No abstract

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture. To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS). Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs. This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

No abstract

Our results show that (i) the MVM-IL2 parvoviral vector efficiently transduces tumour cells; and (ii) the low multiplicity of infection (MOI = 1) used in our experiments was sufficient to elicit an anti-tumour effect on distant cells, which supports further studies on this vector as a new tool for cancer gene therapy.

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1a) that interacts with a 'hypoxia-responsive element' (HRE), the consensus sequence of which ( A / G CGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.The parvovirus Minute virus of mice, MVM(p), replicates preferentially in oncogenic-transformed cells, where it completes a lytic life cycle (Rommelaere & Cornelis, 1991). This property is exploited in gene-therapy vectors derived from autonomous parvoviruses (Russell et al., 1992;Cornelis et al., 2004). The MVM(p) genome is composed of two transcription units, encoding two sets of viral proteins, NS (non-structural) and VP (capsid), under the control of the P4 and P38 promoters, respectively (Cotmore et al., 1983). The non-structural protein NS1 plays an important role in viral DNA replication (Nüesch et al., 1998), is toxic in transformed cells (Caillet-Fauquet et al., 1990) and is required for transactivation of the P38 promoter, which is otherwise only expressed at a very low basal level (Rhode, 1985).The mechanisms underlying the preferential expression of MVM(p) in transformed cells are only poorly understood, but it is at least in part linked to the upregulation of the P4 promoter in these cells compared with normal cells. We therefore wanted to determine whether P4 activity could be further enhanced and whether this could improve vector production. We have explored the possibility of producing MVM vectors under low oxygen tension (hypoxia). Gene expression and production of the human erythrovirus B19 virus are increased under these conditions (Caillet-Fauquet et al., 2004;Pillet et al., 2004). Regulation of promoter activity in hypoxia occurs through the interaction of the transcriptional regulator hypoxia-inducible factor (HIF-1) with a 'hypoxia-responsive element' (HRE) (Semenza, 2001). The MVM(p) promoter contains four putative HREs (consensus sequence A / G CGTG), two in each of the sense and antisense orientations.The presence of the HRE in the promoter region is necessary, but not sufficient, for promoter regulation. Other cellular elements are probably...