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Supplemental Figure 1. Vav1 is expressed in a subset of tumor cell lines. Lysates for the
indicated pancreatic cancer or breast cancer (CA1D) cell lines were immunoblotted to detect
Vav1 and actin as a loading control.
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Supplemental Figure 2. Azathioprine does not inhibit migration by pancreatic tumor cells.
Vav1-expressing (DanG, Panc04.03, CFPAC, A) or non-expressing (BxPC3, PANC1, B) cells
were grown to confluence, pre-treated with azathioprine for 1.5 days, and assayed for cell
migration using a wound-healing assay. Representative photomicrographs are shown of
migratory endpoints (t=24 hours, or t=7 hours for Panc04.03 cells), with the white line indicating
the starting point. The distance migrated was quantified and is graphed as the mean +/- SEM of
at least 3 independent experiments. * p<0.05.
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Supplemental Figure 3. Vav1 is expressed in tumors from a genetic model of pancreatic
cancer. (A) Azathioprine inhibits invasive migration induced by mouse Vav1. PANC1 cells
were transfected with empty vector or mouse Vav1, treated with vehicle or azathioprine for 2
days, and seeded in a transwell migration assay. The percent of cells invaded across the filter was
scored. Mouse Vav1 promotes invasive migration, and is sensitive to azathioprine’s anti-invasive
effects. Graphed data represent the mean +/- SEM of 4 independent experiments. * p≤0.05, ns:
no statistically significant difference. (B) Primary tumors from the p48Cre/+; KRasG12D; p53Flox/+
mice were solubilized and immunoblotted for Vav1 and actin as a loading control. Panc1 cells
were used as a negative control. Positive controls were DanG cells, and Panc1 cells transfected
with Vav1. Vav1 is present in the majority of mouse pancreatic tumors.
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Supplemental Figure 4
A. Flank xenograft model
Cell line n Treatment Primary tumor sizeDanG(Vav1 positive)
17 0 mg/kg 766 +/- 74 mm3
17 5 mg/kg 672 +/- 81 mm3
BxPC3(Vav1 negative)
6 0 mg/kg 915 +/- 200 mm3
6 5 mg/kg 962 +/- 276 mm3
B. Orthotopic xenograft model
Cell line n Treatment Primary tumor sizeDanG(Vav1 positive)
10 0 mg/kg 810 +/- 122 mm3
9 5 mg/kg 611 +/- 88 mm3
L3.6(Vav1 negative)
8 0 mg/kg 1.00 +/- 0.08 g8 5 mg/kg 0.97 +/- 0.11 g
C. Genetic mouse model: p48Cre/+; KRasG12D/+; p53Flox/+
n Primary tumor size (g) Survival (weeks)0 mg/kg 22 1.20 +/- 0.15 22.4 +/- 0.75 mg/kg 19 1.55 +/- 0.21 22.9 +/- 1.010 mg/kg 23 1.61 +/- 0.21 24.5 +/- 1.1
D. Genetic mouse model: PDX-Cre; KRasG12D; p53Flox/Flox
n Primary tumor size (g) Survival (weeks)0 mg/kg 12 0.9 +/- 0.09 8.6 +/- 0.45 mg/kg 12 0.8 +/- 0.04 8.0 +/- 0.315 mg/kg 12 0.9 +/- 0.13 8.3 +/- 0.2
E. Genetic mouse model: PDX-Cre; KRasG12D; p53Flox/R172H
n Primary tumor size (g) Survival (weeks)0 mg/kg 4 0.5 +/- 0.08 7.6 +/- 0.55 mg/kg 4 0.4 +/- 0.10 9.0 +/- 0.7
All data represent the mean +/- S.E.M.
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Supplemental Figure 4. Azathioprine treatment does not impact tumor size in mouse
models of pancreatic cancer. (A) DanG or BxPC3 tumor cells were implanted subcutaneously
into the flank of athymic nude mice, and mice were treated three times weekly with vehicle or
azathioprine for 3 weeks (DanG) or 5 weeks (BxPC3), and the volume of the primary tumor was
monitored. (B) DanG or L3.6 tumor cells were injected into the head of the pancreas of athymic
nude mice in an orthotopic tumor model, and mice were treated three times weekly with vehicle
or azathioprine for 3 weeks (DanG) or 5 weeks (L3.6). Upon necropsy, primary tumor size was
measured by volume (DanG) or mass (L3.6). (C-E) Multiple genetic mouse models for
pancreatic cancer, treated with vehicle or azathioprine three times weekly beginning at 5 weeks
of age until sacrifice. (C) While metastasis was inhibited in the p48Cre/+; KRasG12D; p53Flox/+ mice,
azathioprine did not impact primary tumor size. (D-E) The more aggressive mouse models PDX-
Cre; KRasG12D; p53Flox/Flox or PDX-Cre; KRasG12D; p53Flox/R172H had a mean survival time of only
approximately 8 weeks, and did not develop metastases. Azathioprine had no significant effect
on primary tumor size or survival time. All data represent the mean +/- SEM of the indicated
number of mice (n).
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Supplemental Figure 5. Azathioprine reduces cell propagation in a Vav1-independent
manner. (A) Cultured pancreatic cancer cells were incubated with azathioprine for 4 days, then
counted on a hemocytometer in the presence of trypan blue. Azathioprine treatment reduced the
total viable cell number after four days (normalized to vehicle-treated cells), in both Vav1-
positive and Vav1-negative cells. (B) The percent of trypan blue-positive dead cells was
determined. Azathioprine did not induce significant cell death after four days in either the Vav1-
positive or Vav1-negative cells. (C) Cells were treated as described in A, but after 4 days were
analyzed by MTS assay. The absorbance at 490 was measured in duplicate samples and
normalized to vehicle-treated cells for each cell type. All graphed data represent the mean +/-
SEM of 3 independent experiments. * p<0.05, ** p<0.01.
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