## 4 Discussion
### Sensitivity of EGFR and HER-2 Positive Cells to Gefitinib and Caneritinib  
We also show that the anti-tumour effects of gefitinib and canertinib are affected by the levels of EGFR and HER-2 in the ovarian cancer cell line SKOV-3. Furthermore, canertinib shows a greater effect on cell growth and apoptosis than gefitinib in this cell line. Ascitic fluid-derived EGFR positive cells respond to both gefitinib and canertinib, but these responses are distinctive among cell samples. For instance, the measurement of cellular viability is inconsistent among cell samples. The cells that respond to gefitinib also tend to respond to canertinib. A different scenario is observed in the EGFR and HER-2 positive cells, with only canertinib showing growth inhibitory potential in these cells. However, some cells do not respond to canertinib, even though they have the receptors. This data provides us with valuable information, suggesting that the non-responsiveness of cells to both gefitinib and canertinib could be attributed to additional compensatory pathways that overcome the anti-tumour activities of tyrosine kinase inhibitors (TKIs). However, it should be noted that this study has a small sample size and requires further investigation. Gefitinib has been used extensively to treat non-small cell lung carcinoma (NSCLC), which harbours an EGFR mutant protein [38]. The monotherapy of gefitinib has been evaluated in several trials of other solid tumours of various origins, including cancer of the ovary, but has not shown any effect [21; 39; 40; 41; 42]. Canertinib is a dual TKI, that has been used as a proof-of-concept compound in many preclinical studies of various tumour types [43; 44; 18; 45]. Its anti-tumour activities are far superior to the reversible EGFR inhibitor gefitinib. Gefitinib-resistant cancer cells are effectively treated with canertinib in the both wild-type and EGFR mutant cancer cells [46]. However, the anti-tumour activities of canertinib in clinical trials are much less promising. In a phase I clinical trial with canertinib in patients with solid tumours, anti-tumour activity of the inhibitor was notable in only a few individuals [47]. Another phase I clinical evaluation of canertinib in 53 cancer patients did not show any of the objective responses, but the inhibitor down-regulated EGFR, HER-2 and Ki67 [48]. A randomised phase II trial of oral canertinib did not show activity in unscreened patients with advanced ovarian cancer [49]. In our EGFR positive cell populations, both gefitinib and canertinib equally reduced both pERK and pAKT activation. This is, however, rather expected, because both gefitinib and canertinib effectively block EGFR. However, interestingly, some patients with EGFR positive cells do not respond to the inhibitors. It is possible that these cells might have additional pathways that could be activated during the course of the inhibitor treatment. Another plausible explanation is that cells in clusters have a lower cell division rate, and therefore any response to gefitinib and canertinib will be delayed or even non-existent, as these treatments inhibit dividing cells rather than induce apoptosis. In the EGFR and HER-2 positive cells, canertinib shows wider inhibition than gefitinib, especially in the responsiveness of pERK. The decrease in pAKT is also prominent with the canertinib treatment, but this is less than the decrease in pERK. There are a few preclinical studies investigating the activation of the ERK and AKT signalling pathway in ovarian cancer cells in 3D cell clusters. The non-receptor tyrosine kinase Src is reported to be an essential oncogenic protein that is active in the survival of the mouse ovarian cancer cell line ID-8. Cell aggregates in both vitro and in vivo conditions of a syngeneic mouse model have activated ERK and AKT via Src activation [26].