The amount of late-apoptotic or necrotic cells did not vary significantly (Table?2). Table 2 Percentage of early- and late-apoptotic/necrotic IGROV1 cells after treatment with MK1775 and 177Lu-DOTA-chCE7 thead th rowspan=”1″ colspan=”1″ IGROV1 /th th rowspan=”1″ colspan=”1″ Untreated control /th th rowspan=”1″ colspan=”1″ MK1775 [300?nM] /th th rowspan=”1″ colspan=”1″ 177Lu-DOTA-chCE7 [5.0?MBq/ml] /th th rowspan=”1″ colspan=”1″ Combined treatment /th /thead Early-apoptosis4%??18%??229%??1040%??4Late-apoptosis/necrosis9%??210%??213%??212%??1 Open in a separate window The data were generated from three independent flow cytometry experiments Immunohistology reveals significant higher levels of DNA-DSBs in SKOV3ip xenografts administered with MK1775 as a single agent or in combination with 177Lu-DOTA-chCE7 Two in vivo experiments were performed in order to assess whether the promising results from the in vitro work could be confirmed. GUID:?235E369A-B877-47AA-9BEF-0A98DC196E1D Data Availability StatementThe datasets generated and/or analysed during the current study are available on request. Abstract Background 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, Mouse monoclonal to HK2 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. Methods PKIs cytotoxicity was determined via cell colony-forming assays. Biomarker of DNA double-strand breaks (DSBs, H2A.X) was analysed by western blot and fluorescence CMP3a 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. Results 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%; and are the concentrations of A and B contained in combination that provide the same effect. Synergy is determined for CI? ?1, additivity for CI?=?1 and antagonism for CI? ?1 . Results Antibody radiolabelling and ligand substitution In order to determine the DOTA-to-mAb (chCE7) ratio a mass spectroscopic analysis was performed. Results showed that an average of 7.6 chelators was coupled to one intact antibody molecule for RICs used in in vitro experiments. For RICs utilized in the in vivo study an average of 2.7C3.1 chelators was coupled. Specific activity ranged from 2000 to 2850?MBq/mg for RICs with 7.6 chelators and 240C560?MBq/mg for RICs with 2.7C3.1 chelators. Lindmo method  was used to prove the immunoreactive fraction of the radiolabelled mAbs (60C83%). Cytotoxicity of selected PKIs towards IGROV1 and SKOV3ip cells First we investigated the sensitivity of the IGROV1 and SKOV3ip OC cell lines towards the selected PKIs. Respective dose-response curves are shown in Fig.?1a-e and resulting IC50-values are summarised in Table?1. PKIs alisertib and MK1775 showed IC50-ideals in the medium nanomolar range for both OC cell lines. Similar sensitivities of SKOV3ip cells were observed towards PKIs temsirolimus and MK2206. In comparison, IC50-ideals for temsirolimus and MK2206 against IGROV1 cells could only be estimated within in the micromolar range, since highest applied PKI concentration of 1 1?M was not sufficient enough to reach IC50. No cytotoxicity was recognized for both cell lines based on the treatment with PKI saracatinib (Fig. ?(Fig.1b1b). Open in a separate windows Fig. 1 IC50-ideals for any Alisertib, b Saracatinib, c MK1775, d Temsirolimus and e MK2206. IC50-ideals were determined by colony-forming assays. IGROV1 and SKOV3ip cells were incubated for 48?h with accordant PKI concentrations ranging from 0.1C1000?nM Table 1 IC50-values for PKIs against IGROV1 and SKOV3ip cells CMP3a thead th rowspan=”1″ colspan=”1″ /th th rowspan=”1″ colspan=”1″ Alisertib /th th rowspan=”1″ colspan=”1″ Saracatinib /th th rowspan=”1″ colspan=”1″ MK1775 /th th rowspan=”1″ colspan=”1″ Temsirolimus /th th rowspan=”1″ colspan=”1″ MK2206 /th /thead IGROV150??3?nMn.a.306??4?nMn.a.n.a.SKOV3ip158??3?nMn.a.133??4?nM120??4?nM131??3?nM Open in a separate windows em Abbreviation /em : em n.a /em . not available MK1775 sensitizes IGROV1 cells towards treatment with 177Lu-DOTA chCE7 in vitro PKIs saracatinib, MK2206 and temsirolimus showed only limited cytotoxicities against both OC cell lines and were therefore not regarded as for further investigations. First in vitro combination experiments were performed CMP3a using the PKIs alisertib CMP3a and MK1775 combined with 177Lu-DOTA-chCE7. Both PKIs shown their ability to radiosensitize IGROV1 cells by decreasing the inhibitory concentration of 177Lu-labelled mAb chCE7 necessary to reduce colony-forming ability to 60% of an untreated control (IC60). However, MK1775 decreased the IC60-value of 177Lu-DOTA-chCE7 15-collapse compared to a 6-collapse decrease observed for mixtures including PKI alisertib (Additional file 1: Number S2). With this experiment the cells were incubated for only 4?h instead of 8?h with 177Lu-labelled chCE7. The colony-forming ability was reduced by a maximum of 60% in comparison to the untreated cells even with the highest amount of radioactivity. Based on these findings MK1775 shown the most encouraging radiosensitizing effect and was consequently further investigated..