Ovarian cancer is the eight leading cause of cancer related deaths within the female population worldwide (1, 2). The most dominant subtype is high-grade serous ovarian cancer (HGSOC), which has an epithelial origin (3, 4). Due to the absence of symptoms at earlier stages of the disease, patients are often diagnosed at an advanced disease stage (International Federation of Gynecology and Obstetrics (FIGO) stage III and IV). In first line, the current standard of care of advanced ovarian cancer consists of a cytoreductive debulking surgery combined with platinum-based chemotherapy and eventually bevacizumab and/or poly (ADP-ribose) polymerase (PARP) inhibitors (5). The carboplatin-paclitaxel chemotherapy combination is the preferred regimen in a first-line treatment setting. However, the choice of chemotherapy at recurrence depends on tumor characteristics and whether platinum is again an option or not. At the time of recurrence, both platinum and non-platinum agents such as paclitaxel, gemcitabine, pegylated liposomal doxorubicin (PLD) and topotecan, as well as targeted therapies such as PARP inhibitors and bevacizumab can be used as single agents or in combination schedules (6). Nevertheless, HGSOC patients who get diagnosed in an advanced disease stage have a poor 5 year survival of only 20%-41% (7).

In most patients, recurrence of the disease involves the presence of metastases confined to the peritoneal cavity. In the ongoing search for more effective treatment strategies to locally target this peritoneal disease, the rapidly evolving research field of radionuclide therapy is of interest. Historically, the main focus in the context of ovarian cancer was on the investigation of β-particle emitters, with various success rates. In the past, radiocolloids containing ³²P or ¹⁹⁸Au have been used for IP treatment of patients with ovarian cancer (8–11). However, its use was limited in time due to the increased incidence of adverse effects, and replacement with chemotherapy treatment was recommended (9). Additionally, antibody guided ⁹⁰Y has been explored in the context of ovarian cancer, but it did not proceed from phase III clinical trials (12, 13). Alternative types of radiotherapy options that have been explored for ovarian cancer include proton beam therapy and carbon ion therapy with successful outcomes in two case reports of patients with recurrent ovarian cancer (14, 15).

The use of α-particle emitters is assumed to have advantages over the prior β-therapies. They are particularly of interest for the treatment of micrometastatic cancer dissemination in body cavities, one of the characteristics of peritoneal carcinomatosis in patients with ovarian cancer (16). Alpha-emitters are highly cytotoxic for the cancer cells residing in the abdominal cavity, while sparing surrounding radiosensitive organs because of their short penetration depth, thus limiting toxicities compared to β-emitters. To date, Xofigo^® (²²³RaCl₂) remains the only α-emitting radiopharmaceutical approved by the European Medicines Agency and the Food and Drug Administration, and is currently used for the treatment of skeletal metastases of castration-resistant prostate cancer (17). However, α-emitters, such as ²¹²Pb and ²¹¹At have been investigated for ovarian cancer in phase I clinical trials, where feasibility of this type of treatment was confirmed without apparent signs of dose-limiting toxicities (18, 19).

The α-emitter ²²⁴Ra, when adsorbed onto the microparticle drug carrier CaCO₃, has shown therapeutic potential in immunodeficient ovarian cancer xenograft mouse models, when administered as an IP treatment (20, 21). Based on these promising data, the ²²⁴Ra-CaCO₃-MP are currently being assessed in phase I clinical trials for both ovarian cancer (22) and colorectal carcinoma (23), two cancer types characterized by the presence of a widespread metastatic disease within the peritoneal cavity.

The current paper focusses on the evaluation of ²²⁴Ra-CaCO₃-MP in terms of its potential to treat peritoneally disseminated ovarian cancer in an immune competent mouse model. The aim was to examine the tolerability and efficacy of combinations with chemotherapy regimens commonly used in patients with ovarian cancer.

In the search for more effective treatment strategies for ovarian cancer, α-emitting radionuclide therapies are emerging. The high energy deposition in combination with limited penetration depth can be exploited to target residual microscopic disease without affecting the surrounding radiosensitive organs. These micrometastases remain present within the peritoneal cavity after cytoreductive debulking surgery and are often related to the high recurrence rate of the disease. In this study, we specifically investigated the therapeutic potential of a newly developed α-emitting radiopharmaceutical which consists of ²²⁴Ra adsorbed onto CaCO₃ microparticles, and the safety to use this in combination with chemotherapy regimens commonly used in clinical practice. We provide proof-of-principle of the therapeutic potential of ²²⁴Ra-CaCO₃-MPs in a syngeneic model of ovarian cancer in immune competent mice. Furthermore, the sequential administration of ²²⁴Ra-CaCO₃-MPs with two different standard of care chemotherapy regimens indicated that a synergistic effect can be obtained, however, the synergism was more pronounced with carboplatin-PLD compared to carboplatin-paclitaxel. In general, the various treatment combinations appeared well-tolerated in the mice.

The therapeutic potential of ²²⁴Ra-CaCO₃-MPs in the immune compromised ES-2 and SKOV3 mouse model for ovarian cancer have previously been demonstrated. Different product formulations of ²²⁴Ra-CaCO₃-MPs were able to prolong survival with MSRs ranging between 1.5 and 2.8 (20, 21, 28), while the MSRs observed in the current study ranged between 1.1 and 1.3. An important factor that might negatively influence the therapeutic efficacy in the immune competent ID8-fLuc mouse model for ovarian cancer is the reaction of the tumor immune microenvironment to the particle drug carrier (CaCO₃ microparticles). It has been shown that IP injections of microparticle drug carriers, including but not limited to CaCO₃ microparticles, elicit an immune suppressive and tumor promoting effect in the ID8-fLuc model, mediated by innate immune suppressive cells such as myeloid-derived suppressor cells and M2-like macrophages (30). Both cell types are known to be involved in ovarian cancer development and progression [recently reviewed (31)]. We believe that the slight survival benefit in the ID8-fLuc model can be explained by the fact that the immune-related tumor promoting mechanisms in response to the CaCO₃ microparticles partially counteract the therapeutic effect of the ²²⁴Ra. In another syngeneic mouse model of disseminated peritoneal disease, albeit of colorectal origin (CT26.WT), treatment with the ²²⁴Ra-CaCO₃-MPs was able to significantly prolong survival (MSR of 1.8) (28), indicating that the tumor-promoting mechanisms are not universal among different disease models.

One novelty with the current study is the use of the fully immune competent ID8-fLuc mouse model for ovarian cancer. Previously published work on the ²²⁴Ra-CaCO₃-MPs in ovarian cancer models was performed in immune compromised mouse models (ES-2 and SKOV3). Since the strong immune suppressive tumor microenvironment in patients with ovarian cancer is an important factor in the disease progression (31), we provide additional proof of the therapeutic potential of the ²²⁴Ra-CaCO₃-MPs in a mouse model that closely resembles this clinical situation. The study design was aimed to mimic the clinically relevant standard of care chemotherapy regimens, although, it should be noted that the IP administration route of the different chemotherapeutics differs from the standard administration route in patients with ovarian cancer (intravenous administration).

However, we recognize that our study encounters some limitations. With the current data, we are not able to provide a mechanistic explanation as to why two chemotherapy chemotherapy regimens result in a different outcome when combined with ²²⁴Ra-CaCO₃-MPs. Several mechanisms can be responsible for creating synergistic or additive effects between chemotherapy and radiation therapy. The mechanism of action for the specific chemotherapeutic drug may radiosensitize tumor cells to α-radiation to a varying degree. In addition, it is known that different chemotherapy regimens have different effects on the ovarian cancer immune microenvironment in mice (32, 33). Hence, the immune response caused by the ²²⁴Ra-CaCO₃-MPs and the chemotherapeutics may favor some but not all combinations and dosages. A future characterization of both the cytotoxic mechanisms and immunological responses of the combined ²²⁴Ra-CaCO₃-MPs and chemotherapy treatment might therefore aid with identifying the most optimal combination regimens.

In the past, other applications with α-particle emitters have been evaluated for the treatment for ovarian cancer. Preclinical evaluation of IP treatment with ²¹¹At-labeled monoclonal antibodies showed a high therapeutic efficacy in treating micrometastatic growth in the OVCAR-3 ovarian cancer mouse model (34, 35). Additionally, the α-emitter ²¹²Pb has been evaluated as an IP treatment in the immunodeficient ES-2 and A2780cp20 mouse models for ovarian cancer showing a therapeutic potential when labeled to a monoclonal antibody or CaCO₃ microparticles (36, 37). Additionally, ²¹²Pb and ²¹¹At colloids have also been investigated previously in a preclinical setting for IP ovarian cancer dissemination, where they have proven their therapeutic potential (38, 39). No immediate and/or late signs of local radiation-induced toxicities were observed in the phase I clinical evaluation of ²¹¹At- or ²¹²Pb-labeled antibody treatments in patients with ovarian cancer (18, 19, 40, 41). These results are as expected with the limited range of tissue penetration of α-emitters, preventing irradiation of other radiosensitive organs within the peritoneal cavity.

Furthermore, the combined effects of α-therapies and chemotherapeutics on weight development in mice as a measure for toxicity have been evaluated previously. Milenic and colleagues reported a modest weight loss in mice treated sequentially with ²¹²Pb-trastuzumab and gemcitabine compared to mice that received gemcitabine alone in a model for colon carcinoma (LS-174T) (42), which is in contrast to what we observed in our study. However, the same group reported no difference in weight development between mice that received paclitaxel and ²¹³Bi-trastuzumab or paclitaxel alone in the same tumor model (43), indicating a differential response to combinations of different types and dosages of chemotherapy and radionuclides. Both combination regimens described above also produced synergistic therapeutic effects that could not be reached by these therapeutics separately (42, 43).

We provide proof-of-principle for the therapeutic efficacy ²²⁴Ra-CaCO₃-MPs in an immune competent mouse model for ovarian cancer, both alone and in combination with chemotherapy. Furthermore, the results indicate a safe sequential administration with two different chemotherapy regimens often used in clinical practice. The results support further evaluation of ²²⁴Ra-CaCO₃-MPs in patients with ovarian cancer. However, further investigations remain to identify the most optimal chemotherapy regimen to combine with ²²⁴Ra-CaCO₃-MPs and the sequence of therapies to fully exploit a potential synergistic effect.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Katholieke Universiteit Leuven.

Conceptualization and interpretation of results: RW, SW, AC, and TB. Design of experiments: RW and SW. In vivo experiments: RW, YB, and MR. Analysis of results: RW and MR. Statistics: RW and JC. Manuscript writing: RW. Manuscript proof-reading: JC, YB, MR, SW, TB, IV, and AC. Supervision: SW, TB, IV, and AC. Funding acquisition: TB and AC. All authors have read and agreed to the published version of the manuscript.

This research was funded by the Norwegian Research Council (with project number 304591) and Oncoinvent AS.

RW was employed by Oncoinvent AS. AC was a contracted researcher for Oncoinvent AS and Novocure and a consultant for Sotio a.s. SW was employed by and a shareholder of Oncoinvent AS. TB was employed by and a shareholder of Oncoinvent AS. IV is a consultant for Agenus, Akesobio, AstraZeneca, Bristol Myers Squibb, Deciphera Pharmaceuticals, Eisai, Elevar Therapeutics, F. Hoffmann-La Roche, Genmab, GSK, Immunogen, Jazzpharma, Karyopharm, Mersana, MSD, Novocure, Novartis, Oncoinvent, OncXerna, Sanofi, Seagen, Sotio, Verastem Oncology and Zentalis, was a contracted researcher for Oncoinvent AS, performs corporate sponsored research for Amgen and Roche, and receives accommodation and travel expenses from Karyopharm, Genmab and Novocure. The funders (The Norwegian Research Council (project 304591) and Oncoinvent AS) provided support in the form of salaries for authors RW, SW, and TB but did not have any additional role in the study design, data collection, analysis or interpretation, preparation of the manuscript, or decision to publish. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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