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Mascarenhas et al. J Cancer Metastasis Treat 2020;6:22 I http://dx.doi.org/10.20517/2394-4722.2020.52 Page 3 of 7

Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the Molecular
Medicine Research Institute. At the end of the study, period animals were euthanized by decapitation as
approved by MMRI IACUC guidelines, the NIH’s Office of Laboratory Animal Welfare (OLAW), and
AVMA recommendations.

For the lung metastasis study, 7-week old female B6D2F1 mice (Charles River; 18-22 gram body weight)
received implantation via tail vein of 1x10e5 B16MET-e100 tumor cells per animal. Beginning on Day 2,
animals received either saline or nephrilin peptide (1.2 mg/kg) daily via subcutaneous bolus injection, for
3 weeks (n = 6 per group). At the end of the treatment period, animals were sacrificed and lung metastases
in each lobe of the right lung were counted.

For biodistribution studies, 10-week old male C57BL6 mice (Jackson Laboratories; 30 grams body weight)
were used. Animals received either saline or nephrilin peptide (4 mg/kg) + FITC-labelled peptide tracer
(1:100 molar ratio to nephrilin) via subcutaneous bolus injection once daily for seven days (n = 3 per
group). The concentration of tracer peptide administered is too low to generate measurable biological
effects, of itself. At the end of the treatment period, animals were sacrificed and left lung, left kidney, brain
(left hemisphere), and liver (left half) were immediately stored in formalin for further histological analysis.
Brain (right hemisphere), right kidney, liver (right half), plasma, and each lobe of the right lung were
immediately frozen at -80 °C for further analysis.

Plasma cytokine/chemokine analysis
Analysis of plasma was performed using a mouse cytokine 44-plex immunoassay (Custom Plex Discovery
Assay, Eve Technologies, Calgary, AB). Statistically significant differences (Student’s t-test, P < 0.05)
between saline-treated and nephrilin-treated animals in the biodistribution experiment were found in the
plasma levels of two of the 44 analytes tested.

Tissue extracts
Frozen tissue extracts were prepared using the CellLyticMT extraction buffer (Sigma-Aldrich, St. Louis,
MO) as specified by the manufacturer. Extracts were stored at -20 degrees C before assay by ELISA (for
nephrilin peptide, using in-house polyclonal antibody #2501) or for succinate assay using a kit purchased
from Megazyme, Inc (Chicago, IL).

Statistical analysis
Data are presented as means ± SD unless otherwise indicated. Probability values (P values) were computed
using Student’s t-test and expressed relative to the saline-treated group.

RESULTS
Biodistribution of nephrilin
Figure 1 shows tissue sections of the brain, kidney, left lung, and liver from mice injected with either saline
or nephrilin (4 mg/kg) + 1:100 FITC-tracer peptide (see methods). A fairly even distribution of peptide
across tissues is visible in the slides, and corroborated by the biodistribution of nephrilin peptide in the
brain (0.81 μg nephrilin/mg total tissue protein), kidney (0.97) and liver (0.74), as measured by ELISA.
The concentration of nephrilin in the left lung was not measured, as that tissue was fixed in formalin.
The concentration of nephrilin and tracer peptide in the right lung was measured. Table 1 shows the
distribution of nephrilin in the four lobes of the right lung. There is a gradient of accumulation of peptide
(μg/mg total tissue protein) in the deep lobes: accessory (5.07) > inferior (3.45) > middle (2.84) > superior
[17]
(2.12). This gradient is reminiscent of that previously shown for lung elastase in rodent lung . We also
measured the concentration of succinic acid in tissue extracts of each lobe of the right lung. Interestingly,