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Background: TC-71 Ewing sarcoma cells overexpress vascular endothelial growth factor (VEGF) with a shift from the 189 to the 165 isoform.Methods: The effect of CAPER-α on the expression of the VEGF isoforms, tumor growth, and vessel density was analyzed after transfection of TC-71 cells with CAPER-α cDNA or siRNA.Results: CAPER-α correlated inversely with the VEGF(165) /VEGF(189) mRNA ratio. Up-regulation of CAPER-α resulted in decreased tumor growth, tumor vessel density, and chemotactic activity of the cell's supernatant. CAPER-α expression was regulated by EWS/FLI-1 through a protein-protein interaction.Conclusions: Increased VEGF(165) expression is secondary to the down-regulation of CAPER-α by EWS/FLI-1. CAPER-α mediates alternative splicing and controls the shift from VEGF(189) to VEGF(165) . [ABSTRACT FROM AUTHOR]

Background: The addition of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) to chemotherapy has been shown to improve overall survival in patients with nonmetastatic osteosarcoma (OS). The authors report the results of addition of liposomal MTP-PE to chemotherapy for patients with metastatic OS.Methods: Intergroup-0133 was a prospective randomized phase 3 trial for the treatment of newly diagnosed patients with OS. The authors compared 3-drug chemotherapy with cisplatin, doxorubicin, and high-dose methotrexate (Regimen A) to the same 3 drugs with the addition of ifosfamide (Regimen B). The addition of liposomal MTP-PE to chemotherapy was evaluated.Results: Five-year event-free survival (EFS) for patients who received liposomal MTP-PE (n = 46) was 42% versus 26% for those who did not (n = 45) (relative risk for liposomal MTP-PE, 0.72; P = .23; 95% confidence interval [CI], 0.42-1.2). The 5-year overall survival for patients who received MTP-PE versus no MTP-PE was 53% and 40%, respectively (relative risk for liposomal MTP-PE, 0.72; P = 0.27; 95% CI, 0.40-1.3). The comparison of Regimen A with Regimen B did not suggest a difference for EFS (35% vs 34%, respectively; relative risk for Regimen B, 1.07; P = .79; 95% CI, 0.62-1.8) or overall survival (52% vs 43%, respectively; relative risk for Regimen B, 1.1, P = .75; 95% CI, 0.61-2.0).Conclusions: When the metastatic cohort was considered in isolation, the addition of liposomal MTP-PE to chemotherapy did not achieve a statistically significant improvement in outcome. However, the pattern of outcome is similar to the pattern in nonmetastatic patients. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the anti-angiogenic family formed by distal splice site selection in the terminal exon, termed VEGFxxxb, where xxx is the amino acid number. The most studied isoforms, VEGF165 and VEGF165b have been shown to be present in tumour and normal tissues respectively. VEGF165b has been shown to inhibit VEGF- and hypoxia-induced angiogenesis, and VEGF-induced cell migration and proliferation in vitro. Here we show that overexpression of VEGF165b by tumour cells inhibits the growth of prostate carcinoma, Ewing's sarcoma and renal cell carcinoma in xenografted mouse tumour models. Moreover, VEGF165b overexpression inhibited tumour cell-mediated migration and proliferation of endothelial cells. These data show that overexpression of VEGF165b can inhibit growth of multiple tumour types in vivo indicating that VEGF165b has potential as an anti-angiogenic, anti-tumour strategy in a number of different tumour types, either by control of VEGF165b expression by regulation of splicing, overexpression of VEGF165b, or therapeutic delivery of VEGF165b to tumours. [ABSTRACT FROM AUTHOR]

Cellular resistance to chemotherapeutic agents is attributable to several mechanisms, including alteration of topoisomerase IIa gene expression. Our previous studies have shown that transient transfection with a vector containing either Drosophila or human topoisomerase IIalpha gene into drug-resistant tumor cells enhanced their drug sensitivity. Furthermore, we constructed a recombinant adenovirus, Ad-hTopoIIalpha, containing the human topoisomerase IIa gene that was able to selectively increase etoposide sensitivity in drug-resistant tumor cells. We also examined Ad-hTopoIIalpha for therapeutic efficacy in vitro using additional etoposide-resistant cell lines, including a mouse breast cancer cell line and a human leukemia cell line. The etoposide-resistant mouse breast cancer cell line FvP, which is derived from FM3A, and etoposide-resistant human breast cancer cell line, MDA-VP, which derived from MDA-P cells showed increased sensitivity to etoposide as well as increased expression of human Topoisomerase IIa mRNA, but this was not seen in FM3A and MDA-P cells. On the other hand, the etoposide-resistant human leukemia cell line K562/MX2 and the parental cell line K562/P did not show enhanced sensitivity against etoposide or an increase in human Topoisomerase IIa mRNA. Using a recombinant adenovirus containing beta-galactosidase gene (Ad-beta-gal), K562 cells were not transducted by the recombinant adenovirus, while both etoposide-sensitive FM3A cells and etoposide resistant FvP cells were transducted by recombinant adenovirus. Ad-hTOP2alpha and etopside treatment showed reduced inoculated tumor weight in the mice. We concluded that a recombinant adenovirus containing the human Topoisomerase IIalpha gene might be a powerful tool for overcoming drug resistance in breast cancer cells, but not in leukemia cells. [ABSTRACT FROM AUTHOR]

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, IL1β, 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., (©2023 American Association for Cancer Research.)

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., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bhagat, Shrestha, Jeyabal, Peng, Watowich and Kleinerman.)

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.

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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Among sarcomas, which are rare cancers, many types are exceedingly rare; however, a definition of ultra-rare cancers has not been established. The problem of ultra-rare sarcomas is particularly relevant because they represent unique diseases, and their rarity poses major challenges for diagnosis, understanding disease biology, generating clinical evidence to support new drug development, and achieving formal authorization for novel therapies., Methods: The Connective Tissue Oncology Society promoted a consensus effort in November 2019 to establish how to define ultra-rare sarcomas through expert consensus and epidemiologic data and to work out a comprehensive list of these diseases. The list of ultra-rare sarcomas was based on the 2020 World Health Organization classification, The incidence rates were estimated using the Information Network on Rare Cancers (RARECARENet) database and NETSARC (the French Sarcoma Network's clinical-pathologic registry). Incidence rates were further validated in collaboration with the Asian cancer registries of Japan, Korea, and Taiwan., Results: It was agreed that the best criterion for a definition of ultra-rare sarcomas would be incidence. Ultra-rare sarcomas were defined as those with an incidence of approximately ≤1 per 1,000,000, to include those entities whose rarity renders them extremely difficult to conduct well powered, prospective clinical studies. On the basis of this threshold, a list of ultra-rare sarcomas was defined, which comprised 56 soft tissue sarcoma types and 21 bone sarcoma types., Conclusions: Altogether, the incidence of ultra-rare sarcomas accounts for roughly 20% of all soft tissue and bone sarcomas. This confirms that the challenges inherent in ultra-rare sarcomas affect large numbers of patients., (© 2021 American Cancer Society.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)

Introduction: We clarified the renal uptake and urinary secretion mechanism of [ 99m Tc]dimercaptosuccinic acid ([ 99m Tc]DMSA) via drug transporters in renal proximal tubules., Methods: [ 99m Tc]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 [ 99m Tc]DMSA with and without probenecid were established using LLC-PK 1 cells. Biodistribution and single photon emission computed tomography (SPECT) imaging in mice were conducted using [ 99m Tc]DMSA with and without probenecid., Results: [ 99m Tc]DMSA uptake was significantly higher in Flp293/OAT3 than in mock cells. Uptake via OAT3 was inhibited by probenecid. [ 99m Tc]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 [ 99m Tc]DMSA in LLC-PK 1 cells, probenecid loading inhibited accumulation from the basolateral side into LLC-PK 1 cells, whereas accumulation from the apical side into cells gradually increased. Transport of [ 99m Tc]DMSA from both sides was low. Biodistribution and SPECT imaging studies showed that [ 99m Tc]DMSA with probenecid loading resulted in significantly higher accumulation in blood, heart, liver, and bladder after [ 99m Tc]DMSA injection compared with control mice. Probenecid induced significantly lower accumulation in the kidney after [ 99m Tc]DMSA injection., Conclusions: [ 99m Tc]DMSA accumulates in renal proximal tubular epithelial cells from blood via OAT3 on the basolateral side, and then a small volume of [ 99m Tc]DMSA will be excreted in urine via MRP2. ADVANCES IN KNOWLEDGE: [ 99m Tc]DMSA accumulates via OAT3 in renal proximal tubular epithelial cells and is slightly excreted from the cells via MRP2. IMPLICATIONS FOR PATIENT CARE: [ 99m Tc]DMSA may be useful for measuring renal transport function with OAT3 in patients., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: 131 I-labeled m-iodobenzylguanidine ([ 131 I]MIBG) has been used to treat neuroblastoma patients, but [ 131 I]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 [ 131 I]MIBG in neuroblastomas and improve accumulation by inhibition of the transporter in neuroblastomas., Methods: [ 131 I]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 [ 131 I]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 [ 123 I]MIBG with and without probenecid in SK-N-SH-bearing mice., Results: [ 131 I]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. [ 131 I]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 [ 131 I]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: [ 131 I]MIBG exports via MRP1 and MRP4 in neuroblastoma. The accumulation and tumor-to-blood or muscle ratios of [ 131 I]MIBG are improved by inhibition of MRPs with probenecid in neuroblastoma. ADVANCES IN KNOWLEDGE: [ 131 I]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 [ 131 I]MIBG accumulation., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103 + cDC1s in mice, CD141 + cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied., Methods: We generated murine CD103 + cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103 + cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103 + cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103 + cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103 + cDC1s., Results: In vitro-generated CD103 + cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103 + cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103 + cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ + CD8 + T cells, and suppressed melanoma and osteosarcoma growth. CD103 + cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103 + cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases., Conclusions: Our data indicate an in vitro-generated CD103 + cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103 + cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Fas-associated protein with death domain (FADD) was first identified for its role in linking death receptors to the apoptotic signaling pathway with subsequent cell death. Later studies reported non-apoptotic functions for FADD in normal cells and cancer cells. Non-apoptotic functions for FADD in osteosarcoma (OS) have not been reported. In this study, FADD protein expression was knocked down in human CCHOSD, LM7, and SaOS2 OS cell lines followed by assessment of sensitivity to TNFα- or TRAIL-induced cell death. Knock down of FADD significantly increased TNFα-induced cell death in LM7 and SaOS2 cell lines. The mode of TNFα-induced cell death was apoptosis and not necroptosis. Inhibition of nuclear factor kappa B (NFκB) in wildtype cells increased TNFα-induced cell death to similar levels observed in FADD knockdown cells, suggesting a role for FADD in NFκB pro-survival cell signaling. In addition, knock down of FADD increased SMAC mimetic-mediated TNFα-induced cell death in all cell lines studied. The results of this study indicate that FADD has a pro-survival function in OS following TNFα treatment that involves NFκB signaling. The results also indicate that the pro-survival function of FADD is associated with XIAP activity., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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.

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.

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.

Recent studies in mouse models of cancer have shown that exercise improves tumor vascular function, thereby improving chemotherapy delivery and efficacy. However, the mechanisms underlying this improvement remain unclear and the effect of exercise on Ewing sarcoma (ES), a pediatric bone and soft tissue cancer, is unknown. The effect of exercise on tumor vascular hyperpermeability, which inversely correlates with drug delivery to the tumor, has also not been evaluated. We hypothesized that exercise improves chemotherapy efficacy by enhancing its delivery through improving tumor vascular permeability. We treated ES-bearing mice with doxorubicin with or without moderate treadmill exercise. Exercise did not significantly alter ES tumor vessel morphology. However, compared to control mice, tumors of exercised mice had significantly reduced hyperpermeability, significantly decreased hypoxia, and higher doxorubicin penetration. Compared to doxorubicin alone, doxorubicin plus exercise inhibited tumor growth more efficiently. We evaluated endothelial cell sphingosine-1-phosphate receptors 1 and 2 (S1PR1 and S1PR2) as potential mediators of the improved vascular permeability and increased function afforded by exercise. Relative to tumors from control mice, vessels in tumors from exercised mice had increased S1PR1 and decreased S1PR2 expression. Our results support a model in which exercise remodels ES vasculature to reduce vessel hyperpermeability, potentially via modulation of S1PR1 and S1PR2, thereby improving doxorubicin delivery and inhibiting tumor growth more than doxorubicin alone does. Our data suggest moderate aerobic exercise should be tested in clinical trials as a potentially useful adjuvant to standard chemotherapy for patients with ES., (© 2019 The Authors. Pediatric Blood & Cancer Published by Wiley Periodicals, Inc.)

Studies of cardiac function in adolescent and young adult survivors have been performed at least a decade after anthracycline exposure, with little knowledge of short-term changes in cardiac function in this age group. To this end, we evaluated cardiac function within a 2-year period among 18 patients who received high-dose anthracyclines for osteosarcoma treatment. At 2 years, there was a significant decline in left ventricular ejection fraction ( p  = 0.005), with 8 of 18 patients having a >10% reduction. There was a significant change in E-wave velocity ( p  = 0.04). To our knowledge, this is the first study assessing short-term change in systolic and diastolic function in osteosarcoma patients receiving anthracyclines.

Indications for immunotherapies are still unclear, and there is a great need for real-time patient immune status monitoring. In this study, we confirmed that the local and systemic immune profiles of an orthotopic osteosarcoma model with or without luciferase transfection were statistically equivalent. Next, we used flow cytometry to describe systemic immune cell populations influenced by osteosarcoma disease progression. When compared to vehicle-inoculated sham mice, it was found that tumor-bearing mice had significant immunophenotype disturbances at approximately 11 weeks after inoculation (at which time 90% of primary tumor-bearing mice have fulminant pulmonary metastases). Percent populations of natural killer cells and T regulatory cells were increased in the spleens of tumor-bearing mice ( p < 0.0083) compared to shams. Additionally, T lymphocytes from spleens of tumor-bearing mice showed increased Tim-3/PD-1 exhaustion status ( p < 0.0083). There were also increases in the percent populations of myeloid cells and overall M1/M2 macrophage marker expression on tumor-bearing mice spleens versus controls ( p < 0.00714). Finally, treatment with 20  μ g α -PD-L1 decreased T-cell exhaustion back to sham status, with a corresponding increase in CTLA-4 expression on cytotoxic T cells in the majority of mice tested. Checkpoint inhibition also increased splenic monocyte maturation and returned macrophage M1/M2 marker expression back to sham status. These data suggest that cancer induces systemic immune dysregulation and that these changes may be elucidated and utilized for treatment purposes by sampling the systemic immune environment via the spleen. In addition, treatment with the checkpoint inhibitor α -PD-L1 may neutralize but not overcome the systemic immunological changes induced by a progressing malignancy.

Osteosarcoma (OS) pulmonary metastasis translates into poor patient survival. The implication of PD-1-PD-L1 pathway in the context of NK cells and/or macrophages in OS is unknown. We investigated the effect of anti-PD-1 in OS lung metastasis and the role of NK cells and/or macrophages in anti-PD-1 responses. A human LM7 OS mouse model was used. Immunohistochemistry for tissues (PD-L1, caspase-3, Ki-67, NK cells, macrophages), and Western blotting for OS lung tumors (p-Stat3, p-Erk1/2) was performed. NK and macrophages were assessed using flow cytometry. NK cell and macrophage depletion were conducted using anti-asialo GM1 and clodrosome, respectively. PD-L1 expression was observed in human OS cells and OS patient lung metastases. Anti-PD1 antibody led to a significant decrease in the number of OS lung metastases, enhanced tumor apoptosis, decreased tumor cell proliferation, and p-STAT-3/p-Erk1/2 signaling blockade in OS lung tumors. NK cells and macrophages in OS lung tumors expressed PD-1 and anti-PD1 increased NK cell and macrophage tumor infiltration. Increased numbers of antitumor M1 macrophages and decreased pro-inflammatory M2 macrophages were seen. NK depletion did not affect therapeutic effect of anti-PD-1, suggesting that NK cells were not directly involved. However, macrophage depletion significantly compromised anti-PD1 efficacy, confirming their role in efficacy of anti-PD-1 against OS lung metastasis. Our findings suggest that OS lung metastases regression by anti-PD1 can be attributed to activated tumor M1 macrophages and reduced M2 macrophages. Owing to the co-relation of M1 macrophages with OS patient outcome, we provide a novel mechanism of PD-1 blockade and a basis for future clinical trials for anti-PD-1 antibodies in OS., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Chemotherapy-induced autophagy is a proposed mechanism of chemoresistance and potential therapeutic target in osteosarcoma. We evaluated heat shock protein 27 (HSP27) and autophagy-related proteins as predictors of pathologic treatment response and prognostic markers among osteosarcoma patients who received standard chemotherapy. We analyzed 394 tumor specimens (pre-treatment, post-treatment, and metastases) from 260 osteosarcoma patients by immunohistochemistry for cytoplasmic light chain 3B (LC3B)-positive puncta, sequestosome 1 (SQSTM1), high mobility group box 1 (HMGB1), and HSP27 expression. The staining percentage and intensity for each marker were scored and the extent to which marker expression was correlated with pathologic response, relapse-free survival (RFS), and overall survival (OS) was assessed. LCB3 + puncta in post-treatment primary tumors (50%) and metastases (67%) was significantly higher than in pre-treatment biopsy specimens (30%; P = 0.023 and <0.001). Among 215 patients with localized osteosarcoma, both pre-treatment [multivariate hazard ratio (HR), 26.7; 95% confidence interval (CI), 1.47-484; P = 0.026] and post-treatment HSP27 expression (multivariate HR, 1.85; 95% CI, 1.03-3.33; P = 0.039) were associated with worse OS. Lack of LC3B + puncta at resection was an independent poor prognostic marker in both univariate (HR, 1.78; 95% CI, 1.05-3.03; P = 0.034) and multivariate models (HR, 1.75; 95% CI, 1.01-3.04; P = 0.045). Patients with LC3B + /HSP27 - tumors at resection had the best 10-year OS (75%) whereas patients with LC3B - /HSP27 + tumors had the worst 10-year survival (25%). Neither HSP27 expression nor the presence of LCB3 + puncta was correlated with pathologic treatment response. Our findings establish HSP27 expression and LC3B + puncta as independent prognostic markers in osteosarcoma patients receiving standard chemotherapy and support further investigation into strategies targeting HSP27 or modulating autophagy in osteosarcoma treatment. Mol Cancer Ther; 17(6); 1315-23. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: In newly diagnosed osteosarcoma (OS) patients, the time between surgery and resumption of chemotherapy is 2-7 weeks. Delays > 16 days are associated with increased risk of relapse and decreased overall survival. Identifying an effective therapy that can be used postoperatively may prevent relapse. We investigated whether aerosol gemcitabine (GCB) initiated after tumor resection inhibited the growth of OS lung metastases without affecting the wound-healing process., Methods: Mice were injected intratibially with OS cells. Amputation was performed when the tumor reached 1.5 cm. Full-thickness excisional wounds were also made on the dorsal skin and tail. Aerosol GCB or PBS was initiated 48 hours after amputation (3 times/week for 3 weeks). Wound sections were evaluated by immunohistochemistry for Ki-67 (proliferation), CD31 (vessels), VEGF, IL-10, bFGF, mast cells, macrophages, and M1/M2 macrophage ratios. The lungs were analyzed for macro- and micrometastases., Results: Aerosol GCB inhibited the growth of the lung metastases but had no effect on the 3 phases of wound healing in the dorsal skin, tail, or bone. Production of cytokines at the wound sites was the same., Conclusion: These data indicate that initiating aerosol GCB postoperatively may kill residual lung metastases thereby preventing relapse and improve survival.

Obesity at diagnosis is a negative prognostic indicator for several pediatric cancers including acute leukemia and bone tumors. Incidence of obesity in children has increased three-fold over the past 2 decades, and causes for this include poor diet, excessive caloric intake, and lack of physical activity, which are collectively referred to as energy balance-related behaviors. Few energy balance interventions have been implemented in pediatric cancer patients during treatment, and here we will probe the rationale for pursuing such studies. The need to modify composition of calories consumed and to identify specific beneficial exercise regimens will be discussed, relative to weight reduction or management.

The metastatic potential of osteosarcoma cells is inversely correlated to cell surface FAS expression. Downregulation of FAS allows osteosarcoma cells to escape FAS ligand-mediated apoptosis when they enter a FAS ligand-positive microenvironment such as the lung. We have previously demonstrated that miR-20a, encoded by the miR-17-92 cluster, downregulates FAS expression in osteosarcoma. We further demonstrated an inverse correlation between FAS expression and miR-20a expression. However, the mechanism of FAS regulation by miR-20a was still unclear. The purpose of the current study was to evaluate the mechanism of FAS regulation by miR-20a in vitro and test the effect of targeting miR-20a in vivo We investigated whether miR-20a's downregulation of FAS was mediated by binding to the 3'-untranslated region (3'-UTR) of FAS mRNA with the consequent induction of mRNA degradation or translational suppression. We identified and mutated two miR-20a binding sites on the FAS mRNA 3'-UTR. Using luciferase reporter assays, we demonstrated that miR-20a did not bind to either the wild-type or mutated FAS 3'-UTR. In contrast, overexpression of miR-20a resulted in downregulation of FAS promoter activity. Similarly, the inhibition of miR-20a increased FAS promoter activity. The critical region identified on the FAS promoter was between -240 bp and -150 bp. Delivery of anti-miR-20a in vivo using nanoparticles in mice with established osteosarcoma lung metastases resulted in upregulation of FAS and tumor growth inhibition. Taken together, our data suggest that miR-20a regulates FAS expression through the modulation of the FAS promoter and that targeting miR-20a using anti-miR-20a has therapeutic potential. Mol Cancer Ther; 17(1); 130-9. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Autophagy is a catabolic process involved in cellular homeostasis. Autophagy is increased above homeostatic levels by chemotherapy, and this can either promote or inhibit tumor growth. We previously demonstrated that aerosol gemcitabine (GCB) has a therapeutic effect against osteosarcoma (OS) lung metastases. However, some tumor cells failed to respond to the treatment and persisted as isolated lung metastasis. Here, we examined the mechanisms underlying the dual role of chemotherapy-induced autophagy in OS and sought to identify biomarkers to predict OS response to treatment. In this study, we demonstrate that treatment of various OS cells with GCB induced autophagy. We also showed that GCB reduces the phosphorylation of AKT, mTOR and p70S6K and that GCB-induced autophagy in OS can lead to either cell survival or cell death. Blocking autophagy enhanced the sensitivity of LM7 OS cells and decreased the sensitivity of CCH-OS-D and K7M3 OS cells to GCB. Using a kinase array, we also demonstrated that differences in the phosphorylated heat shock protein 27 (p-HSP27) expression in the various OS cell lines after treatment with GCB, correlates to whether chemotherapy-induced autophagy will lead to increase or decrease OS cells sensitivity to therapy. Increased p-HSP27 was associated with increased sensitivity to anticancer drug treatment when autophagy is inhibited. The results of this study reveal a dual role of autophagy in OS cells sensitivity to chemotherapy and suggest that p-HSP27 could represent a predictive biomarker of whether combination therapy with autophagy modulators and chemotherapeutic drugs will be beneficial for OS patients., Competing Interests: CONFLICTS OF INTEREST There are no conflicts of interest to disclose.

Osteosarcoma occurs predominantly in children and young adults. High-grade tumors require multidisciplinary treatment consisting of chemotherapy in the neoadjuvant and adjuvant settings, along with surgical intervention. Despite this approach, death from respiratory failure secondary to the development and progression of pulmonary metastases remains a significant problem. Here, we identify the IL-11 receptor α subunit (IL-11Rα) as a cell surface marker of tumor progression that correlates with poor prognosis in patients with osteosarcoma. We also show that both IL-11Rα and its ligand, IL-11, are specifically up-regulated in human metastatic osteosarcoma cell lines; engagement of this autocrine loop leads to tumor cell proliferation, invasion, and anchorage-independent growth in vitro. Consistently, IL-11Rα promotes lung colonization by human metastatic osteosarcoma cells in vivo in an orthotopic mouse model. Finally, we evaluate the IL-11Rα-targeted proapoptotic agent bone metastasis-targeting peptidomimetic (BMTP-11) in preclinical models of primary intratibial osteosarcomas, observing marked inhibition of both tumor growth and lung metastases. This effect was enhanced when BMTP-11 was combined with the chemotherapeutic drug gemcitabine. Our combined data support the development of approaches targeting IL-11Rα, and establish BMTP-11 as a leading drug candidate for clinical translation in patients with high-risk osteosarcoma., Competing Interests: Conflict of interest statement: R.P. and W.A. are founders and equity stockholders of Alvos Therapeutics (Arrowhead Pharmaceuticals Research Corporation). Arrowhead Pharmaceuticals has licensed rights to patents and technologies described in this article. R.P. and W.A. are inventors on patent applications and intellectual property related to this work and will be entitled to standard royalties if licensing and/or commercialization occurs. The University of New Mexico Health Sciences Center manages and monitors these arrangements, along with the University of Texas M.D. Anderson Cancer Center.

There is a crucial need for a new therapeutic approach for osteosarcoma (OS) lung metastasis since this disease remains the main cause of mortality in OS. We previously demonstrated that natural killer (NK) cell therapy has minimal efficacy against OS metastasis. This study determined whether the histone deacetylase inhibitor entinostat could immunosensitize OS cells to NK cell lysis and increases the efficacy of NK cell therapy for OS lung metastasis. Entinostat upregulated ligands for NK cell-activating receptors (major histocompatibility complex [MHC] class I polypeptide-related chain A [MICA] and B [MICB]; UL16 binding proteins 1, 2, 5, and 6; and CD155) on OS cells both in vitro and in vivo and led to more susceptibility to NK cell-mediated cytotoxicity in vitro . Importantly, entinostat did not change NK cell viability, receptor expression, or function within the 24-h treatment. We also demonstrated two potential mechanisms by which entinostat enhanced expression of MICA and MICB on OS cells. Although entinostat upregulated ligands for the NK cell activating receptor on OS lung metastasis, it failed to augment the efficacy of NK cell therapy in our nude mouse human OS lung metastasis model. This can be partly explained by our finding that although the infused NK cells were active and functional and could penetrate into the lungs, they failed to infiltrate into the lung nodules. These challenges regarding cellular immunotherapy against solid tumors may be overcome by combination therapy, such as adding a NK cell-activating cytokine (IL-2 or IL-21).

Osteosarcoma (OS) is the most common primary malignancy of bone and patients with metastatic disease or recurrences continue to have very poor outcomes. Unfortunately, little prognostic improvement has been generated from the last 20 years of research and a new perspective is warranted. OS is extremely heterogeneous in both its origins and manifestations. Although multiple associations have been made between the development of osteosarcoma and race, gender, age, various genomic alterations, and exposure situations among others, the etiology remains unclear and controversial. Noninvasive diagnostic methods include serum markers like alkaline phosphatase and a growing variety of imaging techniques including X-ray, computed tomography, magnetic resonance imaging, and positron emission as well as combinations thereof. Still, biopsy and microscopic examination are required to confirm the diagnosis and carry additional prognostic implications such as subtype classification and histological response to neoadjuvant chemotherapy. The current standard of care combines surgical and chemotherapeutic techniques, with a multitude of experimental biologics and small molecules currently in development and some in clinical trial phases. In this review, in addition to summarizing the current understanding of OS etiology, diagnostic methods, and the current standard of care, our group describes various experimental therapeutics and provides evidence to encourage a potential paradigm shift toward the introduction of immunomodulation, which may offer a more comprehensive approach to battling cancer pleomorphism.

Natural killer (NK) cells have therapeutic potential for cancer due to their capacity for targeting tumor cells without prior sensitization. Our laboratory has developed an NK cell expansion protocol that generates large quantities of NK cells for therapeutic infusion that secret 20 times the amount of interferon gamma (IFNγ) than resting NK cells. IFNγ can upregulate major histocompatibility complex (MHC)-class I, an inhibitory ligand for NK cells, but can also upregulate intercellular adhesion molecule 1 (ICAM-1) which promotes NK:target cell interaction for an efficient lysis. Due to the opposing effects reported for IFNγ on tumor sensitivity to NK cells, we evaluated a panel 22 tumor cell lines from the pediatric preclinical testing program corresponding to different tumor types. We determined the impact of IFNγ on their expression of NK cell activating and inhibitory ligands, death receptors, and adhesion molecules using mass cytometry. We also evaluated the effect of IFNγ on their sensitivity to NK cell-mediated lysis. Our results show upregulation of PD-L1, ICAM-1, MHC-class I, HLA-DR, CD95/FasR, and CD270/HVEM after IFNγ treatment, this upregulation is variable across different tumor types. We also observed a variable impact of IFNγ in NK cell-mediated lysis. For six of the cancer cell lines IFNγ resulted in increased resistance to NK cells, while for three of them it resulted in increased sensitivity. Modeling of the data suggests that the effect of IFNγ on NK cell-mediated tumor lysis is mostly dependent on changes in MHC-class I and ICAM-1 expression. For three of the cell lines with increased resistance, we observed higher upregulation of MHC-class I than ICAM-1. For the cell lines with increased sensitivity after IFNγ treatment, we observed upregulation of ICAM-1 exceeding MHC-class I upregulation. ICAM-1 upregulation resulted in increased conjugate formation between the NK cells and tumor cells, which can contribute to the increased sensitivity observed. However, the effects of MHC-class I and ICAM-1 are not readily predictable. Due to the high IFNγ secretion of NK cell infusion products, a better understanding of the NK ligands on tumor cells and how they are affected by IFNγ is essential to optimize NK cell immunotherapy.

NKG2D-mediated immune surveillance is crucial for inhibiting tumor growth and metastases. Malignant tumor cells often downregulate NKG2D ligands to escape from immune surveillance. High-profile studies have shown that restoring NKG2D ligand expression via genetic engineering inhibits tumor formation and progression. However, no effective in vivo approaches are available to restore these ligands across different types of solid tumors because the classic stress signal-dependent induction of this ligand in vitro is transient and has rarely been duplicated in solid tumors in vivo We found that coadministration of an immune stimulatory signal (IL12) and chemotherapy (doxorubicin) restored the NKG2D ligand Rae-1 in multiple tumor types, including a human tumor model. The restored expression of NKG2D ligands was associated with tumor cell death and delay of tumor progression in vivo Induction of tumor-specific NKG2D ligands required the engagement of CD8 + T cells and was regulated by the histone acetyltransferases GCN5 and PCAF. The tumor-specific restoration of NKG2D ligands in a variety of tumor models, including a human tumor model, resulted in NKG2D-dependent tumor regression and extended survival time. The elucidation of a CD8 + T cell-dependent mechanism suggests that activated NKG2D + CD8 + T-cell therapy alone may be able to restore the NKG2D ligand in tumors. Cancer Immunol Res; 5(4); 300-11. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: p53 is mutated in about 50% of human cancers, mostly through missense mutations. Expression of mutant p53 is associated with poor clinical outcomes or metastasis. Although mutant p53 is inherently instable, various stressors such as DNA damage or expression of the oncogenic Kras or c-myc affect the oncogenic properties of mutant p53. However, the effects of inflammation on mutant p53 are largely unknown. IL27 is an important immunomodulatory cytokine, but its impact on mutant p53-driven tumorigenesis has not been reported., Experimental Design: IL27RA(-/-) mice were bred with mutant p53 heterozygous (p53(R172H/+)) mice to obtain IL27RA(-/-)p53(H/+) and IL27RA(-/-)p53(H/H) mice. Mouse survival and tumor spectra for the cohort were analyzed. Stability of p53 protein was analyzed via IHC and Western blot analysis., Results: This study unraveled that lack of IL27 signaling significantly shortened the survival duration of mice with tumors expressing both copies of the mutant p53 gene (Li-Fraumeni mouse model). Interestingly, in mice that were heterozygous for mutant p53, lack of IL27 signaling not only significantly shortened survival time but also doubled the incidence of osteosarcomas. Furthermore, lack of IL27 signaling is closely associated with increased mutant p53 stability in vivo from early age., Conclusions: These results suggest that IL27 signaling modulates the oncogenic properties of mutant p53 in vivo Clin Cancer Res; 22(15); 3876-83. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: Natural killer (NK) cell cytotoxicity correlates with the ligation of activating receptors (e.g., NKG2D) by their ligands (e.g., MHC class I-related chains [MIC] A and B) on target cells. Histone deacetylase inhibitors (HDACi) at high concentrations inhibit tumor growth and can increase NKG2D ligand expression on tumor targets, but are widely regarded as toxic to NK cells., Methods: We investigated the mechanism of entinostat, a benzamide-derivative narrow-spectrum HDACi, in augmenting the cytotoxicity of NK cells against human colon carcinoma and sarcoma by assessing gene and protein expression, histone acetylation, and cytotoxicity in in vitro and murine models., Results: We observed that entinostat dose- and time-dependent increase in MIC expression in tumor targets and NKG2D in primary human NK cells, both correlating with increased acetylated histone 3 (AcH3) binding to associated promoters. Entinostat pretreatment of colon carcinoma and sarcoma cells, NK cells, or both led to enhanced overall cytotoxicity in vitro, which was reversed by NKG2D blockade, and inhibited growth of tumor xenografts. Lastly, we showed decreased expression of MICA and ULBP2 transcription in primary human osteosarcoma., Conclusions: Entinostat enhances NK cell killing of cancer cells through upregulation of both NKG2D and its ligands, suggesting an attractive approach for augmenting NK cell immunotherapy of solid tumors such as colon carcinoma and sarcomas.

Despite successful primary tumor treatment, the development of pulmonary metastasis continues to be the most common cause of mortality in patients with osteosarcoma. A conventional drug development path requiring drugs to induce regression of established lesions has not led to improvements for patients with osteosarcoma in more than 30 years. On the basis of our growing understanding of metastasis biology, it is now reasonable and essential that we focus on developing therapeutics that target metastatic progression. To advance this agenda, a meeting of key opinion leaders and experts in the metastasis and osteosarcoma communities was convened in Bethesda, Maryland. The goal of this meeting was to provide a "Perspective" that would establish a preclinical translational path that could support the early evaluation of potential therapeutic agents that uniquely target the metastatic phenotype. Although focused on osteosarcoma, the need for this perspective is shared among many cancer types. The consensus achieved from the meeting included the following: the biology of metastatic progression is associated with metastasis-specific targets/processes that may not influence grossly detectable lesions; targeting of metastasis-specific processes is feasible; rigorous preclinical data are needed to support translation of metastasis-specific agents into human trials where regression of measurable disease is not an expected outcome; preclinical data should include an understanding of mechanism of action, validation of pharmacodynamic markers of effective exposure and response, the use of several murine models of effectiveness, and where feasible the inclusion of the dog with naturally occurring osteosarcoma to define the activity of new drugs in the micrometastatic disease setting., (©2014 American Association for Cancer Research.)

Background: We have previously shown that aerosol interleukin-2 (IL-2) increased the number of intravenously injected human natural killer (NK) cells in the lungs. In this study we investigated whether this increase was secondary to NK cell proliferation and determined the site of the proliferation., Materials and Methods: Nude mice with osteosarcoma lung metastases were injected with NK cells and treated with aerosol IL-2 or aerosol PBS. BrdU was injected prior to euthanasia to identify proliferating NK cells. The percentage of proliferating NK cells in the lung, bone marrow, spleen, and liver was determined using flow cytometry. Survival studies for mice with osteosarcoma lung metastasis treated with aerosol PBS, aerosol IL-2 alone, aerosol PBS plus NK cells, and aerosol IL-2 plus NK cells were also performed., Results: Treatment with aerosol IL-2 induced the proliferation of injected NK cells in the lung. Aerosol IL-2 did not increase the proliferation of NK cells in the spleen and liver. Treatment with aerosol IL-2 and aerosol IL-2 plus NK cells increased the overall survival of mice with osteosarcoma lung metastasis., Conclusion: Aerosol IL-2 increases lung NK cell numbers by stimulating local NK cell proliferation. Aerosol IL-2's effect on NK cell proliferation is organ specific, which makes it ideal for the specific targeting of lung metastasis. Aerosol IL-2 plus NK cell therapy induced metastatic regression and increased overall survival demonstrating the potential of this therapeutic approach for patients with osteosarcoma., (© 2014 Wiley Periodicals, Inc.)

Background: Children (patients ≤ 18 years of age) are not usually included on pharmaceutical industry sponsored Phase I trials., Methods: We reviewed the medical records of 40 patients ≤ 18 years treated in ≥ 1 phase I trial at MD Anderson., Results: The median OS was 8.5 months (95% CI, 5.5-13.2 months). In the multivariate analysis, age ≥15 only predicted increased OS (P = 0.0065), and >3 prior therapies (P = 0.053) predicted decreased OS. The median PFS was 2.8 months (95% CI, 2.3-4.1 months). In the multivariate analysis, independent factors that predicted increased PFS were age ≥15 years (P < 0.001) and prior radiation therapy (P = 0.049); performance status >1 (P < 0.001) and >3 prior therapies (P = 0.002) predicted decreased PFS. RMH score ≥ 2 and MDACC score ≥ 3 were associated with decreased median OS (P = 0.029 and P = 0.031 respectively)., Conclusions: It is feasible to conduct phase I studies in pediatric patients based on adult protocols. In the era of targeted therapy more trials should allow pediatric patients earlier in the drug development especially if deemed safe in adults in early phase trials., Translational Relevance: Most pharmaceutical industry sponsored trials exclude patients less than 18 years in phase I clinical trials. Even in the era of targeted therapy pediatric patients usually have to wait for most phases of trials to be completed in adults before being allowed to enroll in clinical trials of new therapies, even in the advanced metastatic and relapsed setting. Some investigator initiated phase 1 trials of combinations of US FDA approved agents allow patients less than 18 years. We report the preliminary analyses of the outcomes of pediatric patients enrolled in phase I studies initially designed for adults, but allowing for enrollment of patients under 18.

Background: Survival of patients with osteosarcoma lung metastases has not improved in 20 years. We evaluated the efficacy of combining natural killer (NK) cells with aerosol interleukin-2 (IL-2) to achieve organ-specific NK cell migration and expansion in the metastatic organ, and to decrease toxicity associated with systemic IL-2., Procedure: Five human osteosarcoma cell lines and 103 patient samples (47 primary and 56 metastatic) were analyzed for NKG2D ligand (NKG2DL) expression. Therapeutic efficacy of aerosol IL-2 + NK cells was evaluated in vivo compared with aerosol IL-2 alone and NK cells without aerosol IL-2., Results: Osteosarcoma cell lines and patient samples expressed various levels of NKG2DL. NK-mediated killing was NKG2DL-dependent and correlated with expression levels. Aerosol IL-2 increased NK cell numbers in the lung and within metastatic nodules but not in other organs. Therapeutic efficacy, as judged by tumor number, size, and quantification of apoptosis, was also increased compared with NK cells or aerosol IL-2 alone. There were no IL-2-associated systemic toxicities., Conclusion: Aerosol IL-2 augmented the efficacy of NK cell therapy against osteosarcoma lung metastasis, without inducing systemic toxicity. Our data suggest that lung-targeted IL-2 delivery circumvents toxicities induced by systemic administration. Combining aerosol IL-2 with NK cell infusions, may be a potential new therapeutic approach for patients with osteosarcoma lung metastasis., (© 2013 Wiley Periodicals, Inc.)

To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse., (©2014 AACR.)