ANGPTL4 Negatively Regulate the Progression of Osteosarcoma by Remodeling Branched-Chain Amino Acid Metabolism

BackgroundAngiopoietin-like-4 (ANGPTL4), a secreted glycoprotein that is mainly recognized as a regulator in lipid metabolism, now, is implied in the regulation of the growth and metastasis of various carcinomas. However, less is known about its functions in the progression of sarcomas, let alone osteosarcoma (OS), which is the most common malignant diagnosed in musculoskeletal system.MethodsThe expression of ANGPTL4 in clinical OS samples and cell lines paired with their controls were analyzed in both mRNA and protein levels. Cell functional analysis including proliferation and colony formation were carried out to detect the roles ANGPTL4 takes in the progress of OS using stable ANGPTL4 overexpression and knockdown HOS/MNNG cell lines. The RNA-Seq and bioinformatics analysis were then employed to discover the BCAA metabolism related signaling which is involved in ANGPTL4 functioning on HOS/MNNG cell growth. Furthermore, BCAAs content measurement, and BCATs rescue experiments were performed to confirm the BCAA/mTOR signaling axis that ANGPTL4 triggered in HOS/MNNG cells. Finally, a xenograft mouse model was carried out to further verify the ANGPTL4 /BCAA/mTOR signaling axis discovered. ResultsWe found that the expression of ANGPTL4 is reduced in clinical OS tissues and cell lines compared to cancellous bone tissues and BMSCs, respectively. The knockdown of ANGPTL4 in HOS/MNNG cells results in enhanced cell growth and clone formation. Moreover, BCAA/mTOR signaling axis were discovered to be triggered by ANGPTL4 down regulation in HOS/MNNG cell using RNA-seq. It was also verified that the accumulation of BCAAs activates the mTOR signaling pathway, and in turn promotes HOS/MNNG cell growth using BCAAs content measurement, and BCAT inhibition. Finally, the IHC results of xenograft mouse model also confirmed this ANGPTL4/BCAA/mTOR signaling axis in vivo.ConclusionsTaken together, our results demonstrate that the expression of ANGPTL4 were negatively related to OS progress. Moreover, it was found the down-regulation of ANGPTL4 promoted OS cell growth via BCAAs/mTOR axis.

Histopathological Tumor and Normal Tissue Responses after 3D-Planned Arc Radiotherapy in an Orthotopic Xenograft Mouse Model of Human Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. Innovative treatment concepts may enhance oncological outcome. Clinically relevant tumor models are essential in developing new therapeutic strategies. In the present study, we used two human PDAC cell lines for an orthotopic xenograft mouse model and compared treatment characteristics between this in vivo tumor model and PDAC patients. Tumor-bearing mice received stereotactic high-precision irradiation using arc technique after 3D-treatment planning. Induction of DNA damage in tumors and organs at risk (OARs) was histopathologically analyzed by the DNA damage marker γH2AX and compared with results after unprecise whole-abdomen irradiation. Our mouse model and preclinical setup reflect the characteristics of PDAC patients and clinical RT. It was feasible to perform stereotactic high-precision RT after defining tumor and OARs by CT imaging. After stereotactic RT, a high rate of DNA damage was mainly observed in the tumor but not in OARs. The calculated dose distributions and the extent of the irradiation field correlate with histopathological staining and the clinical example. We established and validated 3D-planned stereotactic RT in an orthotopic PDAC mouse model, which reflects the human RT. The efficacy of the whole workflow of imaging, treatment planning, and high-precision RT was proven by longitudinal analysis showing a significant improved survival. Importantly, this model can be used to analyze tumor regression and therapy-related toxicity in one model and will allow drawing clinically relevant conclusions.

Development of Anti-CD99scFvs for the Treatment of Acute Myeloid Leukemia

CD99 has gained much attention in recent years as a novel therapeutic target in hematological malignancies, due to its upregulation in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). We have recently shown that targeting CD99 with a knockdown approach or with commercial antibodies results in antileukemia activity in AML cells. We have also developed anti-CD99 nanoparticles and demonstrated excellent in vitro and in vivo antileukemia activity. Here we report the development of human single chain variable fragment targeting CD99 (anti-CD99 scFv) and the preclinical activity in AML cells and in AML xenograft mouse model. The anti-CD99 scFv was developed by inserting CD99scFv DNA sequence into the pFUSE vector, encoding pATL103-CD99 scFvs. To produce anti-CD99 scFv, the plasmids were transfected in Expi293 (HEK293T) cells. Then the medium was harvested and underwent two cycles of dialysis. To determine product purity, proteins were analyzed on SDS-PAGE gel stained with Coomassie blue. For each 240ml cell culture media with recombinant CD99 plasmids, we obtained about 1-2mg anti-CD99 scFv. The binding affinity of CD99 scFv to CD99 surface protein was assessed in 293T cells (CD99 null cells) which exhibited no binding and in MOLM-13 cells and MV4-11 (CD99 positive AML cells) which demonstrated strong binding. Treatment with 5uM of anti-CD99 scFv significantly reduced cell viability in both leukemic cell line MOLM-13 and MV4-11 (MOLM-13: 35.14%, P= 0.008; MV4-11: 30.17%, P=0.002) and primary AML patient blasts (29.37%, P=0.048) compared with control cells. Colony forming assay showed that anti-CD99 scFv treated AML blasts exhibited less number of colonies compared with control cells (plating efficiency (PE): 0.035% vs 0.12%). We also established the in vivo antileukemia activity of anti-CD99 scFv using MOLM-13 cells (FLT3-ITD positive AML cells) NOD scid gamma (NSG) xenograft mouse model. MOLM-13 (2.5x10^6) cells were engrafted into NSG mice via IV tail injection (N=4 mice per group). Mice were treated with PBS (group 1) or 4mg/kg of anti-CD99 scFV on days 10, 14, 18 and 22 post cell engraftment. Mice were euthanized on day 24 and levels of leukemia engraftment were assessed by flow cytometry measurements of huCD45 staining of cells collected from the bone marrow and the peripheral blood and compared between the two groups. Mice treated with four doses of 4mg/kg CD99scFv demonstrated significant reduction in leukemia engraftment in the bone marrow assessed by flow cytometry measurements of huCD45 staining compared with the PBS mice group (huCD45%: 39.7 vs 56, P = 0.0017). In conclusion, we report the development of anti-CD99 single chain variable fragments for the treatment of AML. Our study demonstrates good binding affinity and specificity and a promising preclinical antileukemia activity both in AML cells and in xenograft mouse model. Disclosures Yaghmour: Jazz: Consultancy, Honoraria; Astellas: Consultancy; Takeda: Consultancy; Incyte: Consultancy.