Investigator: PnP Research Corporation, Drury, MA
PnP Research Corporation LLC is a Massachusetts based company, developing ultrasensitive antigen detection platform using plasmonic nanoparticles.
Mucins are complex glycoproteins that line the peritoneum, breast ducts, and the respiratory, digestive, and genital tracts. While mucins serve to protect the underlying tissue from environmental stressors [1], inflammatory conditions, including neoplasms, can cause them to degrade and shed into immunogenic forms, such as MUC16 (CA125) in ovarian cancer [2]. To detect and diagnose ovarian cancer, serum assays for the CA125 antigen were developed and tested in the 1980s [3]. However, other conditions, such as pregnancy, endometriosis, and pelvic infection, can also cause elevated CA125 serum levels, which confound results. Furthermore nearly 20% of ovarian cancer patients never present with elevated CA125 serum levels [4]. Thus, it is believed that other mechanisms must exist to remove CA125 from circulation. Recent studies have revealed that antigens such as CA125 can interact and bind with immune cells. Once bound, the CA125 markers may become undetected by traditional serum assays. More importantly, this binding has been found to affect the behavior of certain subclasses of immune cells. For example, the anti-tumor activity of NK cells is blunted by the binding of MUC16 [5].
Current cytometric analysis methods, which mainly rely on fluorescence, have a lower detection threshold of ~500 molecules per cell due to factors including fluorophore emission cross-talk and the autofluorescence background of the cell. As alteration of immune cell activity, - such as the interaction between NK cells and CA125 - occurs at binding levels on the order of tens of molecules per cell, new methods and contrast agents are needed for the sensitive and accurate quantification of bound cancer antigens. Such a toolkit would have significant applications in the early detection, monitoring, and immunotherapy of cancer.
Using the bright light scattering of plasmonic nanoparticles (PNPs), we are creating a digital cytometric analysis method that can quantify molecular binding events near the single-molecule limit in whole blood. This ability will enable the study and development of immunotherapies and enable personalized treatments to aid in the treatment of cancer. PNPs, 60-80 nm in size, are functionalized with antibodies either via adsorption or through covalent attachment, with the final construct validated using dynamic light scattering methods. These antibody-functionalized PNPs are administered either to whole blood in an accelerant formulation designed to promote rapid binding or to FICOLL-prepared mononuclear frozen white blood cells. The binding of PNPs to cellular surfaces is read out using high numerical aperature dark field microscopy, which can detect individual PNP as well as their aggregates through plasmon-coupling induced color shifts. This color-corrected detection enables a digital method for the precise enumeration of individual PNP with detection sensitivity on the order of individual molecules of bound antigen (Fig 1). In preliminary studies, this approach has enabled the detection and quantification of CA125 bound to the surface of different white blood cell types, including B cells, monocytes, and NK cells. Importantly, in these studies, patients with ovarian cancer or ovarian masses show distinctive patterns of CA125 binding that may be representative of different disease classes.
By adapting the plasmonic vertical nanopillar platform (being developed the LBRC), the Raman Lab will build a catalogue of spectral and biophysical markers that constitute each metastatic site and validate/correlate them against immunohistochemistry as well as metabolomic and proteomic analyses. Through this collaboration, we will also determine the chemotherapeutic efficacy of four major classes of FDA-approved drugs - antimetabolites (gemcitabine), anti-mitotics (taxol), antibiotic / DNA replication disruptor (doxorubicin) and alkylating agents (cyclophosphamide) - by monitoring the identified mechanical and molecular markers and testing against standard MTS and clonogenic assays.
PnP Research Corporation is developing an ultrasensitive antigen detection platform using plasmonic nanoparticles. In parallel, the LBRC is also developing new plasmonic nanoparticles using click chemistry to generate PEGylated particles with covalently attached antibodies for reduced nonspecific binding and greater sensitivity. PnP Research currently enumerates molecular binding events using dark field microscopy, but searching for rare events in whole blood requires faster imaging methods. In response to this need, the LBRC is develping a novel wide-field photothermal microscopy setup for rapid, flow-based quantification of individual plasmonic nanoparticles on the cell surface.