The role of complement factor C5a and its receptor; C5aR in the development of canine mammary tumours

It was hypothesised that complement may play a role in the pathogenesis of canine mammary cancer. Complement activation is an integral pathway in the inflammatory process and recent studies had shown that selective inhibition of the C5a peptide had inhibited the progression of cervical cancer in mice. Before an effect of a mediator or pharmacological agent can be investigated, it should be determined if receptors for the mediator of interest are, indeed, found within the target tissue. Cultured cell lines were established from normal canine mammary tissue to focus experiments on the mammary epithelial cell from where mammary tumours primarily originate via neoplastic transformation of these particular cells. The use of cell lines would also restrict interference from other cell types normally found within the mammary gland, including fibroblasts and inflammatory cells. Immunohistochemical techniques were then applied to the cultured canine mammary epithelial cells and evidence of the expression of a C5a receptor (C5aR or CD88) was demonstrated. However, these results were inconclusive since the antibodies used for immunostaining had been validated for mice and could potentially bind to receptors other than for C5a in canine tissue. It was therefore decided to investigate whether C5a had direct effects on these isolated mammary cells using cell proliferation assays. A C5aR antagonist (ACF – [opdChaWR]; PMX53) resulted in a significant and dose-dependent inhibition of proliferation, while a C5aR agonist (YSFKPMPLar; EP54) had no effect on cell growth. Following on from the cell proliferation studies in normal mammary epithelial cells, it was decided to investigate if there was any change in C5aR expression in mammary tumour cells. Several mammary tissue samples from normal dogs (n=8) and dogs with various tumours (n=7) were collected from veterinary clinics. Immunohistochemistry was performed but, again, was inconclusive (described in Chapter 4). A quantitative reverse transcriptase-Polymerase Chain Reaction (qRT-PCR) assay was therefore developed using primers specific for canine C5aR. There was low but significant up-regulation of messenger RNA (mRNA) in all tumour samples, compared to no expression in the normal mammary samples. This was the first time that C5a had been shown to be involved in the pathogenesis of canine mammary cancer. The next logical step would have been using the C5a agonist and antagonist peptides in dogs. However, an unproven therapy would be unethical in dogs with spontaneously occurring mammary tumours, while the costs of these molecules would be prohibitive. It was therefore decided to explore a mouse model. Two mouse mammary tumour cells lines, 4T1 and EMT6, were selected and expression of C5aR/CD88 was confirmed by using an immunofluorescence assay with a monoclonal anti-mouse C5aR antibody. The effects of C5a on cell growth and viability were then investigated by using the agonist, EP54, and antagonist, PMX53, peptides. Treatment of the 4T1 cell line with EP54 lead to the death of cells in culture as showed by the Trypan Blue exclusion test and the ratio of live:dead cells using the Alamar Blue assay. Neither agonist nor antagonist treatment affected the growth or viability of the EMT6 cells. This was an unexpected result and showed that an agonist of C5a had a direct and deleterious effect on selected mammary tumour cells. The in vitro results differed with what had been reported in the literature and it was therefore uncertain if the anti-tumour activity of a C5a agonist was a direct or toxic effect on the tumour cells and if these results would persist in vivo. A model was developed where mammary tumours could reliably and predictably be induced by injecting either of the two mouse cell lines in BalbC mice (which is where these cell lines were originally isolated from). Mice then had tumours induced with injection of either EMT6 or 4T1 cells and, concurrently, were administered one of the following treatments daily for 14 days: (1) PMX53; (2) EP54; (3) meloxicam (representative of the oxicam’ group being investigated for tumour control by other groups), or (4) saline (control). An important outcome was that the agonist peptide significantly inhibited mammary tumour development in the mice. However, meloxicam and PMX53 did not inhibit tumour growth, compared to control, while PMX53 appeared to enhance tumour development. The study was repeated, although this time blinded to the lead investigator (Noor) and the following treatments administered: (1) PMX53; EP54; (3) EP141, or (4) saline (control). The EP141 peptide was used since it was an agonist of C3aR only, while EP54 was an agonist for both C5aR and C3aR, so it was hoped that a specific response to C5a could be differentiated. Again, EP54 inhibited tumour growth, while EP141 was not significantly different with control tumours. Furthermore, mice treated with EP54 generally appeared healthier, as judged by a health index based on appearance and activity, compared to control animals. These findings have never been reported previously and represent a major and important advance in cancer therapy