Preparation and Characterization of HET0016 Formulation

HPßCD (2-hydroxy Propyl-β-Cyclodextrin) was purchased from Sigma-Aldrich (St. Louis, MO), cell culture media was from Thermo Scientific (Waltham, MA), and fetal bovine serum was purchased from Hyclone (Logan, Utah). HET0016 was made by Dr. Levedyeva in the Department of Chemistry, Augusta University with a purity of more than 97%, and was prepared for animal treatment according to our previously described method [8 (link)]. Additional information for HET0016 synthesis strategies is provided in the S1 File. Cell culture grade DMSO was purchased from Fischer Scientific (Pittsburg, PA). the complex of HET0016 plus HPßCD was made as per previous publication [8 (link)]. VEGFR2 tyrosine kinase inhibitor (Vatalanib) and colony-stimulating factor 1 receptor (CSF1R) inhibitor (GW2580) were purchased from LC Laboratories, Woburn, MA. SB225002 (CXCR2 inhibitor) was purchased from Selleckchem, Houston, TX. Navarixin was purchased from MedKoo bioscience Inc, Morrisville, NC. All flow antibodies are from Bio Legend, San Diego, CA. All antibodies for western blotting, immunohistochemistry, and immunofluorescence were purchased from Santa Cruz (total-CXCR2 and anti-GAPDH), R&D Systems (anti-hCXCR2), Thermo Scientific (anti-Laminin), and Sigma Aldrich (β-actin and FITC-conjugated tomato lectin). All culture media were purchased from Corning and GE Healthcare Life Sciences.