High-multiplexing quantitative CodePlex proteomic profiling of platelets in triple-negative breast cancer (TNBC) patients and healthy subjects.

e15017 Background: Platelets play significant roles in inflammation, immune regulation, and intracellular communication processes involved in tumor pathogenesis. Aberrant platelet activation has been associated with tumor progression and metastasis, however, cancer patient platelet deep profiling remains underutilized. New tools are needed for rapid high throughput quantification of potential altered platelet secretome in cancer patients. Such technology may provide novel insights into impactful cancer-associated biomarkers. Methods: We have developed a high throughput multiplexed proteomic technology to profile platelet cytokine content and releasates and performed a pilot study in healthy patients versus triple negative breast cancer (TNBC). Macrochambers of the CodePlex chip were pre-patterned with a 23-plex antibody IsoPlexis Human Adaptive Immune Panel: GM-CSF, Granzyme B, IFN-g, IL-10, IL-13, IL-15, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MIP-1a, MIP-1b, perforin, sCD137, TGF-b1, TNF-a, TNF-b. 2% BSA/PBS was used as background control. The chips are loaded into an IsoLight automation system, releasates measured by fluorescence ELISA, and analysis performed by IsoSpeak software. In a pilot study to test feasibility, platelets were meticulously harvested from triple negative breast cancer patients (n=2) and healthy controls (n=2) and stimulated for 30 minutes (ADP or TRAP-6) to promote cytokine release. Both the supernatants and platelet lysates were then loaded on CodePlex chips and analyzed. Results: The methodology of bulk proteomic measurements has previously been validated in a variety of biofluids such as serum/plasma, cerebrospinal fluid, tracheal washes, and cell culture supernatants. Freshly isolated platelets activated by TRAP6 generally released more cytokines than those activated by ADP stimulation in both healthy controls and disease samples. Interestingly, platelet releasates in supernatants from TNBC patients demonstrated increased pro-tumor IL-8 and IL-9, and reduced antitumor IFN-g compared to healthy controls. Higher levels of IL-8 and IL-9 and minimal IFN-g were detected at similar activation concentrations, in platelet lysates from both healthy and TNBC platelets. This suggests that TNBC may alter platelet content and/or activation. Increased numbers are being performed to validate these initial results. Conclusions: From this small pilot study, multiplexing quantitative proteomic analysis is potentially useful in detecting differential cytokine profile in platelet releasates and lysate contents between TNBC patients and healthy controls. Quantifying the platelet proteomes in cancer patients using high throughput proteomics may provide valuable insights to dissect cancer-platelet crosstalk and potential pathogenesis mechanisms.