Abstract
Simple SummaryHigh-grade serous ovarian cancer is a fatal disease typically detected at an advanced stage when options for effective treatment are significantly limited. The lack of a screening modality to identify ovarian cancer in its early stage continues to be a major impediment in the management of this malignancy. The serum biomarker CA125, a repeating peptide epitope present in the sialomucin, MUC16, is unsuitable as a screening test. We have demonstrated that immune cells of ovarian cancer patients capture miniscule amounts of CA125 on their cell surface. Here, we report an automated, sensitive, alignment-free microscopy platform to qualitatively and quantitatively assess the low-abundance binding of CA125 to circulating leucocyte subsets. Through a comparison of the CA125 levels on immune cell subsets of ovarian cancer patients versus healthy donors, we demonstrate that our new technique can serve as a novel diagnostic platform for detection and monitoring of ovarian cancer.MUC16, a sialomucin that contains the ovarian cancer biomarker CA125, binds at low abundance to leucocytes via the immune receptor, Siglec-9. Conventional fluorescence-based imaging techniques lack the sensitivity to assess this low-abundance event, prompting us to develop a novel “digital” optical cytometry technique for qualitative and quantitative assessment of CA125 binding to peripheral blood mononuclear cells (PBMC). Plasmonic nanoparticle labeled detection antibody allows assessment of CA125 at the near-single molecule level when bound to specific immune cell lineages that are simultaneously identified using multiparameter fluorescence imaging. Image analysis and deep learning were used to quantify CA125 per each cell lineage. PBMC from treatment naïve ovarian cancer patients (N = 14) showed higher cell surface abundance of CA125 on the aggregate PBMC population as well as on NK (p = 0.013), T (p < 0.001) and B cells (p = 0.024) compared to circulating lymphocytes of healthy donors (N = 7). Differences in CA125 binding to monocytes or NK-T cells between the two cohorts were not significant. There was no correlation between the PBMC-bound and serum levels of CA125, suggesting that these two compartments are not in stoichiometric equilibrium. Understanding where and how subset-specific cell-bound surface CA125 takes place may provide guidance towards a new diagnostic biomarker in ovarian cancer.
Highlights
IntroductionCA125 is a repeating protein epitope of the >2 million Da MUC16 molecule [1,2]
CA125 is a repeating protein epitope of the >2 million Da MUC16 molecule [1,2].This sialomucin is overexpressed by high-grade serous ovarian tumors [3,4]
Using plasmonic nanoparticles (PNP) conjugated to the anti-CA125 antibody, we recently demonstrated in a small patient cohort that cell-bound CA125 is more abundant on peripheral blood mononuclear cells (PBMC) of serous invasive cancer patients than in healthy controls [18]
Summary
CA125 is a repeating protein epitope of the >2 million Da MUC16 molecule [1,2]. This sialomucin is overexpressed by high-grade serous ovarian tumors [3,4]. MUC16 is shed from the cell surface, and as a result, can be detected in serum using antibodies directed against the CA125 epitope [5]. It has been established that MUC16 binds to peripheral blood mononuclear cells (PBMC) [11] and, in the case of NK cells, this binding blunts its anti-tumor response [12,13,14]. Our previous studies showing MUC16 binding to PBMC relied on the use of flow cytometry Quantitative levels of serum CA125 have proven useful to define progression-free survival of serous ovarian cancer patients after they have received first-line therapy [6] and in the management of serous ovarian cancer in patients past their cytoreductive surgery and chemotherapy [7,8,9,10].
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