We next examined whether a fusion protein could have biological effects in vivo. For these experiments, we used a system developed previously, in which tumour cells injected intraperitoneally rapidly and preferentially attach and grow initially on the milky spots, learn more a series of organized immune aggregates found on the omentum.38 This system offers a convenient way to examine the effects of fusion protein

treatment on tumour growth because fusion protein can be delivered intraperitoneally multiple times and tumour growth can be analysed by examining the dissociated omental cells. For these experiments we used the Colon 38 cell line, a rapidly growing tumour cell line that expresses both MMP2 and MMP9 in vitro (Fig. 6a). The omental tissue normally expresses a relatively small amount of

MMP2 and MMP9 but when Colon 38 tumour is present on the omentum, MMP levels increase (Fig. 6b). Using this tumour model, we examined the ability of the IL-2/MMPcs/IL-2Rα fusion protein to affect tumour growth. Colon 38 cells were injected intraperitoneally, allowed to attach and GSI-IX chemical structure grow for 1 day, and then treated daily with fusion protein intraperitoneally. At day 7 the animals were killed and the omenta were examined for tumour growth using flow cytometry and by a colony-forming assay (Fig. 6c–e). Figure 6(c) illustrates the gating scheme employed to analyse the tumour population present on the omentum by flow cytometry and panels I, II and III represent plots of single mice from each of the three test groups studied. Figure 6(d) illustrates the compiled flow cytometry data obtained from the individual mice. We found that treatment with the fusion protein can reduce tumour growth in vivo. In the mice that received

tumour and fusion protein treatment (group I), there was a significant decrease (P < 0·01) in the percentage of tumour cells detected on the omenta compared with the mice, which were inoculated with tumour but not treated with fusion protein (group II Fig. 6d). As expected, there was a substantial fraction of cells in the tumour gate in mice that received tumour but were not treated with fusion protein (Fig. 6c panel II) and a very low fraction of cells in the tumour gate of mice that did not receive tumour (Fig. 6c panel III). Similar results were obtained when the presence Dapagliflozin of tumour cells was assessed using a colony-forming assay33 in which cells isolated from the omentum were tested for their ability to form colonies in vitro. These compiled data are shown in Fig. 6(e). Again, a significant difference was observed (P = 0·0119) between the fusion-protein-treated mice and the vehicle-treated mice in the number of viable tumour cells present on the omenta. Hence, in both the flow cytometry and the colony-forming assays there was a clear decrease in the tumour burden with fusion protein treatment although it should be noted that the decrease was not evident in all the treated animals.