The murine PDAC cell line KPC4580P, a gift from Dr. Jen Jen Yeh of University of North Carolina, was established from male LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1Cre/+; LSL-Rosa26 Luc/+ mouse as previously described . The cells were cultured with DMEM : F12 media in 50:50 ratio with 10% FBS and 1% penicillin-streptomycin antibiotics at 37° C with 5% CO2. Cells were used within three passages of thawing between experiments.
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, San Diego (UCSD). Wild type (WT) 12-14 week old adult male C57BL/6 (Jackson Laboratories, Bar Harbor, ME) were utilized in an orthotopic pancreatic tumor model as previously described . Briefly, 500,000 KPC-4580P cells were injected SQ and allowed to grow for 10 days. The tumors were harvested, and small pieces (~ 2 mm3) were implanted onto the tail of the mouse pancreas. Once the tumors reached 5 mm diameter, the mice underwent PRVI as previously described . In short, PRVI oxaliplatin and PRVI saline mice underwent laparotomy followed by temporary pancreatic venous isolation with microvascular clamps (Roboz Surgical, Boston, MA), and portal vein cannulation. Mice were randomized to receive either oxaliplatin 2 mg/kg in 100 µL saline or saline (100 µL), infused at a rate of 5 mL per minute. The venous isolation was released after 5 min of clamp time. The portal vein puncture site was covered with Surgicel (Ethicon, Raritan, NJ). Intraperitoneal injections of oxaliplatin at concentrations of 2 mg/kg, 20 mg/kg, or 40 mg/kg were used as systemic controls. Mice (n = 3) from the PRVI oxaliplatin and systemic oxaliplatin groups were randomly selected for euthanasia at 3 h post-infusion to evaluate platinum concentrations within tumors as well as in plasma. All mice were euthanized on day 7, and tumors and foot skin (peripheral nerves) were harvested and evaluated for treatment response.
Tissue (tumor and foot skin) specimens collected from euthanized mice 7 days after infusion underwent histologic evaluation. Slides containing serial tissue sections of paraffin-embedded tissue blocks were deparaffinized with xylene and hydrated using ethanol gradient. For IHC staining using the neuronal marker PGP 9.5, slides were deparaffinized with xylene, hydrated using ethanol gradient, and quenched with methanol/hydrogen peroxide, followed by antigen retrieval with citrate buffer (pH=6). Tissue sections were blocked with horse serum and incubated with primary antibody anti-mouse PGP 9.5 (ProteinTech) followed by HRP-conjugated secondary antibody. DAB (3,3’-diaminobenzidine) solution was used to develop the staining and counterstained with hematoxylin. The slides were imaged using an Olympus SC100 microscope at 20 × magnification. The number of PGP 9.5 stained foci were counted independently within the epithelium and the dermal layers as Intra-Epidermal Nerve Fibers (IENF) and Sub-epidermal Neural Plexi (SNP), respectively (19) in 5 mice/group with each data point representing the mean value from 3 regions of interest per mouse.
Measurement Of Platinum Concentration
Tumor tissue from animals was collected into 15 mL conical tubes, documenting actual tissue weights, and washed with 70% ethanol. In the fume hood, 571 µL of Metal Free Nitric Acid (37%) was added to the tube containing the tissue and vortexed at high speed for 10 seconds. Samples were left in a fume hood overnight for tissue digestion. Once tissue was fully dissolved, the total weight of the mixture was determined and increased to a final volume of 10 mL with ultrapure distilled water. Samples were centrifuged and the supernatant was filtered and collected for Inductively Coupled Plasma- Mass Spectroscopy analysis. The ICP-MS analysis was done on a Thermo Scientific iCAP RQ ICP-MS in the Environmental and Complex Analysis Laboratory on the UC San Diego campus. The analysis was conducted in standard (STD) mode monitoring 159Tb and 209Bi as internal standards. The results were reported as ppb of platinum relative to the initial tumor weight .
Motor Nerve Conduction Velocity
Mice were anesthetized using 2.5 to 4 ppm isoflurane in oxygen and transferred to a circulating water–heated pad, with anesthesia maintained via a face mask connected to the isoflurane regulator. Two recording electrodes (platinum-tipped sub-dermal needle electrodes, Grass Technologies) were inserted into the interosseous muscles between the animal’s second, third, and fourth toes, and secured to the heating pad with lab tape. A grounding electrode was placed into the skin at the neck. A PowerLab stimulator (AD instruments) was set to deliver a 200-mV, 50-µsec-duration square-wave stimulus every 2 sec. The stimulating electrode was inserted into the ankle near the Achilles tendon and adjusted until the resulting M waves were clear and maximal. The electrode was removed and inserted into the sciatic notch at the hip and resulting M waves were recorded, as previously described . The distance between the stimulation sites at the hip and ankle was measured and divided by the time (latency) between the peaks of proximal and distal sites to obtain the motor nerve conduction velocity (MNCV).
Statistical Analysis And Data Availability
All data were analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc, La Jolla, CA). Student’s t test with 2-tailed hypothesis was used to compare 2 groups or 1-way analysis of variance with Tukey’s multiple comparison test was used for multiple groups. Survival was compared between groups by Kaplan-Meier analysis. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.