br Introduction br Pancreatic ductal adenocarcinoma PDAC is
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality in the United States . In 2017, there were 53,670 new cases and 43,090 deaths resulting from PDAC, which ac-counted for approximately 3% of all new cancer cases and 7% of all cancer deaths . The estimated overall PDAC 5-year survival rate is 8%, and the 5-year survival in advanced stages is only 3% . Early diagnosis significantly improves the survival of PDAC, the 5-year sur-vival rate for patients with minute tumors smaller than 10 mm (TS1a) following surgery can reach 80–86% . Therefore, a precision diag-nostic platform for early detection and localization of minute PDAC tumors is urgently needed.
Pancreatic cancer is one of the most aggressive cancers. It is common for metastasis to have already occurred by the time a patient
receives a diagnosis. However, metastases are frequently missed, and many patients with undetectable metastasis undergo surgery that does not provide significant survival benefit. On the other hand, benign pancreatic masses are often treated as minute PDAC tumors. Therefore, unnecessary surgeries are performed because traditional imaging techniques cannot discriminate minute PDAC tumors from benign pancreatic masses and cannot detect small metastases [3,4]. Non-invasive reporter gene imaging (NRGI) can provide noninvasive as-sessments of endogenous biologic processes in patients and can be performed using diﬀerent imaging modalities , however, insuﬃcient transgene expression and lack of tumor specificity limit clinically ef-fective NRGI. Non-imaging based diagnostic modalities for PDAC have included circulating biomarkers based on “omics” analyses, such as proteins, nucleic acids, circulating tumor CAY 10444 (CTCs), and exosomes, and are promising, but have not yet reached clinical applicability.
∗ Corresponding author. Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, OH, 43614, USA. E-mail address: [email protected] (F.C. Brunicardi).
33–46% of patients treated with pancreatic surgery have undetectable metastases or benign pancreatic masses that do not justify surgical in-tervention. Therefore, a precision diagnostic platform that can diﬀer-entiate minute resectable PDAC from currently undetectable metastases or benign masses is needed to improve survival for patients with re-sectable PDAC and avoid unnecessary surgical risk. One strategy for that is an integrated technology using a multiple-stage amplification strategy of dual reporter genes to enhance the specificity and sensitivity of detection and localization of minute PDAC tumors and currently
undetectable disease. The sensitivity and specificity of this precision diagnostic platform was tested in in vitro and in vivo human PDAC
models as a feasibility study.
2. Materials and methods
2.1. Cell lines, vectors, and antibodies
Human pancreatic cancer cell lines (PANC-1, Mia PaCa2, Capan2, and AsPc1) were purchased from the American Type Culture Collection (ATCC, Bethesda, MD) every 6 months. Human embryonic kidney cells, HEK 293FT, were purchased from ThermoFisher Scientific (ThermoFisher Scientific, MA). The cell lines were maintained in Dulbecco's modified Eagle medium (ThermoFisher Scientific, MA) supplemented with 100,000U/L of penicillin, 100,000μg/L of strepto-mycin, and 10% fetal bovine serum (FBS). Human primary pancreatic cells epithelial cells (HPPE) were purchased from Cell Biologics, Inc. (Chicago, IL). Human pancreatic ductal epithelial (HPDE) cell line was kindly provided by Ming-Sound Tsao (University of Toronto, Toronto, Ontario, Canada) and authenticated by short tandem repeat analysis (STR) using Promega kit with 16 STR loci every 6 months. Patient derived cell lines (PDCL-15, original name TKCC-15-Lo) was kindly provided by Dr. Andrew Biankin from Wolfson Wohl Cancer Research Centre, UK with authentication by STR. PDCL-15 was maintained in M199/F12 (Ham mixture) media mixed 1:1 with Ham's F-12 and sup-plemented with 15 mM HEPES, 1x MEM vitamin solution, 20 mM glu-tamine, 25 μg/mL human apo-transferrin, 20 ng/mL human re-combinant EGF, 0.2IU/mL Insulin, 0.5 pg/mL Triiodothyronine, 40 ng/ mL hydrocortisone, 2 μg/mL Ophosphorylethanolamine, 0.06% glucose solution, and 7.5% Fetal Calf Serum (FCS).
AAVpro Helper Free System (AAV2) and AAVpro™ Extraction
Feng Zhang (Addgene).
2.2. Construction and preparation of lentiviral vectors
LentiCas9-tdtomato-blast (LentiCas9-tdT) was generated by ligation of Cas9 and tdTomoto through T2A peptide. A LentiGuide vector was generated by cloning GFP in pCDH-CMV-MCS-EF1-Neo (System Biosciences, Mountain View, CA) followed by insertion of a human KrasG12D homology directed repair (HDR) donor (for human) or a mouse HDR donor (for mouse). Gene-specific sgRNA oligos were cloned into the lentiGuide-Puro. The sgRNA sequences targeting KrasG12D, TP53, P16, and SMAD4 were designed by the CRISPR Design Tool (http://crispr.mit.edu/) and synthesized by IDT. All the sgRNA se-quences were listed. All constructs were sequence verified. Lentiviruses were produced by transfecting HEK 293T cells with the vector, pMD2.G and psPAX2 DNAs in 5:2:3 ratio with PEI. After 48 h of culturing, the viral supernatant was collected and passed through a 45 μm filter. Lentiviral particles were precipitated and concentrated with PEG-it by centrifugation at 1,500g for 30 min at 4 °C. The lentiviruses were re-suspended in 500 μl PBS and frozen in a −80 °C freezer.