Cabozantinib

Cisplatin was purchased from Selleckchem (via Absource Diagnostics GmbH, Munich, Germany) and osimertinib from MedChemExpress (via Hycultec, Beutelsbach, Germany). Cabozantinib and tinvantinib, both obtained from Selleckchem (via Absource Diagnostics GmbH, Munich, Germany), were used for pharmacological inhibition. Cabozantinib, a receptor tyrosine kinase inhibitor (TKI) that targets multiple tyrosine kinases including VEGFR2, RET, AXL, FLT3, c-KIT, and c-MET, is clinically approved for the treatment of medullary thyroid cancer, renal cell carcinoma, and hepatocellular carcinoma [21 (link)]. Tivantinib, a selective inhibitor of unactivated c-MET and its self-phosphorylation, has been successful in phase II trials; however, it failed in two phase III trials for second-line treatment of advanced high-Met hepatocellular carcinoma [22 (link)]. Drugs were used at non-toxic concentrations evaluated by trypan blue staining.

TPC-1 (ATCC, Manassas, VA) and 8505C (ATCC) were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 U of penicillin and 100 μg of streptomycin per ml. C643 (ATCC) was maintained in the same medium additionally supplemented with 292 mg/L glutamine. FTC133 was obtained from B. Nelkin (Johns Hopkins Medical Institute) and maintained in DMEM/Ham’s F-12 medium (Invitrogen). These media were supplemented with serum and antibiotics as above. The progenies of PTC-1 pTRIPZ-dox-shMort and C643 pTRIPZ-dox-shMort were generated by clonal selection after prolonged cell culture in the presence of puromycin.
Mito-CP [33 (link)] was obtained from B. Kalyanaraman (Medical College of Wisconsin). Methyl-triphenyl-phosphonium (TPP), puromycin, G418, and doxycycline were purchased from Sigma-Aldrich (St Louis, MO, USA). Vandetanib was purchased from LC Laboratories (Woburn, MA, USA). PLX4032 and cabozantinib were purchased from Selleck Chemicals (Houston, TX, USA). Chemical structures of Mito-CP and TPP are depicted in Figure S2 of ref [18 (link)].

All mouse studies were approved by the University of Cincinnati Institutional Animal Care and Use Committee (IACUC) (protocol: 07-01-11-01, approval date: 17 September 2018) that maintains an American Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC) facility. C57BL/6 mice purchased from Jackson Laboratories were orthotopically transplanted with 500,000 7940B cells derived from a primary spontaneous PDAC tumor arising in the body of the pancreas (C57BL/6) of a male transgenic Kras^LSL-G12D/+, Trp53^LSL-R172H/+, Pdx1-Cre (KPC) mouse [39 (link),40 (link)] (kindly donated by Dr. Gregory Beatty, University of Pennsylvania). Nod scid gamma mice were orthotopically transplanted with 500–1000 pancreatic cancer organoids derived from PDAC patients.
In a separate series of experiments, 7 days post-orthotopic transplantation, C57BL/6 mice (Jackson Laboratories) were treated with gemcitabine (Selleckchem, S1149) (325 μg/mouse, i.p.) every 2 weekdays and Epothilone A (abaraxane, Selleckchem, S1297) (13 μg/mouse, i.v.) every 5 days, InVivoPlus anti-mouse PD-1 (BioXCell, BP0146) (200 μg/mouse, i.p.) every 5 days, Cabozantinib (cabo, Selleckchem, S1119) (780 μg/mouse, oral gavage) every weekday or a combination of 3 or 4 drugs for 7 days. The mice were sacrificed, tumors were removed and weighed.