br Minimum Essential Medium Roswell Park Memorial Institute RPMI
Minimum Essential Medium α, Roswell Park Memorial Institute (RPMI) 1640 Medium, Dulbecco's Modified Eagle Medium, Leibowitz’ L-15 Medium, EGM-2 Endothelial Cell Growth Medium, M-199 Me-dium, Endothelial Cell Growth Suspension (ECGS), Penicillin/strepto-mycin solution, 2% gelatin solution, Opti-MEM Reduced Serum Medium, RNAiMAX Lipofectamine, 10× Tris-buffered saline (TBS), Scott's Bluing Solution, Fisherfinest Histoplast paraffin, MX35 Ultra low-profile cryotome blades, polylysine slides, HPLC grade ethyl acetate, acetonitrile, ACS grade glacial acetic acid, 2-propanol, recombinant human TNF-α, recombinant human OPG, recombinant murine RANKL, Vybrant™ DiD lipophilic dye, CellTrace™ Calcein Green dye and 7-AAD viability assay dye were purchased from Fisher Scientific. 4% paraformaldehyde (PFA) solution was purchased from Santa Cruz Bio-technology. Recombinant human TRAIL was obtained from Peprotech. Fetal bovine serum (FBS), sterile phosphate-buffered saline (PBS), ACK
② TUMOUR KILLING
PSGL-1/SLEX P- and E-selectins
③ INHIBITION OF
BONE TUMOUR Tumour
Fig. 1. Combinatorial targeting of cancer bone metastasis using mRNA engineered mesenchymal stem cells. Proposed strategy for bone metastasis treatment by targeted delivery of multiple factors using mRNA-engineered mesenchymal stem cell (MSC) equipped with functions for a) specific and efficient bone metastases homing through P-selectin glycoprotein ligand-1 (PSGL-1) and Sialyl-Lewis X (SLEX), b) local cancer killing through 2-NBDG deaminase (CD)/pro-drug 5-Fluorocytosine, and c) osteolysis inhibition within the tumour niche through a modified version of RANKL decoy receptor, osteoprotegerin (OPG).
lysis Buffer, Tissue-Tek Optimal Cutting Temperature (OCT) embedding medium and Superfrost Plus slides were purchased from VWR. Puromy-cin powder, crystalline bovine serum albumin (BSA), Tween 20, Triton X-100, as well as donkey and goat normal sera, Harris' Haematoxylin, Eosin Y, 2-methylbutane, 10% Formalin solution, sucrose, EDTA, 5-Fluorouracil (5-FU), and 5-Fluorocytosine (5-FC) were purchased from MilliporeSigma. Isoflurane was purchased from Piramal Healthcare. Ketamine, xylazine, and buprenorphine were purchased from Western Medical Supply. D-luciferase was purchased from Perkin Elmer. Histo-Clear II was used as a xylene substitute for dehydration of tissue samples and was purchased from National Diagnostics. Water was purified using the Millipore Milli-Q system.
MDA-MB-231 human ductal adenocarcinoma cells, 4T1 murine breast cancer, RAW264.7 macrophages, and HL-60 promyeloblasts were purchased from American Type Culture Collection, Inc. (ATCC) and respectively cultured in Leibovitz’ L-15 medium, RPMI medium, Dulbecco's Modified Eagle Medium and RPMI medium supplemented with 10% FBS. MDA-MB-231 and 4T1 cells were transduced to express both firefly luciferase enzyme and RFP (LucF/RFP) using CMV-Luc3-2A-RFP (Puro) lentiviral particles (GenTarget, San Diego, CA, USA) per the manufacturer's protocol. Briefly, 1 × 104 cells were seeded in a 24-well plate and infected with a multiplicity of infection of 10 particles/ cell. 72 hours post-transduction, media was replaced with fresh media containing 20 μg/mL puromycin for selection. 5 μg/mL puromycin was used for routine culture to maintain transduction. LucF/RFP 4T1 CLL1 cells were in vivo selected from a mouse femur metastasis, which arose from a mammary fat pad injection of LucF/RFP 4T1 cells. 4T1 CLL1 were cultured similarly to parental 4T1 cells. Human mesenchymal stem cells (MSC), obtained under principles of informed consent from the bone marrow of a healthy donor (#8011L), were purchased from Texas A&M Institute for Regenerative Medicine (Bryan, TX, USA), a NIH-funded non-profit organisation for MSC isolation, characterisation and distribution. These cells were fully characterised per established guidelines [35,36]: MSC were tested for presence of viruses (HIV,
hepatitis, etc.), characterised by their ability to differentiate into bone and fat, and by their negative expression for CD45, CD19, CD34, CD11b, CD79a, HLA-II:DR DQ DP, CD14 and positive expression for CD90, CD105, CD73a. MSC were cultured in Minimum Essential Medium-α enriched with 15% FBS, 2% L-glutamine, and 1% penicillin-streptomycin solution. To further characterise if engineering affects MSC function, we performed additional osteogenic and adipogenic differentiations of our engineered MSC (Supplementary Fig. 1). Specifically, PSGL-1/SLEX/ CD/OPG MSC were seeded onto 24-well tissue culture plates at a density of 6 × 104 cells/well and cultured for 2–3 weeks. The media was made from Osteogenesis or Adipogenesis Differentiation Kits (EMD Millipore) and changed according to the manufacturer's instructions. At the end-point, cells were fixed and stained with Alizarin Red-S or Oil-Red-O, ac-cording to the manufacturer's instructions. Human umbilical vein endo-thelial cells (HUVEC) were kindly provided by Dr. Hughes (Department of Cellular and Molecular Biosciences, University of California, Irvine) and cultured in M-199 medium supplemented with 10% FBS, 2% L-gluta-mine, 1% penicillin-streptomycin solution, and 50 μg/mL ECGS. MDA-MB231 LucF/RFP were cultured without CO2.