Ovarian cancer (OC) is one of the most lethal gynecologic malignancies. Due to the lack of specific symptoms and screening methods, this disease is usually diagnosed only at an advanced and metastatic stage. The gold-standard treatment for OC patients consists of debulking surgery followed by taxane combined with platinum-based chemotherapy. Most patients show complete clinical remission after first-line therapy, but the majority of them ultimately relapse, developing radio- and chemoresistant tumors. It is now proposed that the cause of recurrence and reduced therapy efficacy is the presence of small populations of cancer stem cells (CSCs). These cells are usually resistant against conventional cancer therapies and for this reason, effective targeted therapies for the complete eradication of CSCs are urgently needed. In this review article, we highlight the mechanisms of CSC therapy resistance, epithelial-to-mesenchymal transition, stemness, and novel therapeutic strategies for ovarian CSCs.

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.

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Background: Today's standard treatment of advanced-stage ovarian cancer, including surgery followed by a paclitaxel-platinum-based chemotherapy, is limited in efficacy. Recently, we could show that radioimmunotherapy (RIT) with ¹⁷⁷Lu-labelled anti-L1 cell adhesion molecule (L1CAM) monoclonal antibody chCE7 is effective in ovarian cancer therapy. We investigated if the efficacy of anti-L1CAM RIT can be further improved by its combination with paclitaxel (PTX). Methods: In vitro cell viability and cell cycle arrest of human ovarian cancer cells were assessed upon different treatment conditions. For therapy studies, nude mice (n = 8) were injected subcutaneously with IGROV1 human ovarian carcinoma cells and received a single dose of 6 MBq ¹⁷⁷Lu-DOTA-chCE7 alone or in combination with 600 μg PTX (31.6 mg/kg). Tumour growth delay and survival were determined. To investigate whether PTX can influence the tumour uptake of the radioimmunoconjugates (RICs), a biodistribution study (n = 4) and SPECT/CT images were acquired 120 h post injections of 2 MBq ¹⁷⁷Lu-DOTA-chCE7 alone or in combination with 600 μg PTX. Results: Lu-DOTA-chCE7 in combination with PTX revealed a significantly decreased cell viability of ovarian carcinoma cells in vitro and was effective in a synergistic manner (combination index < 1). PTX increased the RIT efficacy by arresting cells in the radiosensitive G2/M phase of the cell cycle 24 h post treatment start. In vivo combination therapy including ¹⁷⁷Lu-DOTA-chCE7 and PTX resulted in a significantly prolonged overall survival (55 days vs. 18 days/PTX and 29 days/RIT), without weight loss and/or signs of toxicity. Biodistribution studies revealed no significant difference in tumour uptakes of ¹⁷⁷Lu-DOTA-chCE7 72 h post injection regardless of an additional PTX administration. Conclusions: Combination of anti-L1CAM ¹⁷⁷Lu-RIT with PTX is a more effective therapy resulting in a prolonged overall survival of human ovarian carcinoma-bearing nude mice compared with either monotherapy. The combination is promising for future clinical applications.

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.

Cancer stem cells (CSCs) are highly tumorigenic, self-renewable cells with a key role in tumor relapse, metastasis, and therapy resistance. Effective CSC-targeted therapies remain an unmet clinical need, strongly dependent on the selection of suitable targets and thorough validation of therapeutic agents. L1 cell adhesion molecule (L1CAM) is a targetable CSC-associated biomarker aberrantly expressed in various malignancies, including ovarian cancer (OC). ¹⁶¹Tb is attractive for clinical application because of its substantial emission of conversion electrons/Auger electrons as well as β^– emission. Leveraging the high cytotoxicity of conversion electrons/Auger electrons, ¹⁶¹Tb is promising for radioimmunotherapy against radioresistant tumor cells such as CSCs. The aim of this study was to confirm the presence of L1CAM⁺/CD133⁺ ovarian CSCs in patient samples and preclinically investigate, in a tumor prevention mouse model, ¹⁶¹Tb-based anti-L1CAM radioimmunotherapy as a new therapeutic modality against CSCs compared with ¹⁷⁷Lu-based anti-L1CAM radioimmunotherapy. Methods: L1CAM⁺/CD133⁺ CSCs were examined in OC samples by immunofluorescence. After radiolabeling anti-L1CAM DOTA-chCE7 with ¹⁷⁷Lu or ¹⁶¹Tb and purification, we assessed radioimmunoconjugate quality by determining the radiochemical purity and the immunoreactive fraction. The internalized and membrane-bound fractions and the radiocytotoxicity of radiolabeled DOTA-chCE7 were evaluated with cell uptake and cell proliferation assays. Ovarian L1CAM⁺/CD133⁺ CSCs were sorted via fluorescence-activated cell sorting from OVCAR8 and SKOV3ip cells and inoculated into immunocompromised mice, who then received treatment with [¹⁷⁷Lu]Lu-DOTA-chCE7 or [¹⁶¹Tb]Tb-DOTA-chCE7. Results: L1CAM⁺/CD133⁺ CSCs (0.3%–21%) were confirmed in samples from patients who were chemotherapy-naïve or had relapsed OC. [¹⁷⁷Lu]Lu-DOTA-chCE7 and [¹⁶¹Tb]Tb-DOTA-chCE7 were produced with high radiochemical purity and retained 76%–96% immunoreactivity. Cell uptake after 15 h ranged from 50% to 75% for both radioimmunoconjugates. [¹⁶¹Tb]Tb-DOTA-chCE7 showed significantly increased cytotoxicity, eliminating all ovarian CSCs and tumor cells differentiated from the CSCs in vivo, compared with [¹⁷⁷Lu]Lu-DOTA-chCE7 (3 tumors in OVCAR8 group and 1 tumor in SKOV3ip group). Follow-up tumor analysis confirmed that sorted ovarian L1CAM⁺/CD133⁺ CSCs regenerated the tumor heterogeneity in vivo. Conclusion: This work addresses the critical need for CSC-specific therapies in the clinics by establishing ¹⁶¹Tb-based anti-L1CAM radioimmunotherapy as a novel therapeutic modality against CSCs. We found that ¹⁶¹Tb-based anti-L1CAM radioimmunotherapy eliminated ovarian CSCs more efficiently than ¹⁷⁷Lu-based anti-L1CAM radioimmunotherapy, emphasizing its promising therapeutic potential. Visual Abstract