functional groupings of differentially expressed genes with known roles in skeletal development were identified across the time-course of bone bridge formation, including Wnt signalling (SFRP1, SFRP4, ß-catenin, Csnk2a1, Tcf7, Lef1, Fzd1, Fzd2, Wisp1 and Cpz), BMP signalling (BMP-2, BMP-6, BMP-7, Chrd, Chrdl2 and Id1), osteoblast differentiation (BMP-2, BMP-6, Chrd, Hgn, Spp1, Axin2, ß-catenin, Bglap2) and skeletal development (Chrd, Mmp9, BMP-1, BMP-6, Spp1, Fgfr1 and Traf6). These studies provide insight into the molecular pathways which act cooperatively to regulate bone formation following growth plate cartilage injury and highlight potential therapeutic targets to limit bone bridge formation.

Attenuated Wnt/ß-catenin signalling mediates methotrexate chemotherapy-induced bone loss and marrow adiposity in rats.

Cancer chemotherapy often causes significant bone loss, marrow adiposity and haematopoietic defects, yet the underlying mechanisms and recovery potential remain unclear. Wnt/ß-catenin signalling is integral to the regulation of osteogenesis, adipogenesis and haematopoiesis; using a rat model, the current study investigated roles of this signalling pathway in changes to bone marrow stromal and haematopoietic cell differentiation after chemotherapy with methotrexate (MTX), a commonly used antimetabolite. MTX treatment in rats (5 daily administrations at 0.75 mg/kg) has previously been found to decrease bone volume and increase marrow fat, which was associated with increased osteoclastogenesis in haematopoietic cells and with an osteogenesis to adipogenesis switch in bone marrow stromal cells of treated rats. In the current study, on day 6 after the first MTX dose we found that accompanying these changes as well as a suppressed haematopoietic cellularity but increased granulocyte/macrophage differentiation potential, there was an increase in mRNA expression of Wnt antagonists sFRP-1 and Dkk-1 in bone, a reduction in nuclear ß-catenin protein in bone marrow stromal cells, and decreased mRNA levels of ß-catenin target genes lef-1, cyclin D1 and survivin, suggesting reduced activation of Wnt/ß-catenin signalling in the bone during MTX-induced damage. Concurrent administration of BIO, a GSK-3ß inhibitor that stabilises ß-catenin, partially abrogated the MTX-induced transient changes in osteogenic/adipogenic commitment, granulocyte/macrophage lineage differentiation and osteoclast number. These findings demonstrate a potentially important role of Wnt/ß-catenin signalling in MTX chemotherapy-induced cellular changes to the bone marrow microenvironment.