Title: The trafficking and tissue localisation of FAPá+ stromal cells in cancer.
Funder: Wellcome Trust

Abstract: The first part of the project will be to validate the existing transcriptomic data regarding chemokine receptor expression by FAP+ cells in tumour and peripheral tissues. Tissues will be collected and digested to obtain single cell suspension, they will then be stained for FAP expression and sorted by FACS before mRNA analysis by qRT-PCR. Protein expression of CXCR7 will also be examined by flow cytometry and histology. Once chemokine receptor expression has been confirmed, function will then be analysed. Preliminary data suggest that there is little turnover of FAP+ cells within the tumour microenvironment (Kraman & Fearon, unpublished data); however as the tumour grows, the proportion of resident FAP+ cells remains constant. It is therefore possible that FAP+ cells from peripheral sites populate the tumour. FAP+ cells from the subcutaneous LL2/OVA tumour produce high levels of SDF-1 (Roberts, Kraman and Fearon, unpublished data), and it has been demonstrated in humans that serum SDF-1 can become elevated in cancer patients (Woo et al. 2008, Macoska et al. 2008) and inflammation, including pre-eclampsia (Schanz et al. 2011), Duchenne muscular dystrophy (Abdel-Salam et al. 2010), burn injury (Drukala et al. 2011) and stroke (Paczkowska et al. 2009). This may therefore be a mechanism by which FAP+ cells are recruited to sites of tissue injury. Haematopoietic stem cells are maintained in the bone marrow by interaction between CXCR4 and SDF-1 (Ma et al. 1999), if interrupted by treatment with AMD3100, reversible release of CD34+ haematopoietic stem cells occurs (Liles et al. 2003). By analogy, it is possible that CXCR7 plays a role in the retention of FAP+ cells in the tissues by interaction with matrix-bound SDF-1 and that in an environment with high circulating levels of free circulating SDF-1, this retention is out-competed. In order to investigate this, serum levels of SDF-1 will be examined by ELISA in tumour bearing KPC mice and compared to non-tumour bearing counterparts and littermates lacking one of the three KPC mutations (Hingorani et al 2005). It has been shown in a model of acute liver failure that intraperitoneal injection of SDF-1 promotes migration of bone marrow mononuclear cells to the liver (Jin et al. 2009). Should SDF-1 levels be raised in the KPC mice, this data will be used to inform a subcutaneous osmotic pump, which will deliver a dose of SDF-1 sufficient to maintain the same serum level; the question may then be asked as to whether high circulating SDF-1 can recapitulate the cachectic phenotype observed in tumour bearing mice. Mobilisation or death of FAP+ cells will be analysed by flow cytometry and histology of tissues in combination with analysis of FAP+ cell numbers in peripheral blood. If they are found to be mobilised, a subcutaneous matrigel plug either injected with SDF-1 or surrounding the osmotic pump could also be used to trap and identify any cells migrating toward the chemokine gradient. Given that SDF-1 is also the ligand for CXCR4 (Bleul et al. 1996, Oberlin et al. 1996) specific blockade of CXCR7 is required to understand any phenotype resulting from SDF-1 administration. A recent paper by Berahovich et al. (2010) reviews the commercially available antibodies to CXCR7, and there are several small molecule inhibitors of CXCR7 available from ChemoCentryx. These will be trialled in vitro using iodinated or FITC conjugated SDF-1 and CXCR7- expressing cells in order to select the most effective blockade of CXCR7 function and then used in vivo to assess the impact of CXCR7 blockade on FAP+ cell localisation and function with and without exogenous SDF-1. Additionally, currently in development in the Fearon lab is a transgenic mouse containing an inducible Tet-off expression system under the control of the FAP regulatory elements with a TetO operator controlling the expression of Cre; this allows inducible FAP+ cell-specific expression of Cre. This will be crossed with Cxcr7(flox/-) mice (Yu et al. 2011) in order to produce conditional knockout of CXCR7 in FAP+ cells. The effect of CXCR7 blockade or knockout will be analysed by FACS and histology of tissues and blood, with particular attention paid to evidence of cell migration or apoptosis. CXCR7 blockade with small molecule inhibitors in murine cancer models have been performed and demonstrated reduced tumour volume (Burns et al. 2006), however, the studies were conducted on immunodeficient mice or using syngeneic tumours. Using the cachexia-inducing subcutaneous C26 tumour model (Zhou et al. 2010), in combination with CXCR7 knockout in FAP+ cells, we will be able to specifically observe the effect of CXCR7 function on FAP+ cells and its impact on cachexia and anaemia in allogeneic tumours. It will be investigated as to whether blockade of CXCR7 is sufficient to prevent the population of the tumour with FAP+ cells, or to prevent peripheral symptoms such as cachexia. Validation of chemokine receptor expression by FAP+ cells will take approximately six months, during which time STU 06/11 Science 7 reagents required for CXCR7 blockade will be tested and optimised in vitro and the subcutaneous C26 tumour model will be established. Following this, preliminary in vivo blockade of CXCR7 will be done and tissues analysed. The FAP-Tet-off; TetO Cre mouse is currently in development and is predicted to be available to cross to the floxed CXCR7 mouse within 6-8 months, and the mice should be ready to use within one year. This will enable characterisation of the effects of CXCR7 knockout in FAP+ cells. Alongside this project, serum will be collected from KPC mice for SDF-1 analysis by ELISA. Analysis of this data should be completed within three months, enabling the osmotic pump experiments to begin and to run in parallel with the CXCR7 project.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS