Of note, elevated OPNa accounted for the majority of the increased total OPN in cancer patients [40]. The KrasG12D-LSLp53fl/fl GEMM (genetically engineered mouse model) represents one of the most relevant models of human NSCLC [41]. Biology of tumor progression and efficacy of therapeutic agents have been extensively studied in this model. Intranasal inhalation of viral particles containing Cre-recombinase results in activation of mutated KrasPG12DP and ablation of p53 that in turn lead to tumor formation and progression in the lung reminiscent of lesions observed in cancer

patients with a similar mutation [42]. Therefore, the availability of these mice prompted us to test efficacy of AOM1 on tumor growth and progression. However, repeat-dose treatment of these immuno-competent mice with AOM1, a fully human IgG2, resulted in rapid clearance of the antibody from plasma possibly due to the development of Cell Cycle inhibitor anti-drug antibodies (no changes in AOM1 clearance was observed following repeated treatment of MGCD0103 purchase LY2109761 nmr immune-compromised mice, data not shown). To circumvent this limitation, we modified this tumor model by de novo isolating tumors from the lung of KrasG12D-LSLp53fl/fl GEMMs and implanting them subcutaneously (without any in

vitro manipulation) in immunodeficient scid mice to create KPT (KrasG12D-LSLp53fl/fl Trocar) mice. All the implanted tumors were capable of growth and proliferation in the immunodeficient recipients (Figure 4A). ELISA data showed elevated levels of OPN in plasma in KPT mice suggesting a role for OPN in tumor progression in this model (Figure 4B). FACS data indicated that both tumor cells and PBMCs isolated from animals bearing these tumors express αvβ3 and CD44 receptors further supporting a rationale for treatment of sc-tumors with AOM1 (Figure 4C). Analysis of sc tumor volumes did not reveal any significant difference at the primary site of tumor growth in any of the treatment groups (including AOM1 as single agent or in combination with Carboplatin)

suggesting that OPN may not play an important role Branched chain aminotransferase in tumor growth at the primary site of tumorigenesis (Figure 4D). Figure 4 Characterizing OPN and its receptors in mouse NSCLCs. A Development of KPT model. KrasG12D-LSLp53fl/fl (KP) mice were inhaled with Adeno-CMV-Cre at approximately 8 weeks after birth. Lung tumors were inspected at approximately 18 weeks post-inhalation. Pieces of lung tumors were taken from transgenic mice and were implanted subcutaneously (without any in vitro manipulation) into Scid/beige mice using trocar to generate KPT (KrasG12D-LSLp53fl/fl trocar) model as described in the Materials and Methods. B Tumor implantation results in increased levels of OPN in the plasma in tumor bearing mice. C Using flowcytometry, expression of CD44v6 and αvβ3 was evaluated in KP cells and mPBMCs.