BACKGROUND: Tumors can influence peripheral immune macroenvironment, thereby creating opportunities for non-invasive serum/plasma immunobiomarkers for immunostratification and immunotherapy designing. However, current approaches for immunobiomarkers' detection are largely quantitative, which is unreliable for assessing functional peripheral immunodynamics of patients with cancer. Hence, we aimed to design a functional biomarker modality for capturing peripheral immune signaling in patients with cancer for reliable immunostratification. METHODS: We used a data-driven in silico framework, integrating existing tumor/blood bulk-RNAseq or single-cell (sc)RNAseq datasets of patients with cancer, to inform the design of an innovative serum-screening modality, that is, serum-functional immunodynamic status (sFIS) assay. Next, we pursued proof-of-concept analyses via multiparametric serum profiling of patients with ovarian cancer (OV) with sFIS assay combined with Luminex (cytokines/soluble immune checkpoints), CA125-antigen detection, and whole-blood immune cell counts. Here, sFIS assay's ability to determine survival benefit or malignancy risk was validated in a discovery (n=32) and/or validation (n=699) patient cohorts. Lastly, we used an orthotopic murine metastatic OV model, with anti-OV therapy selection via in silico drug-target screening and murine serum screening via sFIS assay, to assess suitable in vivo immunotherapy options. RESULTS: In silico data-driven framework predicted that peripheral immunodynamics of patients with cancer might be best captured via analyzing myeloid nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) signaling and interferon-stimulated genes' (ISG) responses. This helped in conceptualization of an 'in sitro' (in vitro+in situ) sFIS assay, where human myeloid cells were exposed to patients' serum in vitro, to assess serum-induced (si)-NFκB or interferon (IFN)/ISG responses (as active signaling reporter activity) within them, thereby 'mimicking' patients' in situ immunodynamic status. Multiparametric serum profiling of patients with OV established that sFIS assay can: decode peripheral immunology (by indicating higher enrichment of si-NFκB over si-IFN/ISG responses), estimate survival trends (si-NFκB or si-IFN/ISG responses associating with negative or positive prognosis, respectively), and coestimate malignancy risk (relative to benign/borderline ovarian lesions). Biologically, we documented dominance of pro-tumorigenic, myeloid si-NFκB responseHIGHsi-IFN/ISG responseLOW inflammation in periphery of patients with OV. Finally, in an orthotopic murine metastatic OV model, sFIS assay predicted the higher capacity of chemo-immunotherapy (paclitaxel-carboplatin plus anti-TNF antibody combination) in achieving a pro-immunogenic peripheral milieu (si-IFN/ISG responseHIGHsi-NFκB responseLOW), which aligned with high antitumor efficacy. CONCLUSIONS: We established sFIS assay as a novel biomarker resource for serum screening in patients with OV to evaluate peripheral immunodynamics, patient survival trends and malignancy risk, and to design preclinical chemo-immunotherapy strategies.

Charles University Praha Czech Republic

Department of Cellular and Molecular Medicine Cell Death Research and Therapy Laboratory KU Leuven Belgium

Department of Development and Regeneration KU Leuven Leuven Belgium

Department of Gynaecology and Obstetrics UZ Leuven Leuven Belgium

Department of Microbiology Immunology and Transplantation Laboratory of Virology and Chemotherapy Rega Institute KU Leuven Leuven Belgium

Department of Oncology Leuven Cancer Institute Laboratory of Gynaecologic Oncology KU Leuven Leuven Belgium

Department of Oncology Leuven Cancer Institute Laboratory of Tumor Immunology and Immunotherapy KU Leuven Leuven Belgium

Dipartimento Scienze della Salute della Donna e del Bambino Fondazione Policlinico Universitario A Gemelli Istituto di Ricovero e Cura a Carattere Scientifico Rome Italy

Dipartimento Scienze della Vita e Sanità pubblica Università Cattolica del Sacro Cuore Rome Italy

JJP Biologics Warsaw Poland

Laboratory for Molecular Digestive Oncology Department of Oncology KU Leuven Leuven Belgium

Laboratory of Cell Stress and Immunity Department of Cellular and Molecular Medicine KU Leuven Leuven Belgium

National Cancer Institute of Milan Milan Italy

Queen Charlotte's and Chelsea Hospital Imperial College London UK

VIB Center for Cancer Biology VIB Leuven Belgium

Ziekenhuis Oost Limburg Genk Belgium

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