Circulating tumor cell (CTC) isolation and analysis

CTC isolation and analyses were performed as previously described⁴⁶. Cardiac blood draws were performed on day 50 of the experiment using heparinized needles and syringes to obtain roughly 1 mL of circulatory volume. Blood samples underwent immediate red blood cell lysis and the resulting supernatant was aspirated and the procedure was repeated. Red blood cells were resuspended in FACs buffer that contained 1 × 10⁵ polystyrene FluoSpheres (Molecular Probes, F8843, 15 µm scarlet) per mL. CTCs in each sample were detected and quantified using flow cytometry and a known concentration of microspheres. Samples were run on an LSR II (BD Biosciences, λ_ex = 645 nm and λ_em = 680 nm) flow cytometer and analysis performed utilizing FlowJO software (BD Biosciences). Results were used to determine circulating tumor cell concentration (CTC/mL) for each mouse by the equation [number of CTCs detected/ (FluoSpheres detected/100,000 FluoSpheres per mL)].

Hepatic tumor cell isolation was performed as previously described⁴⁶. Liver specimens with visible metastatic lesions were used to isolate and grow single tumor cells from mice. Hepatic sections were quickly minced with a scalpel in cold HBSS and centrifuged before the addition of 0.5% w/v collagenase (Gibco, 17100). Cells were plated and grown for further analysis.

The CTC-derived nuclease activity assay was performed as previously described⁴⁰. Plasma samples were collected from subjects with stage 4 metastatic pancreatic cancer enrolled in a phase I trial where P-AscH⁻ was combined with gemcitabine⁷ approved by The University of Iowa Human Institutional Review Board and the Protocol Review and Monitoring Committee of the Holden Comprehensive Cancer Center at The University of Iowa Hospitals and Clinics on May 22, 2008. Informed consent was obtained from all participants and all research was performed in accordance with the approved guidelines and regulations. The trial was listed on www.clinicaltrials.gov under {"type":"clinical-trial","attrs":{"text":"NCT 01049880","term_id":"NCT01049880"}}NCT 01049880. Nuclease activity was also determined in a separate phase I trial involving local–regional pancreatic cancer without distant metastases⁶. This phase I trial was approved by The University of Iowa Human Institutional Review Board and the Protocol Review and Monitoring Committee of the Holden Comprehensive Cancer Center at The University of Iowa Hospitals and Clinics on December 30, 2014 and listed on www.clinicaltrials.gov under {"type":"clinical-trial","attrs":{"text":"NCT 01852890","term_id":"NCT01852890"}}NCT 01852890. Informed consent for both studies were documented by use of a written consent form approved by the Investigational Review Board and The University of Iowa. Nuclease-activated probes were combined with 10 µL of plasma and incubated for 6 h at 37 °C prior to being measured for fluorescence intensity (λ_ex = 485 nM and λ_em = 528 nM).

Data are presented as the mean ± SEM. For statistical analyses of two groups, unpaired two-tailed Student’s t-test were utilized. For statistical analyses of two nonparametric groups, Mann–Whitney tests were used. To study statistical differences between multiple comparisons, significance was determined using either a one-way ANOVA analysis or a two-way ANOVA analysis with Tukey’s or Bonferroni’s multiple-comparisons tests as stated in the figure legends. To determine if non-random associations between two categorical variables existed, a Fisher’s exact test was used. To compare statistical analysis of survival distributions, log-rank tests were utilized. All analyses were performed in GraphPad Prism (GraphPad Software, Inc.).