3D Multicellular Stem-Like Human Breast Tumor Spheroids Enhance Tumorigenicity of Orthotopic Xenografts in Athymic Nude Rat Model

Therapeutic targeting of stem cells needs to be strategically developed to control tumor growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes present, including cancer stem cells (CSCs). The development of 3D stem-like properties of human breast tumor spheroids in stem cell factor conditioned media was investigated in orthotopic xenografts for enhanced tumorgenicity in the athymic nude rat model. MCF-7, ZR-75-1, and MDA-MB-231 breast cancer cell lines were cultured in serum-free, stem cell factor-supplemented medium under non-adherent conditions and passaged to generate 3rd generation spheroids. The spheroids were co-cultured with fetal lung fibroblast (FLF) cells before orthotopic heterotransplantation into the mammary fat pads of athymic nude rats. Excised xenografts were assessed histologically by H&E staining and immunohistochemistry for breast cancer marker (ERB1), proliferation marker (Ki67), mitotic marker (pHH3), hypoxia marker (HIF-2α), CSC markers (CD47, CD44, CD24, and CD133), and vascularization markers (CD31, CD34). Breast cancer cells cultured in stem cell factor supplemented medium generated 3D spheroids exhibited increased stem-like characteristics. The 3D stem-like spheroids co-cultured with FLF as supporting stroma reproducibly and efficiently established orthotopic breast cancer xenografts in the athymic nude rat.

Humanized anti-DEspR monoclonal antibody to improve overall survival in xenograft pancreatic peritoneal carcinomatosis nude rat model.

e16262 Background: Pancreatic adenocarcinoma (PDAC) with peritoneal carcinomatosis (PPC) has the worst median overall survival (mOS) among all PDAC metastasis. Novel therapeutic approaches are needed for PPC. The Dual Endothelin-1/VEGF-signal-peptide Receptor (DEspR) is a cell surface receptor which regulates PDAC tumor cell and cancer stem-like cell (CSC) anoikis resistance, tumorigenicity, and tumoral angiogenesis. To translate these findings into a novel anti-cancer therapy, we evaluated the efficacy, empirical safety, pharmacokinetics, and pharmacodynamics of a recombinant, humanized, anti-DEspR hinge-stabilized IgG4S228P monoclonal antibody, hu-6g8, in a PPC xenograft tumor nude rat model. Methods: We used a Panc1 CSC-derived xenograft tumor model of PPC developed via intraperitoneal injection of two million Panc1 CSCs functionally isolated for anoikis resistance. Isolated CSCs had variable DEspR expression, thus modeling CSC-heterogeneity. For efficacy studies, PPC-rats were randomized to receive a single intravenous injection of either 3mg/kg or 15mg/kg hu-6g8, 100mg/kg gemcitabine, or saline, given 3 weeks after CSC injection with palpable PPC tumors. Blood samples were collected to monitor for hematological adverse events (AEs) and vital organs were collected to evaluate for hu-6g8-induced apoptosis. Pharmacokinetics was assessed in a separate study cohort by sequential blood draws after rats received a single dose of 3 mg/kg or 15 mg/kg hu-6g8. In a 3rd cohort, pharmacodynamics were assessed by vital organ collection and immunohistochemistry after rats received either a single-iv injection of 3mg/kg hu-6g8 or IgG4 isotype control. Results: Single dose 15 mg/kg hu-6g8 treatment significantly improved median overall survival (189 days, n = 0.0007) with 3 mg/kg hu-6g8 being comparable to 100 mg/kg gemcitabine (92 vs 115 days, n = 0.62). No hematological AEs were observed in hu-6g8 treated rats, and there was no evidence of increased apoptosis by hu-6g8 in normal tissues by immunohistochemistry of activated Caspase-3. Hu-6g8 demonstrated an average elimination half-life of 46.1 hrs in a two-compartment model. Biodistribution of the antibody showed predominant uptake, albeit heterogenous, and induction of activated caspase 3+ apoptosis in the peritoneal tumors by 24-hours with minimal off-target binding. Conclusions: Treatment with monoclonal hu-6g8 significantly improved survival in a model of PCC with excellent tumor specificity and minimal off-target effect.

Abstract 14554: Angiogenic Agent Ono-1301 Enhanced Cardiac Contractile Proteins in Hipsc Derived Cardiac Tissue-like Construct With Functional Angiogenesis

Introduction: Transplantation of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) into myocardial infarcted areas has shown potential in healing myocardial infarction (MI). However, the survival rate of transplanted cells is yet considered limited, restricting the therapeutic effects of the transplantation. We assumed that adding angiogenic agent ONO-1301 could promote early stage angiogenesis between host and graft, and benefit the therapeutic effects of hiPSC-CM transplantation. Methods: This study is based on our former study of Cardiac Tissue-Like Construct (CTLC), where 5 x10e6 of hiPSC-CM was seeded on a 1x1cm poly(lactic-co-glycolic acid) (PLGA) nano fiber (Figure A). In addition, angiogenic agent ONO-1301 was mixed into the fibers. After 24h culture in vitro, 3 layers of CTLCs were transplanted into Nude rat MI models with or without ONO-1301. The rats were raised for another 4 or 8 weeks before sacrifice. Results: In vitro experiments have shown that supernatant of iPS-CM with ONO-1301 underwent a rise in VEGF during a 3 to 6 day culture (Figure B). The VEGF concentration in ONO-1301 positive group is much higer than ONO-1301 negative group (P=0.022) on day3. This difference narrows down over time. In vivo experiments on nude rat have shown that capillary density on MI border zone of ONO fiber+iPS-CM group is 29.95% higher than that of PLGA fiber+iPS-CM group (P=0.004) (figure C). HE and immunofluorescence staining shows remaining transplanted hiPSC-CMs in ONO fiber+iPS-CM group shows more significant TNT2 and human nuclear antigen(HNA) expression than that of PLGA fiber+iPS-CM group (Figure D and E). Also ONO fiber+iPS-CM group showed functional recovery compared to the sham.

Cartilaginous and osteochondral tissue formation by human mesenchymal stem cells on three-dimensionally woven scaffolds

The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remain important challenges for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSC) in large, three dimensionally woven poly(ε-caprolactone) (PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations: PCL alone, PCL-bone, “point-of- care” seeded MSC-PCL-bone, and chondrogenically pre-cultured Ch-MSC-PCL-bone constructs were implanted in a dorsal, ectopic pouch in a nude rat. After eight weeks, only cells in the Ch- MSC-PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, while both tissue profiles were present, constructs that had been chondrogenically pre- cultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions, and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.