We report here on matched EOC serous cell lines derived from solid tumor or ascites samples from the same patient at time of diagnosis and following recurrence. Ovarian epithelial cells typically express keratin 7 but lack expression of keratin 20 [31–33]. This pattern was observed in the tumor tissues of all patients by both Western blot and immunohistochemistry (Figure 3; Figure 4 and Additional file 1. Taken together, we were able to confirm the epithelial origin of the cell lines presented here.
The cell lines were characterized in terms of TP53 mutation status and protein expression, BRCA1, BRCA2, KRAS and BRAF mutation status, and HER2 expression. All of the cell lines had a somatic TP53 mutation, which is consistent with the reported frequency in high-grade serous tumors, estimated at 51 to 93% in recent studies [34–37]. An identical mutation was evident in each cell line derived from the same patient, consistent with a common clonal origin for the tumor and ascites derived from each individual patient. As expected, identical TP53 mutations to those found in the cell lines were also observed in the corresponding tumor tissue (data not shown). The expression of TP53 was investigated in all of the cell lines by Western blot and in the primary solid tumors by immunohistochemistry. Interestingly, p53 was not detected by Western blot in all four 3133 cell lines (and confirmed by a low expression by immunohistochemistry). This is consistent with the truncating nonsense mutation in exon 6 in the TP53 sequence found in each of the 3133 cell lines. Expression of the epidermal growth factor receptor gene, HER2, which is implicated in malignant transformation, was also used to characterize the cell lines, as overexpression is reported on average in 20-30% of ovarian tumors and as high as 75% by a variety of techniques including ELISA, immunohistochemistry and RT-PCR [38–41]. Although there is evidence of overexpression of HER2 being associated with a lower sensitivity to platinum-based chemotherapy , there was no indication of differential expression in the ovarian cancer cell lines by Western blot, or in the solid tumors by immunohistochemistry that could relate to the sensitivity to carboplatin detected by the clonogenic assay.
The distinct tumor growth characteristics within the serous cell lines derived here, indicates a diversity reflective of the heterogeneous nature of this histopathological subtype. For example, saturation density, spheroid formation and colony formation in soft agarose differed between cell lines, and also within cell lines derived from the same patient. Differences in spheroid formation between cell lines derived before and after chemotherapy treatment may offer an interesting point of reference, especially as spheroid models may offer a model system more in line with the in vivo tumor setting . For example, the cell line OV2295 formed semi-compact spheroids, compared to the aggregates in TOV2295(R), possibly reflecting differences in cell to cell adhesion. When comparing cell lines derived from a single patient over time, there is no tendency to be more aggressive in terms of the measured characteristics as the disease progresses. Nevertheless, the current model will allow researchers to address biological questions intrinsic to the cell lines such as clonal heterogeneity within tumors, as well as modification occurring for the development of ascites  and the relationship between biological properties of ascites and solid tumors established from the same patient . In addition, the further investigation of genetic and epigenetic changes between the primary tumor and cell lines at discrete time points may provide insight into the evolutionary processes at play in cancer development .
Interestingly, in vivo tumor formation of the cell lines in SCID mice at subcutaneous sites was only observed with OV3133(R), the first ascites taken from patient 3133. The second ascites sample OV3133(R2) that was taken after doxorubicin treatment, at approximately 500 days after the OV3133(R) was sampled, did not form tumors. The tumors formed in SCID mice grew slowly as compared with the previously established TOV112D and TOV2295(R) cell lines, although this observation was consistent with previously studied high grade serous cell lines such as TOV2223 which also did not form tumors at subcutaneous sites in SCID mice . Although the cell lines outlined here may not be amenable to all pre-clinical xenograft models, in the future we may be able to investigate intraperitoneal injections. Note also that the lack of tumorigenicity in mouse xenograft model may not reflect the situation in humans – this should not be used as the sole criteria for cell line utility.
The new cell lines derived in this study were developed from patient samples that were exposed to specific chemotherapeutic agents. This is in contrast to chemoresistant cell lines generated in vitro, which are often derived from clonal variants that survive by escalating dosages of chemotherapeutics. In contrast, the cell lines we describe here may more accurately represent the molecular evolution that occurs within the tumor microenvironment. Additionally, we have already alluded to the potential to further study the cell lines by utilizing the spheroid model, which may more accurately reflect the in vivo tumor environment . For example, L’Esperance et al., 2008 used the spheroid model of ovarian cancer cell lines (OV90, TOV21G, TOV112D) to investigate response to chemotherapy treatment . Interestingly, higher levels of both cisplatin and paclitaxel were required for a similar response in spheroids compared to a similar study using monolayers on the same cell lines . This suggestion of greater drug resistance in spheroids warrants further attention in the present set of cell lines.
Of the patients from which the cell lines were established, two of the three (1369 and 2295) responded initially to first line therapy using Response Evaluation Criteria in Solid Tumors (RECIST) criteria [46, 47]. In general, response rates for chemotherapy in ovarian cancer are reported at 70-80% . Patient 3133 did not show a clear response to chemotherapy, with evidence of progressive disease after 5 months by RECIST criteria. However, CA-125 levels showed a marked decrease from 764 before the initial paclitaxel/carboplatin treatment to 470 units per ml two months following treatment . This decrease of nearly 40% is just outside the level of decrease which would be indicative of a responder, based on the GCIG (Gynecological Cancer InterGroup) acceptable current criteria for CA-125 response, of at least a 50% decrease in CA-125, for at least 28 days[46, 47]. Although no significant differences were observed in response to paclitaxel in the cell lines derived from primary versus recurrent disease, previous studies have indicated otherwise, such as described in a recent report which determined that 35% of solid tumors and 50% of ascites samples were resistant to paclitaxel . It is noteworthy that the IC50 levels of paclitaxel response in TOV1369 and OV1369(R2) are four-to-twenty and two-to-five times higher, respectively, than all other cell lines examined, and this is possibly due to acquired resistance to taxol as a consequence of prior treatment for breast cancer. We also note that cells from patient 1369 also displayed a lower sensitivity to carboplatin, although the clinical profile of this patient does not suggest inherent chemoresistance. Comparison of the OV2295 (derived prior to recurrence) to the OV2295(R2) and TOV2295(R) cell lines derived following recurrence were the only clear example of acquired resistance to carboplatin. Carboplatin resistance is well documented in ovarian cancer. For example, a recent study found 75% of solid tumors and 59% of ascites samples to be resistant to carboplatin . This is likely due to the selective pressure of the chemotherapy regime exerted on a heterogeneous cell population, resulting in an enrichment of a resistant subset of cells by promoting the expression of a resistance pathway or selection for a population bearing a mutation responsible for a decrease in sensitivity.
Mutation status such as TP53, BRCA1 and BRCA2 [50, 51] are also important factors, which may contribute to tumor progression and chemoresistance of an ovarian tumor tissue or cell line, especially in relation to their role in apoptosis. In this report, based on the investigation of common French Canadian mutations, no BRCA1 or BRCA2 mutations were identified, and therefore we cannot comment on the role of BRCA1/2 as a surrogate marker for chemotherapy response. There did not appear to be a difference in the specific type of TP53 mutation (truncating nonsense for 3133, missense for 1369 and 2295), relative to chemosensitivity status. Although there is evidence of overexpression of HER2 being associated with a lower sensitivity to platinum-based chemotherapy, our results did not show differential expression in the ovarian cancer cell lines by Western blot, or in the solid tumors by immunohistochemistry that could relate to the sensitivity to carboplatin detected by the clonogenic assay. Therefore we can suggest that BRCA and HER2 are not linked to the resistance profile presented in our cell lines and that other factors might be involved. In the case of p53, there was a difference in mutant p53 protein expression between TOV1369 and OV1369(R2), but no corresponding difference in chemotherapy response. Although the OV2295 cell line appeared to have a lower expression of the mutant p53 protein then the recurrent OV2295(R2) and TOV2295(R) cell lines, both of which exhibited acquired carboplatin resistance, any relationship between mutant p53 expression and carboplatin resistance would have to be robustly tested using a gene knock-down experiment. Furthermore, a study using paired pre- and post-chemotherapy tumor samples, determined that differences in gene expression profiles between matched samples could be due to factors not only involved in chemotherapy resistance, but also factors related to tumor progression and proliferation [44, 52]. The cell lines described here may serve as a good model to begin to analyze specific candidates identified in these studies.