22. Tumor Homing of Genetically Modified Bone Marrow Cells

Abstract

The clinical benefit of adenovirus-mediated tumor gene therapy has so far been disappointing. A potential reason for this is inaccessibility of tumor nests due to anatomical barriers formed be stromal or fibrotic tissue. The stroma immediately surrounding tumor cells consists of a three-dimensional extracellular matrix (ECM) and stromal cells such as fibroblasts and inflammatory cells and is usually well vascularized. Therefore, approaches to genetically modify (autologous) bone marrow-derived stromal cells, that after transplantation into cancer patients, home to tumor stroma appear to be promising. For example bone marrow cells could be stably transduced ex vivo to inducibly express proteases and be used as a “Trojan horse” to permeabilize the ECM for subsequent chemo- and immunotherapy as well as for treatment with oncolytic viruses. As a model to study such approaches we used SCID/NOD mice bearing liver metastases derived from MDA–MB435 human breast cancer cells. Tumor nests derived from MDA–MB435 cells are surrounded by stroma and ECM, very similar to the histopathology of ductal mammary carcinoma. In preliminary studies, we demonstrated that a significant fraction of stromal cells (mostly of macrophage lineage) surrounding the tumor nests are derived from human CD34+ cells transplanted into myeloablated NOD/SCID mice. Next we performed a similar study with genetically modified human CD34+ cells. In a first study, human umbilical cord blood-derived CD34+ cells were transduced with a RD114-pseudotyped lentivirus vector expressing GFP under the control of the EF1α promoter. Four weeks after transplantation of transduced CD34+ cells into lethally irradiated NOD-SCID mice, 40 to 50% of engrafted human cells expressed GFP in bone marrow aspirates. Eight weeks after the transplantation, mice received an intraportal transplantation of 2×106 MDA–MB435 cells. One group of mice received G–CSF injection daily to stimulate mobilization of bone marrow cells. The livers of mice were examined 3 weeks after tumor cell injection. Immunohistochemistry revealed human-derived CD45+ cells in mouse livers, specifically in the vicinity of blood vessels and around tumor nests. About 40 to 50 percent of CD45+ cells expressed GFP, indicating that transgenes can be efficiently expressed in stromal cells surrounding metastatatic tumor nests. G-CSF treatment had no effect on the recruitment of bone marrow-derived cells to the tumor stroma. Current effort are aimed towards the use of integrating, capsid-modified Adeno-AAV vectors for CD34+ cell transduction and regulated expression of metalloproteinases.