Feasibility of Flow Cytometric Identification and Isolation of Pre-Platelets to Understand Its Function and Role in Cancer Metastasis

Background:It is known that platelets interact with tumors to support malignant cell growth and stimulate epithelial to mesenchymal transition (Li, et al. "The Role of Platelets in Tumor Growth, Metastasis, and Immune Evasion", Platelets, pp. 547-561, Academic Press, 2019). P-selectin which is secreted from alpha granules has been implicated as the major perpetrator of this tumor cell and platelet communication. (Kim et al, PNAS 95(16): 9325-9330, 1998). Higher numbers of alpha granules are found in newly released young platelets from megakaryocytes, also known as pre-platelets, than mature platelets. Given their potential role in cancer metastasis, the ability to identify and isolate pre-platelets from mature plateletsforassessment of overall functionality and biology is important. Of note, identification and measurement of pre-platelets based on higher RNA content has been in clinical use for various clinical conditions for several years. Here we evaluated the feasibility of identifying and sorting pre-platelets by flow cytometric analyses in multiple platelet apheresis samples from heathy donors.

Study Design and Methods Apheresis samples from adult healthy donors at Roswell Park Comprehensive Cancer Center were obtained and analyzed in accordance with an IRB approved protocol. Platelets sampled from apheresis products were collected with the use of an apheresis device known to yield a leuko-reduced platelet concentrate. All components were irradiated with 2500 cGy immediately after the collection and stored on platelet agitator at 22°C. A 4 mL aliquot was withdrawn from each bag for sorting essays.Pre-platelets were distinguished from mature platelets based on the presence of higher RNA levels. The cell permeant RNA dye TO (BD Biosciences, San Jose, CA) was used to identify pre-plapletes by flow cytometry according to published protocol (Ando et al Blood97 (4): p915-921, 2001).The optimal concentration of TO dye was determined by titration at decreasing dilutions and acquisition on a LSR-II flow cytometer (BD Biosciences, San Jose, CA). Confirmation of flow cytometry-based assay was determined by processing 5 healthy donor whole blood samples by both flow cytometry and using an XE-5000 automated hematology analyzer (Sysmex America, Mundelein, IL). The percentage of pre-platelets was measured as a percent of total platelets by both techniques. Platelet sorting was performed on 12 leuko-reduced apheresis platelet products on a FACSAria II (BD Biosciences, San Jose, CA). Platelets were identified by co-staining with CD41a. Platelet activation was assessed before and after sorting based on P-selectin (CD62P) surface expression. Flow cytometry analysis was performed using FCSExpress v6.

Results: We determined that flow cytometric sorting of pre-platelets optimally required TO (0.5 µg/mL) for optimal staining for gating of pre-platelets. A two way, paired simple t test resulted in a p value of 0.09 when compared to standard gating strategy per XE-5000 automated hematology analyzer. Among all 14 samples evaluated, the percentage of preplatelet to apheresis sample pre-platelet ratio was estimated between 2.8% to 8.2%. Interestingly P-selectin ratio of pre-platelets was significantly higher than mature platelets (median: 67% vs 52%).

ConclusionThis study is the first to demonstrate the feasibility of identifying and sorting pre-platelets using a novel flow cytometric method with TO staining. Isolation of these rare platelet precursor cells (pre-platelets) with known higher P-selectin expression are an essential first step to understanding the potential pre-eminent role of these cells in cancer evasion and metastasis.