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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. PDL‐1 is expressed in human OS cells and OS patients, and PD‐1 expression is seen on NK cells and macrophages in OS lung metastatic tumors from mice. Anti‐PD1 leads to tumor cell apoptosis, decreased tumor cell proliferation and p‐STAT‐3 signaling, and regression of OS lung metastases, through activated M1 macrophages and reduced M2 macrophages infiltrating the tumors, thus making it a promising approach for OS lung metastasis therapy.

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. This study indicates that exercise prevents Doxorubicin‐induced vascular damage, maintaining cardiac blood flow. Exercise initiated during or after Doxorubicin therapy inhibited the acute and late cardiotoxicities and increased recovery from an ischemic event.

New therapies are urgently needed for patients with osteosarcoma (OS). STAT3 and CD47 are potential therapeutic target in OS. Here we investigated the therapeutic activity of the orally bioavailable STAT3 inhibitor, WP1066, and anti-CD47 antibody using OS mouse models. Cytotoxic effect of WP1066 against OS cell lines and its immunomodulatory effects were evaluated . Experimental metastasis and orthotopic syngeneic mouse models were used to investigate the therapeutic efficacy of WP1066 and anti-CD47 antibody. Further flow cytometric analysis was performed. STAT3 was constitutively activated in multiple human and mouse OS cell lines. WP1066 suppressed STAT3 activation and induced apoptosis. WP1066 reduced the viability and proliferation of MDSCs and increased the expression level of MHC-II, and CD80 in macrophages. We demonstrated that WP1066 monotherapy prolonged the survival of mice with OS lung metastasis using an experimental metastasis and an orthotopic model. The therapeutic effect was significantly increased when WP1066 was combined with anti-CD47. This was associated with increased frequency of activated CD8 T cells, NK cells and macrophages in the lungs and LDLNs. Our preclinical studies support further investigation of targeting STAT3 and CD47 as novel immunotherapeutic approach against OS lung metastasis.

Adolescent and young adults (AYAs) with sarcoma and our mouse model were used to evaluate early and late effects of doxorubicin (Dox) on skeletal muscle and the impact of physical activity on preventing Dox-induced damage. Skeletal muscle changes in 46 AYAs were quantified using computed tomography scans pre- and post- high-dose Dox treatment. Physical activity was monitored via wearable device. Patient blood samples were analyzed for myo-miRNA changes. In mice, we analyzed acute and late effects of Dox vs. Dox + exercise on skeletal muscle histology, Z-disc morphology, collagen deposition, changes in IL-6 and myo-miRNA, and changes in blood flow. During chemotherapy, AYA patients had poor physical activity levels and experienced an average muscle area and density reduction of -13.2% ± 10.9% ( p  < 0.01) and − 10.9% ± 25.6% ( p  = 0.01), respectively. In mice, there was an increase in number of abnormal mitochondria, autophagosomes, and disruption in Z-discs in the hindlimb at both 2 and 12-weeks post Dox. In patients and mice, miR-133b, miR-206, and miR-126 significantly decreased after Dox exposure. In the mouse model, exercise prevented all Dox-induced morphologic changes and acute Dox-induced decrease in muscle blood flow. Dox therapy resulted in skeletal muscle loss and reduction in muscle-specific miRNAs (miR-133b, 206, and 126) in plasma from mice and AYA patients, as well as in mouse muscle tissue. Changes in muscle morphology were not seen in mice that exercised, thus indicating the potential use of exercise as an intervention to prevent Dox-induced muscle damage.

BackgroundType 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.MethodsWe 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.ResultsIn 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.ConclusionsOur 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.

ABSTRACT 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.

BackgroundOsteosarcoma (OS) lung metastases remain a significant therapeutic challenge. Innate immune activation is a promising therapeutic approach. Innate immune agonists can modulate the tumor immune microenvironment and improve therapeutic response.MethodsUsing an experimental syngeneic OS lung metastasis BALB/c mouse model with K7M3-luc OS cells, we evaluated the antitumor effects of yeast-derived particulate β-glucan in prevention and therapeutic settings. We then assessed whether the CD40 agonist (CD40a) in combination with β-glucan increased therapeutic response in two different immune-competent mouse models of OS lung tumor burden.ResultsIn the pretreatment settings, mice treated with β-glucan prior to OS cell infusion developed significantly fewer lung tumor burdens and had increased survival. Pretreatment with β-glucan prevented tumor cell seeding in the lungs. In tumor-bearing mice, β-glucan treatment significantly suppressed tumor growth and prolonged overall survival. β-glucan treatment increased activated pro-inflammatory M1-like macrophages and natural killer (NK) cells secreting interferon-γ and granzyme B in the lungs. Depletion studies showed that the antitumor effect of β-glucan was dependent on macrophages and NK cells. Additionally, β-glucan treatment also induced myelopoiesis in the bone marrow. The therapeutic benefit of β-glucan was further augmented when combined with CD40a. Combination therapy significantly increased the infiltration of activated macrophages, including tumor necrosis factor-α secreting macrophages, and NK cells into the lungs compared with monotherapy. Bulk RNA sequencing of lung tissue revealed that the combination treatment group exhibited enhanced activation of antitumor innate immune pathways.ConclusionsCollectively, our findings demonstrate the antitumor activity of β-glucan against OS lung tumor burden, that combining β-glucan and CD40a increases therapeutic activity, and that this activity is mediated by activation of innate immunity (macrophages and NK cells).

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.

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.

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.