Background: The completeness of resection is a key prognostic indicator in patients with ovarian cancer, and the application of tumour-targeted fluorescence image-guided surgery (FIGS) has led to improved detection of peritoneal metastases during cytoreductive surgery. CD24 is highly expressed in ovarian cancer and has been shown to be a suitable biomarker for tumour-targeted imaging. Methods: CD24 expression was investigated in cell lines and heterogenous patient-derived xenograft (PDX) tumour samples of high-grade serous ovarian carcinoma (HGSOC). After conjugation of the monoclonal antibody CD24 to the NIR dye Alexa Fluor 750 and the evaluation of the optimal pharmacological parameters (OV-90, n = 21), orthotopic HGSOC metastatic xenografts (OV-90, n = 16) underwent cytoreductive surgery with real-time feedback. The impact of intraoperative CD24-targeted fluorescence guidance was compared to white light and palpation alone, and the recurrence of disease was monitored post-operatively (OV-90, n = 12). CD24-AF750 was further evaluated in four clinically annotated orthotopic PDX models of metastatic HGSOC, to validate the translational potential for intraoperative guidance. Findings: CD24-targeted intraoperative NIR FIGS significantly (473%) improved tumour detection and resection, and reduced the post-operative tumour burden compared to standard white-light surgery in orthotopic HGSOC xenografts. CD24-AF750 allowed identification of minuscule tumour lesions which were undetectable with the naked eye in four HGSOC PDX. Interpretation: CD24-targeted FIGS has translational potential as an aid to improve debulking surgery of ovarian cancer.

Background: The survival rate of patients with advanced high-grade serous ovarian carcinoma (HGSOC) remains disappointing. Clinically translatable orthotopic cell line xenograft models and patient-derived xenografts (PDXs) may aid the implementation of more personalised treatment approaches. Although orthotopic PDX reflecting heterogeneous molecular subtypes are considered the most relevant preclinical models, their use in therapeutic development is limited by lack of appropriate imaging modalities. Methods: We developed novel orthotopic xenograft and PDX models for HGSOC, and applied a near-infrared fluorescently labelled monoclonal antibody targeting the cell surface antigen CD24 for non-invasive molecular imaging of epithelial ovarian cancer. CD24-Alexa Fluor 680 fluorescence imaging was compared to bioluminescence imaging in three orthotopic cell line xenograft models of ovarian cancer (OV-90 luc+ , Skov-3 luc+ and Caov-3 luc+ , n = 3 per model). The application of fluorescence imaging to assess treatment efficacy was performed in carboplatin-paclitaxel treated orthotopic OV-90 xenografts (n = 10), before the probe was evaluated to detect disease progression in heterogenous PDX models (n = 7). Findings: Application of the near-infrared probe, CD24-AF680, enabled both spatio-temporal visualisation of tumour development, and longitudinal therapy monitoring of orthotopic xenografts. Notably, CD24-AF680 facilitated imaging of multiple PDX models representing different histological subtypes of the disease. Interpretation: The combined implementation of CD24-AF680 and orthotopic PDX models creates a state-ofthe-art preclinical platform which will impact the identification and validation of new targeted therapies, fluorescence image-guided surgery, and ultimately the outcome for HGSOC patients.

High-grade serous ovarian cancer (HGSOC) has poor prognosis and new treatment modalities are needed. Immunotherapy, with checkpoint inhibitors, have demonstrated limited impact. To evaluate the suitability for immunotherapeutics, contextualized preclinical models are required to secure meaningful clinical translation. Therefore, we developed and characterized humanized patient-derived xenograft (hu PDX) murine models of HGSOC, which were established by orthotopic implantation of tumor cell suspensions and intravenous injection of CD34+ cells isolated from umbilical cord blood samples. The developing human immune system in NSG and NSGS mice was followed longitudinally by flow cytometry and characterized by mass cytometry with a panel of 34 surface markers. Molecular imaging of tumor burden, survival analysis, and characterization of tumor-infiltrating immune cells was performed to assess the treatment response to anti-PD-1 (nivolumab) monotherapy. Successful generation of hu PDX models was achieved. Mice treated with nivolumab showed a decrease in tumor burden, however no significant survival benefit was identified when compared to untreated controls. No correlation was seen between PD-L1 expression and CD8 T cell infiltration and response parameters. As the characterization showed an immune infiltration of predominantly myeloid cells, similar to what is observed in HGSOC patients, the models may have the potential to evaluate the importance of myeloid cell immunomodulation as well.

Complete cytoreductive surgery is the cornerstone of the treatment of epithelial ovarian cancer (EOC). The application of fluorescence image-guided surgery (FIGS) allows for the increased intraoperative visualization and delineation of malignant lesions by using fluorescently labeled targeting biomarkers, thereby improving intraoperative guidance. CD24, a small glycophosphatidylinositol-anchored cell surface receptor, is overexpressed in approximately 70% of solid cancers, and has been proposed as a prognostic and therapeutic tumor-specific biomarker for EOC. Recently, preclinical studies have demonstrated the benefit of CD24-targeted contrast agents for non-invasive fluorescence imaging, as well as improved tumor resection by employing CD24-targeted FIGS in orthotopic patient-derived xenograft models of EOC. The successful detection of miniscule metastases denotes CD24 as a promising biomarker for the application of fluorescence-guided surgery in EOC patients. The aim of this review is to present the clinical and preclinically evaluated biomarkers for ovarian cancer FIGS, highlight the strengths of CD24, and propose a future bimodal approach combining CD24-targeted fluorescence imaging with radionuclide detection and targeted therapy.

Improved molecular dissection of the tumor microenvironment (TME) holds promise for treating high-grade serous ovarian cancer (HGSOC), a gynecological malignancy with high mortality. Reliable disease-related biomarkers are scarce, but single-cell mapping of the TME could identify patient-specific prognostic differences. To avoid technical variation effects, however, tissue dissociation effects on single cells must be considered. We present a novel Cytometry by Time-of-Flight antibody panel for single-cell suspensions to identify individual TME profiles of HGSOC patients and evaluate the effects of dissociation methods on results. The panel was developed utilizing cell lines, healthy donor blood, and stem cells and was applied to HGSOC tissues dissociated by six methods. Data were analyzed using Cytobank and X-shift and illustrated by t-distributed stochastic neighbor embedding plots, heatmaps, and stacked bar and error plots. The panel distinguishes the main cellular subsets and subpopulations, enabling characterization of individual TME profiles. The dissociation method affected some immune (n = 1), stromal (n = 2), and tumor (n = 3) subsets, while functional marker expressions remained comparable. In conclusion, the panel can identify subsets of the HGSOC TME and can be used for in-depth profiling. This panel represents a promising profiling tool for HGSOC when tissue handling is considered.