e22010 Background: The outcomes for children with osteosarcoma and synovial sarcoma remain poor and are even worse for metastatic or relapsed disease. Those who do survive frequently suffer from long-term toxicities from current standard-of-care therapies. It is evident novel therapeutics are warranted to improve the outcomes for patients with these difficult tumors. Stem cell-like cancer cells (SCLCCs) are a subpopulation of tumor cells thought to be responsible for treatment resistance, development of metastases and tumor recurrence, making the targeting of this cell population critical. We sought to evaluate the effect of lerociclib, a CDK4/6 inhibitor, on pediatric sarcoma cell stemness. Methods: We investigated two established human osteosarcoma cells lines (U2-OS and MG-63), two metastatic synovial sarcoma patient-derived xenografts (PDXs) (COA-30 and COA-79) and a metastatic epithelioid sarcoma PDX (COA-171). Cells were treated with the CDK4/6 inhibitor, lerociclib, at concentrations below the known LD 50 . Synovial sarcoma stemness markers Octamer-binding transcription factor 4 (Oct4), homeobox protein Nanog, SOX 2 and nestin were evaluated by qPCR. CD117, a marker of stemness in osteosarcoma cells, was examined by flow cytometry. Finally, long-term passaged U2-OS and MG-63 cells were placed in low attachment serum-free conditions, and tumorsphere formation was evaluated using extreme limiting dilution assay (ELDA) in all cell lines. Results: Lerociclib treatment significantly decreased abundance of Oct4 (by 54%), Nanog (by 29%), SOX 2 (by 60%) and nestin (by 63%) mRNA in COA-30 synovial sarcoma cells. Cell surface expression of CD117 decreased from 17% to 11% in MG-63 cells when treated with lerociclib 2μM. Treatment of all cell types with lerociclib led to significantly decreased tumorsphere formation (Table). Conclusions: Treatment with lerociclib led to a decrease in mRNA abundance in known synovial sarcoma stem cell markers, a decrease in CD117 cell surface expression in osteosarcoma cells and decreased the ability of cells to form tumorspheres. These findings indicate lerociclib leads to decreased sarcoma cell stemness, which plays a key role in tumor progression and recurrence and should be further investigated for potential translation to the clinical setting.[Table: see text]
Neuroblastoma is a pediatric tumor characterized by histologic heterogeneity, and accounts for ~15% of childhood deaths from cancer. The five-year survival for patients with high-risk stage 4 disease has not improved in two decades. We used whole exome sequencing (WES) to identify mutations present in three independent high-risk stage 4 neuroblastoma tumors (COA/UAB-3, COA/UAB -6 and COA/UAB -8) and a stage 3 tumor (COA/UAB-14). Among the four tumors WES analysis identified forty-three mutations that had not been reported previously, one of which was present in two of the four tumors. WES analysis also corroborated twenty-two mutations that were reported previously. No single mutation occurred in all four tumors or in all stage 4 tumors. Three of the four tumors harbored genes with CADD scores ≥20, indicative of mutations associated with human pathologies. The average depth of coverage ranged from 39.68 to 90.27, with >99% sequences mapping to the genome. In summary, WES identified sixty-five coding mutations including forty-three mutations not reported previously in primary neuroblastoma tumors. The three stage 4 tumors contained mutations in genes encoding protein products that regulate immune function or cell adhesion and tumor cell metastasis.
Background Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. Methods and Findings We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). Conclusions The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.
Abstract Patient-derived xenografts (PDXs) provide an opportunity to evaluate the effects of therapies in an environment that more closely resembles the human condition than that seen with long-term passage cell lines. In the current studies, we investigated the effects of FAK inhibition on two neuroblastoma PDXs in vitro . Cells were treated with two small molecule inhibitors of FAK, PF-573,228 (PF) and 1,2,4,5-benzentetraamine tetrahydrochloride (Y15). Following FAK inhibition, cell survival and proliferation decreased significantly and cell cycle arrest was seen in both cell lines. Migration and invasion assays were used to determine the effect of FAK inhibition on cell motility, which decreased significantly in both cell lines in the presence of either inhibitor. Finally, tumor cell stemness following FAK inhibition was evaluated with extreme limiting dilution assays as well as with immunoblotting and quantitative real-time PCR for the expression of stem cell markers. FAK inhibition decreased formation of tumorspheres and resulted in a corresponding decrease in established stem cell markers. FAK inhibition decreased many characteristics of the malignant phenotype, including cancer stem cell like features in neuroblastoma PDXs, making FAK a candidate for further investigation as a potential target for neuroblastoma therapy.
