Abstract 2971: Human extramedullary bone and bone marrow in mice: First in vivo model of a genetically controlled hematopoietic environment - Role of CTGF and HIF1-α
Authors: Andreeff Michael , Chen Ye , Battula Venkata Lokesh , Hofmann Nicole A. , Reinisch Andreas , Shi Yue Xi , Wang Rui Yu , Konoplev Sergej , Strunk Dirk , Konopleva Marina 
Published: August 2012

The microenvironment contributes and may regulate cancer development, progression and resistance to treatment. Our group reported first the contribution of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) for tumor development and metastasis. Accumulating evidence has shown that the BM microenvironment also plays a pivotal role in the pathophysiology and propagation of leukemia. We here describe a novel, genetically controlled bone and bone marrow model using human BM derived MSCs and endothelial colony-forming cells (ECFCs) subcutaneously injected into the flanks of NOD/SCID/IL-2rαnull mice, where they developed into bone-like tissues with high osteoblast activity after 10 weeks, with bone structures and typical bone marrow cavities constituting a robust hematopoietic environment. In vivo imaging with Osteosense confirmed the presence of hydroxylapatite, and luciferase imaging of firefly-luciferase labeled human leukemic cells demonstrated the engraftment of human MOLM13/Luc/GFP leukemic cells. The extramedullary leukemic BM was markedly hypoxic, as shown by Pimonidazole staining. Factors critical for MSC to support normal and leukemic hematopoiesis are largely unknown and cannot readily be studied since human MSC do not engraft reliably in xenograft models. We therefore investigated the possibility of genetically modifying MSC in this system and found a significant reduction (50 ± 6%, p&lt;0.001) in leukemial engraftment in extramedullary BM generated with HIF1α knockdown MSCs (1449 ± 194 cells/mm2), compared to vector controls (3037 ± 496 cells/mm2). This finding indicates that HIF1α expression in stromal cells is critical for the engraftment of leukemic cells. Surprisingly, shRNA knockdown of connective tissue growth factor (CTGF) in MSC resulted in the 2-3 fold increased expression of embryonic stem cell markers oct-4, nanog and sox2, increased adipogenic differentiation (PPAR-γ and C/EBβ) and in formation of a more spongy bone. Human leukemic Nalm-6 cells showed 3-fold higher migration to CTGF-KD as compared to control BM, a surprising finding that could mechanistically be related to the 3-fold increased expression of SDF-1 in CTGF-KD-MSC. Conclusion: We have developed a genetically controlled human extra-medullary bone marrow system that allows investigations of the role of specific genes on the homing and expansion of normal and leukemic cells/stem cells in vivo. HIF-1α and CTGF were shown to have distinct and unexpected functions in this system. Results gain further relevance by the recent demonstration of genetic abnormalities in human leukemic bone marrow cells (Andreeff 2008, Blau 2011). The system has allows us to established an in vivo bone and bone marrow model with a genetically controlled human microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2971. doi:1538-7445.AM2012-2971