Many solid tumors, including ovarian cancer, contain small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and play a role in disease recurrence. We demonstrated that the L1 cell adhesion molecule (L1CAM) is a new CSC target in ovarian cancer, triggering radioresistance. Using fluorescence-activated cell sorting, specific cell populations expressing L1CAM alone or in combination with the established CSC marker CD133 were isolated from three ovarian cancer cell lines. Double-positive L1CAM+/CD133+ cells displayed higher spherogenic and clonogenic properties in comparison to L1CAM−/CD133− cells. Furthermore, L1CAM+/CD133+ cells retained highest clonogenic capacity after irradiation and exhibited up-regulation of some CSC-specific genes, enhanced tumor-initiating capacity, self-renewal and higher tumor take rate in nude mice when compared with other cell populations. Superior radioresistance by L1CAM expression was confirmed by deletion of L1CAM using CRISPR-Cas9 technology. Moreover, we found expression signatures associated with epithelial-to-mesenchymal transition phenotype in L1CAM deleted cells. These results indicate that L1CAM in combination with CD133 defines a new cancer cell population of ovarian tumor-initiating cells with the implication of targeting L1CAM as a novel therapeutic approach for ovarian CSCs.

Protein kinase inhibitors (PKIs) are currently tested in clinical studies (phase I-III) as an alternative strategy against (recurrent) ovarian cancer. Besides their anti-tumour efficacy, several PKIs have also shown radiosensitizing effects when combined with external beam radiation. Based on these results we asked if the addition of PKIs offers a therapeutic opportunity to improve radioimmunotherapy (RIT) against ovarian cancer. Five PKIs (alisertib, MK1775, MK2206, saracatinib, temsirolimus) were chosen for cytotoxicity screenings based on their current clinical trials in the treatment of ovarian cancer and their influence on cell cycle regulation and DNA damage repair pathways. We combined selected PKIs with 177Lu-labelled anti-L1CAM monoclonal antibody chCE7 for our investigations. PKIs cytotoxicity was determined via cell colony-forming assays. Biomarker of DNA double-strand breaks (DSBs, γH2A.X) was analysed by western blot and fluorescence microscopy. Flow cytometric measurements were performed to evaluate levels of apoptosis based on mono- or combination treatments. The best combination was used for in vivo combination therapy studies in nude mice with SKOV3ip and IGROV1 human ovarian cancer xenografts. Bonferroni correction was used to determine statistical significance for multiple comparisons. The highest cytotoxicity against both cell lines was observed for MK1775 and alisertib. Combinations including 177Lu-labelled mAb chCE7 and MK1775 decreased 177Lu-DOTA-chCE7 IC60-values 14-fold, compared to 6-fold, when the radioimmunoconjugate was combined with alisertib. The most effective PKI MK1775 was further evaluated and demonstrated synergistic effects in combination with 177Lu-DOTA-chCE7 against IGROV1 cells. Significantly higher amounts of DSBs were detected in IGROV1 cells after combination (91%) compared to either treatment alone (MK1775: 52%; radioimmunoconjugate: 72%; p < 0.0125). Early-apoptosis was significantly enhanced in IGROV1 cells correlating with induced DSBs (177Lu-DOTA-chCE7: 8%, MK1775: 28%, 177Lu-DOTA-chCE7 + MK1775: 40%, p < 0.0125). Immunohistochemistry analysis of γH2A.X expression levels after therapy in SKOV3ip xenografts revealed a high sensitivity of the tumour cells to MK1775 and a high radioresistance. A prominent effect of tumour growth inhibition of the RIT and of the combination therapy was observed in vivo in a late stage IGROV1 xenograft model. Our results warrant further evaluation of combination of MK1775 and radioimmunotherapy.