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.

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.

Background: We generated a CD103⁺DC vaccine using K7M3 OS cell lysates (cDCV) and investigated its ability to induce regression of primary tumors, established lung metastases, and a systemic immune response. Methods: A bilateral tumor model was used to assess cDCV therapy efficacy and systemic immunity induction. K7M3 cells were injected into mice bilaterally. Right-sided tumors received PBS (control) or cDCV. Left-sided tumors were untreated. Tumor growth was compared between the vaccine-treated and untreated tumor on the contralateral side and compared to the control group. The immune cell profiles of the tumors, and tumor-draining lymph nodes (TdLNs) and spleen were evaluated. To determine the efficacy of systemic cDCV therapy against established lung metastases, K7M3 cells were injected intratibially. Leg amputation was performed 5 weeks later. Mice were treated intravenously with PBS or cDCV and euthanized 6 weeks later. Lungs, TdLNs and spleen were collected. The number and size of the lung nodules were quantified. The immune cell profile of tumor, and lymph nodes and spleen were also evaluated. Using this same model, we evaluated the effect of cDCV + anti-CTLA-4. Results: cDCV therapy inhibited the treated and untreated tumors and increased the number of T-cells in these tumors and the lymph nodes compared to control-treated mice. Systemic cDCV therapy administered following amputation decreased the size and number of lung metastases, and increased T-cell numbers in the tumor and lymph nodes. Combining anti-CTLA-4 with cDCV therapy increased cDCV efficacy against lung metastases. Conclusions: Intratumor cDCV generated a systemic immune response inhibiting the growth of both the treated and untreated tumors, with increased T-cells in the tumor and lymph nodes. Systemic cDCV was effective against established lung metastases. Efficacy was increased by anti-CTLA4. cDCVs may provide a novel therapeutic approach for relapsed/metastatic OS patients.

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.