Early skeletal muscle loss in adolescent and young adult cancer patients treated with anthracycline chemotherapy.

BACKGROUND: Early skeletal muscle loss has been observed in adolescent and young adult (AYA) sarcoma patients undergoing treatment. Identification of individuals within the AYA populace that are at greatest risk of anthracycline-induced skeletal muscle loss is unknown. Moreover, investigations which seek out underlying causes of skeletal muscle degradation during chemotherapy are critical for understanding, preventing, and reducing chronic health conditions associated with poor skeletal muscle status. METHODS: Computed tomography (CT) scans were used to investigate changes in skeletal muscle of 153 AYA sarcoma and Hodgkin lymphoma patients at thoracic vertebra 4 after anthracycline treatment. Images were examined at three time points during the first year of treatment. In parallel, we used translational juvenile mouse models to assess the impact of doxorubicin (DOX) in the soleus and gastrocnemius on muscle wasting. RESULTS: Significant reductions in total skeletal muscle index and density were seen after chemotherapy in AYA cancer patients (p < 0.01 & p = 0.04, respectively). The severity of skeletal muscle loss varied by subgroup (i.e., cancer type, sex, and treatment). Murine models demonstrated a reduction in skeletal muscle fiber cross-sectional area, increased apoptosis and collagen volume for both the soleus and gastrocnemius after DOX treatment (all p < 0.05). After DOX, hindlimb skeletal muscle blood flow was significantly reduced (p < 0.01). CONCLUSION: Significant skeletal muscle loss is experienced early during treatment in AYA cancer patients. Reductions in skeletal muscle blood flow may be a key contributing factor to anthracycline doxorubicin induced skeletal muscle loss.

Circulating microRNAs and Cytokines as Prognostic Biomarkers for Doxorubicin-Induced Cardiac Injury and for Evaluating the Effectiveness of an Exercise Intervention.

PURPOSE: To define a set of biomarkers that can be used to identify patients at high risk of developing late doxorubicin (DOX)-induced cardiac morbidity with the goal of focused monitoring and early interventions. EXPERIMENTAL DESIGN: Mice received phosphate buffered saline or DOX 2.5 mg/kg 2x/week for 2 weeks. Blood samples were obtained before and after therapy for quantification of miRNAs (6 and 24 hours), cytokines (24 hours), and troponin (24 hours, 4 and 6 weeks). Cardiac function was evaluated using echocardiography before and 24 hours after therapy. To assess the effectiveness of exercise intervention in preventing DOX-induced cardiotoxicity blood samples were collected from mice treated with DOX or DOX + exercise. Plasma samples from 13 DOX-treated patients with sarcoma were also evaluated before and 24 hours after therapy. RESULTS: Elevations in plasma miRNA-1, miRNA-499 and IL1α, IL1ß, and IL6 were seen in DOX-treated mice with decreased ejection fraction and fractional shortening 24 hours after DOX therapy. Troponin levels were not elevated until 4 weeks after therapy. In mice treated with exercise during DOX, there was no elevation in these biomarkers and no change in cardiac function. Elevations in these biomarkers were seen in 12 of 13 patients with sarcoma treated with DOX. CONCLUSIONS: These findings define a potential set of biomarkers to identify and predict patients at risk for developing acute and late cardiovascular diseases with the goal of focused monitoring and early intervention. Further studies are needed to confirm the predictive value of these biomarkers in late cardiotoxicity.

The Innate Immune System in Cardiovascular Diseases and Its Role in Doxorubicin-Induced Cardiotoxicity.

Innate immune cells are the early responders to infection and tissue damage. They play a critical role in the initiation and resolution of inflammation in response to insult as well as tissue repair. Following ischemic or non-ischemic cardiac injury, a strong inflammatory response plays a critical role in the removal of cell debris and tissue remodeling. However, persistent inflammation could be detrimental to the heart. Studies suggest that cardiac inflammation and tissue repair needs to be tightly regulated such that the timely resolution of the inflammation may prevent adverse cardiac damage. This involves the recognition of damage; activation and release of soluble mediators such as cytokines, chemokines, and proteases; and immune cells such as monocytes, macrophages, and neutrophils. This is important in the context of doxorubicin-induced cardiotoxicity as well. Doxorubicin (Dox) is an effective chemotherapy against multiple cancers but at the cost of cardiotoxicity. The innate immune system has emerged as a contributor to exacerbate the disease. In this review, we discuss the current understanding of the role of innate immunity in the pathogenesis of cardiovascular disease and dox-induced cardiotoxicity and provide potential therapeutic targets to alleviate the damage.

Pramlintide: A Novel Therapeutic Approach for Osteosarcoma through Metabolic Reprogramming.

Despite aggressive combination chemotherapy and surgery, outcomes for patients with osteosarcoma have remained stagnant for more than 25 years, and numerous clinical trials have identified no new therapies. p53 deletion or mutation is found in more than 80% of osteosarcoma tumors. In p53-deficient cancers with structurally altered p63 and p73, interfering with tumor cell metabolism using Pramlintide (an FDA-approved drug for type 2 diabetes) results in tumor regression. Pramlintide response is mediated through upregulation of islet amyloid polypeptide (IAPP). Here, we showed that osteosarcoma cells have altered p63, p73, and p53, and decreased IAPP expression but have the two main IAPP receptors, CalcR and RAMP3, which inhibit glycolysis and induce apoptosis. We showed that in osteosarcoma cells with high- or mid-range glycolytic activity, Pramlintide decreased cell glycolysis, resulting in decreased proliferation and increased apoptosis in vitro. In contrast, Pramlintide had no effect in osteosarcoma cells with low glycolytic activity. Using a subcutaneous osteosarcoma mouse model, we showed that intratumoral injection of Pramlintide-induced tumor regression. Tumor sections showed increased apoptosis and a decrease in Ki-67 and HIF-1α. These data suggest that in osteosarcoma cells with altered p53, p63, and p73 and a high glycolytic function, Pramlintide therapy can modulate metabolic programming and inhibit tumor growth.

Retrospective observational studies in ultra-rare sarcomas: A consensus paper from the Connective Tissue Oncology Society (CTOS) community of experts on the minimum requirements for the evaluation of activity of systemic treatments.

BACKGROUND: In ultra-rare sarcomas (URS) the conduction of prospective, randomized trials is challenging. Data from retrospective observational studies (ROS) may represent the best evidence available. ROS implicit limitations led to poor acceptance by the scientific community and regulatory authorities. In this context, an expert panel from the Connective Tissue Oncology Society (CTOS), agreed on the need to establish a set of minimum requirements for conducting high-quality ROS on the activity of systemic therapies in URS. METHODS: Representatives from > 25 worldwide sarcoma reference centres met in November 2020 and identified a list of topics summarizing the main issues encountered in ROS on URS. An online survey on these topics was distributed to the panel; results were summarized by descriptive statistics and discussed during a second meeting (November 2021). RESULTS: Topics identified by the panel included the use of ROS results as external control data, the criteria for contributing centers selection, modalities for ensuring a correct pathological diagnosis and radiologic assessment, consistency of surveillance policies across centers, study end-points, risk of data duplication, results publication. Based on the answers to the survey (55 of 62 invited experts) and discussion the panel agreed on 18 statements summarizing principles of recommended practice. CONCLUSIONS: These recommendations will be disseminated by CTOS across the sarcoma community and incorporated in future ROS on URS, to maximize their quality and favor their use as control data when results from prospective studies are unavailable. These recommendations could help the optimal conduction of ROS also in other rare tumors.

Doxorubicin-induced cardiotoxicity is mediated by neutrophils through release of neutrophil elastase.

The mechanisms by which Doxorubicin (Dox) causes acute and late cardiotoxicity are not completely understood. One understudied area is the innate immune response, and in particular the role of neutrophils in Dox-induced cardiotoxicity. Here, using echocardiography, flow cytometry and immunofluorescence staining, we demonstrated increased infiltration of neutrophils that correlated with decreased heart function, disruption of vascular structures and increased collagen deposition in the heart after Dox treatment. Depleting neutrophils protected the heart from Dox-induced cardiotoxicity and changes in vascular structure. Furthermore, our data using neutrophil elastase (NE) knock-out mice and the NE inhibitor AZD9668 suggest that neutrophils cause this damage by releasing NE and that inhibiting NE can prevent Dox-induced cardiotoxicity. This work shows the role of neutrophils and NE in Doxorubicin-induced cardiotoxicity for the first time and suggests a new possible therapeutic intervention.

Short-Term Changes in Skeletal Muscle Mass After Anthracycline Administration in Adolescent and Young Adult Sarcoma Patients.

Identification of anthracycline-induced muscle loss is critical for maintaining health in adolescent and young adult (AYA) cancer patients. We used routine chest computed tomography (CT) scans to investigate changes in skeletal muscle of 16 AYA sarcoma patients at thoracic vertebrae 4 (T4) after anthracycline treatment. CT images were examined at three time points (prechemotherapy, postchemotherapy, and 1 year). Significant changes in total skeletal muscle index and density were seen after chemotherapy (p = 0.021 and p = 0.016, respectively) and at 1 year versus baseline (both p < 0.05). This study supports the use of T4 as an early indicator of skeletal muscle loss in AYAs.

Nucleus-mitochondria positive feedback loop formed by ERK5 S496 phosphorylation-mediated poly (ADP-ribose) polymerase activation provokes persistent pro-inflammatory senescent phenotype and accelerates coronary atherosclerosis after chemo-radiation.

The incidence of cardiovascular disease (CVD) is higher in cancer survivors than in the general population. Several cancer treatments are recognized as risk factors for CVD, but specific therapies are unavailable. Many cancer treatments activate shared signaling events, which reprogram myeloid cells (MCs) towards persistent senescence-associated secretory phenotype (SASP) and consequently CVD, but the exact mechanisms remain unclear. This study aimed to provide mechanistic insights and potential treatments by investigating how chemo-radiation can induce persistent SASP. We generated ERK5 S496A knock-in mice and determined SASP in myeloid cells (MCs) by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. Candidate SASP inducers were identified by high-throughput screening, using the ERK5 transcriptional activity reporter cell system. Various chemotherapy agents and ionizing radiation (IR) up-regulated p90RSK-mediated ERK5 S496 phosphorylation. Doxorubicin and IR caused metabolic changes with nicotinamide adenine dinucleotide depletion and ensuing mitochondrial stunning (reversible mitochondria dysfunction without showing any cell death under ATP depletion) via p90RSK-ERK5 modulation and poly (ADP-ribose) polymerase (PARP) activation, which formed a nucleus-mitochondria positive feedback loop. This feedback loop reprogramed MCs to induce a sustained SASP state, and ultimately primed MCs to be more sensitive to reactive oxygen species. This priming was also detected in circulating monocytes from cancer patients after IR. When PARP activity was transiently inhibited at the time of IR, mitochondrial stunning, priming, macrophage infiltration, and coronary atherosclerosis were all eradicated. The p90RSK-ERK5 module plays a crucial role in SASP-mediated mitochondrial stunning via regulating PARP activation. Our data show for the first time that the nucleus-mitochondria positive feedback loop formed by p90RSK-ERK5 S496 phosphorylation-mediated PARP activation plays a crucial role of persistent SASP state, and also provide preclinical evidence supporting that transient inhibition of PARP activation only at the time of radiation therapy can prevent future CVD in cancer survivors.

Exercise intervention decreases acute and late doxorubicin-induced cardiotoxicity.

BACKGROUND: Doxorubicin (Dox) is one of the most effective chemotherapy agents used to treat adolescent and young adult sarcoma patients. Unfortunately, Dox causes cardiotoxicities that compromise long-term survival. We investigated whether exercise prevented cardiotoxicity and increased survival following myocardial infarction. METHODS: Juvenile mice received Dox, Dox + exercise (Exer), Dox then exercise or were exercised during and after Dox. Mice were evaluated by echocardiography and histology immediately after therapy and 12 weeks later. Mice subjected to permanent ligation of the left anterior descending artery 90 days after therapy were assessed for survival at 45 and 100 days. RESULTS: Mice treated with Dox, but not Dox + Exer, had decreased ejection fraction (EF) and fractional shortening (FS) immediately after Dox therapy, which continued to deteriorate over 12 weeks with the development of diastolic failure and fibrosis. Acute Dox-induced cardiotoxicity was documented by induction of autophagy and abnormal mitochondria and vascular architecture with decreased pericytes. These abnormalities persisted 12 weeks after therapy. These acute and late changes were not seen in the Dox + Exer group. Initiating exercise after Dox therapy promoted recovery of EF and FS with no functional or histologic evidence of Dox-induced damage 12 weeks after therapy. Survival rates at 100 days after MI were 67% for control mice, 22% for mice that received Dox alone, and 56% for mice that received Dox + Exer. CONCLUSIONS: Exercise inhibited both early and late Dox-induced cardiotoxicity and increased recovery from an ischemic event. Exercise interventions have the potential to decrease Dox-induced cardiac morbidity.

Exercise Inhibits Doxorubicin-Induced Damage to Cardiac Vessels and Activation of Hippo/YAP-Mediated Apoptosis.

Dose-related cardiomyopathy is a major side effect following doxorubicin (Dox). To investigate whether exercise (Ex)-induced vasculogenesis plays a role in reducing Dox-induced cardiotoxicity, GFP+ bone marrow (BM) cells from GFP transgenic mice were transplanted into wild-type mice. Transplanted mice were treated with Dox, Ex, Dox+Ex, or control. We found Dox therapy resulted in decreased systolic and diastolic blood flow, decreased ejection fraction and fractional shortening, and decreased vascular endothelial cells and pericytes. These abnormalities were not seen in Dox+Ex hearts. Heart tissues from control-, Ex-, or Dox-treated mice showed a small number of GFP+ cells. By contrast, the Dox+Ex-treated hearts had a significant increase in GFP+ cells. Further analyses demonstrated these GFP+ BM cells had differentiated into vascular endothelial cells (GFP+CD31+) and pericytes (GFP+NG2+). Decreased cardiomyocytes were also seen in Dox-treated but not Dox+Ex-treated hearts. Ex induced an increase in GFP+c-Kit+ cells. However, these c-Kit+ BM stem cells had not differentiated into cardiomyocytes. Dox therapy induced phosphorylation of MST1/2, LATS1, and YAP; a decrease in total YAP; and cleavage of caspase-3 and PARP in the heart tissues. Dox+Ex prevented these effects. Our data demonstrated Dox-induced cardiotoxicity is mediated by vascular damage resulting in decreased cardiac blood flow and through activation of Hippo-YAP signaling resulting in cardiomyocyte apoptosis. Furthermore, Ex inhibited these effects by promoting migration of BM stem cells into the heart to repair the cardiac vessels damaged by Dox and through inhibiting Dox-induced Hippo-YAP signaling-mediated apoptosis. These data support the concept of using exercise as an intervention to decrease Dox-induced cardiotoxicity.

Up-regulation of pro-angiogenic molecules and events does not relate with an angiogenic switch in metastatic osteosarcoma cells but to cell survival features.

Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children. Metastases represent a major clinical challenge and an estimated 80% would present undetectable micrometastases at diagnosis. The identification of metastatic traits and molecules would impact in micrometastasis management. We demonstrated that OS LM7 metastatic cells secretome was able to induce microvascular endothelium cell rearrangements, an angiogenic-related trait. A proteomic analysis indicated a gain in angiogenic-related pathways in these cells, as compared to their parental-non-metastatic OS SAOS2 cells counterpart. Further, factors with proangiogenic functions like VEGF and PDGF were upregulated in LM7 cells. However, no differential angiogenic response was induced by LM7 cells in vivo. Regulation of the Fas-FasL axis is key for OS cells to colonize the lungs in this model. Analysis of the proteomic data with emphasis in apoptosis pathways and related processes revealed that the percentage of genes associated with those, presented similar levels in SAOS2 and LM7 cells. Further, the balance of expression levels of proteins with pro- and antiapoptotic functions in both cell types was subtle. Interestingly and of relevance to the model, Fas associated Factor 1 (FAF1), which participates in Fas signaling, was present in LM7 cells and was not detected in SAOS2 cells. The subtle differences in apoptosis-related events and molecules, together with the reported cell-survival functions of the identified angiogenic factors and the increased survival features that we observed in LM7 cells, suggest that the gain in angiogenesis-related pathways in metastatic OS cells would relate to a prosurvival switch rather to an angiogenic switch as an advantage feature to colonize the lungs. OS metastatic cells also displayed higher adhesion towards microvascular endothelium cells suggesting an advantage for tissue colonization. A gain in angiogenesis pathways and molecules does not result in major angiogenic potential. Together, our results suggest that metastatic OS cells would elicit signaling associated to a prosurvival phenotype, allowing homing into the hostile site for metastasis. During the gain of metastatic traits process, cell populations displaying higher adhesive ability to microvascular endothelium, negative regulation of the Fas-FasL axis in the lung parenchyma and a prosurvival switch, would be selected. This opens a new scenario where antiangiogenic treatments would affect cell survival rather than angiogenesis, and provides a molecular panel of expression that may help in distinguishing OS cells with different metastatic potential.

Assessment of drug transporters involved in the urinary secretion of [99mTc]dimercaptosuccinic acid.

INTRODUCTION: We clarified the renal uptake and urinary secretion mechanism of [99mTc]dimercaptosuccinic acid ([99mTc]DMSA) via drug transporters in renal proximal tubules. METHODS: [99mTc]DMSA was added to human embryonic kidney 293 cells expressing human multidrug and toxin extrusion (MATE)1 and MATE2-K, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cation transporter (OCT)2; to Flp293 cells expressing human organic anion transporter (OAT)1 and OAT3; and to vesicles expressing P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)2, MRP4, or breast cancer resistance protein with and without probenecid (OAT inhibitor for both OATs and MRPs). Time activity curves of [99mTc]DMSA with and without probenecid were established using LLC-PK1 cells. Biodistribution and single photon emission computed tomography (SPECT) imaging in mice were conducted using [99mTc]DMSA with and without probenecid. RESULTS: [99mTc]DMSA uptake was significantly higher in Flp293/OAT3 than in mock cells. Uptake via OAT3 was inhibited by probenecid. [99mTc]DMSA uptake into vesicles that highly expressed MRP2 was significantly higher in adenosine triphosphate (ATP) than in adenosine monophosphate (AMP), and probenecid decreased uptake to similar levels as that in AMP. In the time activity curves for [99mTc]DMSA in LLC-PK1 cells, probenecid loading inhibited accumulation from the basolateral side into LLC-PK1 cells, whereas accumulation from the apical side into cells gradually increased. Transport of [99mTc]DMSA from both sides was low. Biodistribution and SPECT imaging studies showed that [99mTc]DMSA with probenecid loading resulted in significantly higher accumulation in blood, heart, liver, and bladder after [99mTc]DMSA injection compared with control mice. Probenecid induced significantly lower accumulation in the kidney after [99mTc]DMSA injection. CONCLUSIONS: [99mTc]DMSA accumulates in renal proximal tubular epithelial cells from blood via OAT3 on the basolateral side, and then a small volume of [99mTc]DMSA will be excreted in urine via MRP2. ADVANCES IN KNOWLEDGE: [99mTc]DMSA accumulates via OAT3 in renal proximal tubular epithelial cells and is slightly excreted from the cells via MRP2. IMPLICATIONS FOR PATIENT CARE: [99mTc]DMSA may be useful for measuring renal transport function with OAT3 in patients.

Bempegaldesleukin (BEMPEG; NKTR-214) efficacy as a single agent and in combination with checkpoint-inhibitor therapy in mouse models of osteosarcoma.

Survival of patients with relapsed/refractory osteosarcoma has not improved in the last 30 years. Several immunotherapeutic approaches have shown benefit in murine osteosarcoma models, including the anti-programmed death-1 (anti-PD-1) and anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) immune checkpoint inhibitors. Treatment with the T-cell growth factor interleukin-2 (IL-2) has shown some clinical benefit but has limitations due to poor tolerability. Therefore, we evaluated the efficacy of bempegaldesleukin (BEMPEG; NKTR-214), a first-in-class CD122-preferential IL-2 pathway agonist, alone and in combination with anti-PD-1 or anti-CTLA-4 immune checkpoint inhibitors in metastatic and orthotopic murine models of osteosarcoma. Treatment with BEMPEG delayed tumor growth and increased overall survival of mice with K7M2-WT osteosarcoma pulmonary metastases. BEMPEG also inhibited primary tumor growth and metastatic relapse in lungs and bone in the K7M3 orthotopic osteosarcoma mouse model. In addition, it enhanced therapeutic activity of anti-CTLA-4 and anti-PD-1 checkpoint blockade in the DLM8 subcutaneous murine osteosarcoma model. Finally, BEMPEG strongly increased accumulation of intratumoral effector T cells and natural killer cells, but not T-regulatory cells, resulting in improved effector:inhibitory cell ratios. Collectively, these data in multiple murine models of osteosarcoma provide a path toward clinical evaluation of BEMPEG-based regimens in human osteosarcoma.

[131I]MIBG exports via MRP transporters and inhibition of the MRP transporters improves accumulation of [131I]MIBG in neuroblastoma.

INTRODUCTION: 131I-labeled m-iodobenzylguanidine ([131I]MIBG) has been used to treat neuroblastoma patients, but [131I]MIBG may be immediately excreted from the cancer cells by the adenosine triphosphate binding cassette transporters, similar to anticancer drugs. The purpose of this study was to clarify the efflux mechanism of [131I]MIBG in neuroblastomas and improve accumulation by inhibition of the transporter in neuroblastomas. METHODS: [131I]MIBG was incubated in human embryonic kidney (HEK)293 cells expressing human organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporter (OAT)1 and OAT2, organic cation transporter (OCT)1 and OCT2, and sodium taurocholate cotransporting polypeptide, and in vesicles expressing P-glycoprotein (MDR1), multidrug resistance associated protein (MRP)1-4, or breast cancer resistance protein with and without MK-571 and probenecid (MRP inhibitors). Time activity curves of [131I]MIBG with and without MK-571 and probenecid were established using an SK-N-SH neuroblastoma cell line, and transporter expression of multiple drug resistance was measured. Biodistribution and SPECT imaging examinations were conducted using [123I]MIBG with and without probenecid in SK-N-SH-bearing mice. RESULTS: [131I]MIBG uptake was significantly higher in OAT1, OAT2, OCT1, and OCT2 than in mock cells. Uptake via OCT1 and OCT2 was little inhibited by MK-571 and probenecid. [131I]MIBG uptake into vesicles that highly expressed MRP1 or MRP4 was significantly higher in ATP than in AMP, and these inhibitors restored uptake to levels similar to that in AMP. Examining the time activity curves for [131I]MIBG in SK-N-SH cells, higher expressions of MDR1, MRP1, MRP4, and MK-571, or probenecid loading produced significantly higher uptake than in control at most incubation times. The ratios of tumors to blood or muscle in SK-N-SH-bearing mice were significantly increased by probenecid loading in comparison with normal mice. CONCLUSIONS: [131I]MIBG exports via MRP1 and MRP4 in neuroblastoma. The accumulation and tumor-to-blood or muscle ratios of [131I]MIBG are improved by inhibition of MRPs with probenecid in neuroblastoma. ADVANCES IN KNOWLEDGE: [131I]MIBG, widely used for treatment of neuroendocrine tumors including neuroblastoma, is excreted via MRP1 and MRP4 in neuroblastoma. IMPLICATIONS FOR PATIENT CARE: Loading with probenecid, OAT, and MRP inhibitors improves [131I]MIBG accumulation.

The Fas/FasL Signaling Pathway: Its Role in the Metastatic Process and as a Target for Treating Osteosarcoma Lung Metastases.

Understanding how the tumor microenvironment participates in inhibiting or supporting tumor growth is critical for the development of novel therapies. Osteosarcoma (OS) metastasizes almost exclusively to the lung, an organ where Fas ligand (FasL) is constitutively expressed. This chapter focuses on our studies dedicated to the interaction of OS cells with the lung microenvironment. We will summarize our studies conducted over the past 20 years showing the importance of the Fas/FasL signaling pathway to the establishment and progression of OS metastases in the lung. We demonstrated that the FasL+ lung microenvironment eliminates Fas-positive (Fas+) OS cells that metastasize to the lungs, through apoptosis induced by Fas signaling following interaction of Fas on the tumor cell surface with FasL on the lung epithelial cells. Expression of the Fas receptor on OS cells inversely correlated with the ability of OS cells to form lung metastases. Blocking this pathway interferes with this process, allowing Fas+ cells to grow in the lung. By contrast, upregulation of Fas on Fas- OS cells inhibited their ability to metastasize to the lung. We demonstrated how the FasL+ lung microenvironment can be leveraged for therapeutic intent through the upregulation of Fas expression. To this end, we demonstrated that the histone deacetylase inhibitor entinostat upregulated Fas expression on OS cells, reduced their ability to form lung metastases, and induced regression of established micrometastases. Fas expression in OS cells is regulated epigenetically by the microRNA miR-20a. We showed that expressions of Fas and miR-20a are inversely correlated, and that delivery of anti-miR-20a in vivo to mice with established osteosarcoma lung metastases resulted in upregulation of Fas and tumor regression. Therefore, targeting the Fas signaling pathway may present therapeutic opportunities, which target the lung microenvironment for elimination of OS lung metastases. We have also shown that in addition to being critically involved in the metastatic potential, the Fas signaling pathway may also contribute to the efficacy of chemotherapy. We demonstrated that the chemotherapeutic agent gemcitabine (GCB) increased Fas expression in both human and mouse OS cells in vitro. In vivo, aerosol GCB therapy induced upregulation of Fas expression and the regression of established osteosarcoma lung metastases. The therapeutic efficacy of GCB was contingent upon a FasL+ lung microenvironment as aerosol GCB had no effect in FasL-deficient mice. Manipulation of Fas expression and the Fas pathway should be considered, as this concept may provide additional novel therapeutic approaches for treating patients with OS lung metastases.

Exosomes: Dynamic Mediators of Extracellular Communication in the Tumor Microenvironment.

It is becoming increasingly recognized that the tumor microenvironment significantly contributes to the development, progression, and metastasis of cancer and also plays a role in response to treatment. The tumor microenvironment is a complex and heterogeneous niche comprised of stromal cells, cancer cells, blood vessels, areas of hypoxia and necrotic tissue, fibrosis, and extracellular matrix. Cellular communication takes place within the tumor microenvironment, both via cell to cell contact, and through extracellular mechanisms such as exosomal signaling. Exosomes are very small membrane-bound vesicles that have been shown to play key roles in the progression of cancer including modulation of the tumor microenvironment through the induction of angiogenesis, the transfer of genetic information that confers drug resistance, and increased cell migration, invasion, proliferation, and survival, as well as the modulation of immune cell interactions. The role of exosomes in several different cancers has been investigated. In the context of osteosarcoma, understanding how exosomes may modulate the tumor microenvironment to support metastatic growth particularly in the lung, the most common site of metastases, may identify novel therapeutic targets for relapsed patients.

Neuronal Repressor REST Controls Ewing Sarcoma Growth and Metastasis by Affecting Vascular Pericyte Coverage and Vessel Perfusion.

Survival rates for Ewing sarcoma (ES) patients with metastatic disease have not improved in over 20 years. Tumor growth and metastasis are dependent on tumor vasculature expansion; therefore, identifying the regulators that control this process in ES may provide new therapeutic opportunities. ES expresses high levels of repressor element 1 silencing transcription factor (REST), which is regulated by the EWS-FLI-1 fusion gene. However, the role of REST in ES growth and the regulation of the tumor vasculature have not been elucidated. To study this role, we established REST-knockout human TC71 ES cell lines through CRISPR/Cas9 recombination. While knockout of REST did not alter tumor cell proliferation in vitro, REST knockout reduced tumor growth and metastasis to the lung in vivo and altered tumor vascular morphology and function. Tumor vessels in the REST-knockout tumors had a punctate appearance with significantly decreased tumor vascular pericytes, decreased perfusion, and increased permeability. REST-knockout tumors also showed increased apoptosis and hypoxia. These results indicate that REST plays a critical role in ES vascular function, which in turn impacts the ability of ES tumors to grow and metastasize. These findings therefore provide a basis for the targeting of REST as a novel therapeutic approach in ES.

Anthracycline-Induced Cardiotoxicity: Causes, Mechanisms, and Prevention.

Doxorubicin is an anthracycline and one of the more effective chemotherapy agents used in the treatment of children, adolescents, and adults with osteosarcoma. Despite its effectiveness, cardiotoxicity is a major late effect that compromises the survival and quality of life of survivors of this and other cancers. Cardiotoxicity is the inability of the heart to pump blood through the body effectively. Doxorubicin-induced cardiotoxicity is dose dependent. Additionally, the age of the patients plays a role in susceptibility with younger patients having a greater risk for cardiotoxicity and heart failure years after treatment is complete. The exact mechanism(s) responsible for doxorubicin-induced cardiotoxicity is poorly understood, and further research needs to be done to elucidate the mechanisms. This chapter summarizes the identified mechanisms that may play a role in anthracycline-induced cardiotoxicity. We will also summarize the types of cardiomyopathies that have been described in survivors treated with doxorubicin and the current recommendations for monitoring survivor for the development of cardiomyopathies. Included will be the important search for defining early biomarkers to identify patients and survivors at risk. Finally, we will summarize some of the interventions proposed for decreasing anthracycline-induced cardiotoxicity.

Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions.

Osteosarcoma metastasizes to the lung, and there is a link between the predominance of tumor-promoting immunosuppressive M2 macrophages in the metastases and poor patient survival. By contrast, M1 macrophage predominance correlates with longer survival. M2 macrophages can be induced by various stimuli in the tumor microenvironment, including exosomes, which are 40- to 150-nm vesicles that are involved in intercellular communication and contribute to tumor progression and immune evasion. Recognizing that tumor cells can influence the tumor microenvironment to make it more permissive and because of the link between M2 dominance and curtailed patient survival, we evaluated the effect of exosomes from non-metastatic K7 and Dunn osteosarcoma cells and the metastatic sublines K7M3 and DLM8 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophages with K7M3 and DLM8 exosomes induced expression of IL10, TGFB2, and CCL22 mRNA (markers of M2 macrophages) and decreased phagocytosis, efferocytosis, and macrophage-mediated tumor cell killing. In contrast, exosomes from non-metastatic K7 or Dunn cells did not inhibit phagocytosis, efferocytosis, and macrophage-mediated cytotoxicity or induce increased expression of IL10, TGFB2 or CCL22 mRNA. In addition, metastatic osteosarcoma cell exosomes significantly increased the secretion of TGFB2, a key signaling pathway associated with tumor- mediated immune suppression. Finally, the inhibition of TGFB2 reversed the suppressive activity of alveolar macrophages exposed to metastatic osteosarcoma cell exosomes. Our data suggest that the exosomes from metastatic osteosarcoma cells can modulate cellular signaling of tumor-associated macrophages, thereby promoting the M2 phenotype and creating an immunosuppressive, tumor-promoting microenvironment through the production of TGFB2.