Article Figures & Data
Figures
- Fig. 1.
Inhibition of EGF-induced autophosphorylation of EGF-R in L3.6pl human pancreatic cancer cells by C225. Tyrosine phosphorylation induced in L3.6pl cells by 40 ng/ml EGF for 10 min was inhibited by C225 in a dose-dependent manner. L3.6pl pancreatic carcinoma cells growing in vitro under serum-free conditions were stimulated for 10 min with EGF (40 ng/ml) in the presence or absence of C225 (0–10 μg/ml). Cells were washed and lysed, and insoluble proteins in the lysate were immunoprecipitated with an anti-EGF-R Mab, separated by 7.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and sequentially probed with antisera specific to phosphotyrosine and EGF-R (see “Materials and Methods”). The immunoreactive proteins were detected by incubating the blot with the corresponding peroxidase-conjugated IgG and visualized using the ECL system. Densitometric quantitation of the ratio of the areas between the Mr 170,000 phosphotyrosine- and the Mr 170,000 EGF-R-specific bands were compared in each case with the untreated cells, arbitrarily assigned the value of 1.0.
- Fig. 2.
In vitro growth inhibition of L3.6pl cells treated with C225 alone and in the presence of gemcitabine. L3.6pl tumor cells were plated at 1000 cells/38-mm2 well in DMEM containing 10% FBS. Twenty-four h later, the cells were refed with 1 or 5% FBS-supplemented DMEM containing increasing concentrations of C225 (0–12.5 μg/ml; A) or 5% FBS-supplemented DMEM containing C225 (2.5μ g/ml) with increasing concentrations of gemcitabine (0–64 ng/ml; B). The cells were incubated for an additional 72 h at 37°C, and the number of metabolically active cells was determined using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium assay. Bars, SD.
- Fig. 3.
Immunohistochemical analysis of activated and total EGF-R in tumor sections of L3.6pl cells growing in the pancreas of nude mice 30 days after therapy. Tumor sections were immunostained with Mab antibodies specific to activated EGF-R (tyrosine phosphorylated) or total EGF-R as described (32 33 34 35 36) . Tumor sections from control (Contr.) and gemcitabine (Gem.)-treated mice show immunoreactivity specific for both activated EGF-R (act. EGF-R) and total EGF-R (EGF-R), whereas tumor sections from C225 and C225 plus gemcitabine (C+G)-treated animals show no change in total EGF-R immunostaining but undetectable immunostaining for activated EGF-R.
- Fig. 4.
PCNA and TUNEL immunohistochemistry of L3.6pl human pancreatic tumors growing in nude mice after 18 days of therapy. Tissue sections were analyzed for expression of PCNA (to show cell division) and TUNEL (to show apoptosis). Decreased immunoreactivity to PCNA is observed in tumors from C225 and C225 plus gemcitabine (Gem.) compared with gemcitabine-treated and control (Contr.) animals. Increased DNA fragmentation (apoptosis) identified by localized green fluorescence was observed in specimens from each treatment group compared with controls.
- Fig. 5.
Immunohistochemical staining of L3.6pl pancreatic tumors for VEGF, IL-8, and CD31 after 18 days of therapy. Control (Contr.), gemcitabine (Gem.), C225, and C225 plus gemcitabine treated tumors (after 18 days of therapy) were stained with anti-VEGF and anti-IL-8 antibodies demonstrating higher immunoreactivity in the control and gemcitabine versus C225 and C225 plus gemcitabine (C+G)-treated tumors. These tumors were also analyzed with anti-CD31 antibodies directed against mouse endothelial cells. Note the increased vessel density in the control and gemcitabine-treated tumors compared with that of the C225 and C225 plus gemcitabine-treated specimens.
- Fig. 6.
Immunoflorescence double-staining for CD31 (endothelial cells) and TUNEL (apoptotic cells) in L3.6pl human pancreatic tumors after 18 days of therapy. Frozen tissue sections were fixed, treated with a rat anti-CD31 antibody, and then incubated with goat antirat IgG conjugated to Texas Red. After the sections were washed, TUNEL was performed using a commercial kit with modifications (see “Materials and Methods”). Immunofluorescence microscopy was performed using ×400. Endothelial cells were identified by red fluorescence, and DNA fragmentation was detected by localized green and yellow fluorescence within the nucleus of apoptotic cells.
Tables
- Table 1
Therapy of L3.6pl pancreatic tumors in nude mice with C225 and gemcitabine
Therapya Incidence of macroscopic tumorsb Total pancreas weight mg (range)e Median tumor volume mm3 (range)f Median body weight g (range) Pancreas tumor Liver metastasisc Regional LN metastasisd Control 10 /10 5 /10 10 /10 923 (660–1371) 539 (254–860) 22 (16–24) Gemcitabine 10 /10 3 /10 6 /10 297 (205–485) 152 (59–365) 22 (18–29) C225 5 /10 2 /10 8 /10 119 (97–157) 0.3 (0–13) 25 (21–28) C225+ gemcitabine 0 /9 0 /9 1 /9 130 (93–173) 0 (0) 23 (20–27) -
a L3.6pl cells (1 × 106) were implanted into the pancreas. Therapy began on day 7, when median tumor size was 18 mm3. C225 (1 mg), Gemcitabine (250 mg/kg), alone or in combination, was injected i.p. biweekly for 4 weeks. Control mice received saline. Tumor weight and metastases were evaluated at necropsy at day 32. One experiment of two is shown.
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b Number of tumor-positive mice per number of mice receiving injections.
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c Number of mice with visible nodules (>1 mm in diameter) per number of mice receiving injections.
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d Number of mice with enlarged regional lymph nodes (LN) per number of mice receiving injections.
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e P < 0.0001 (unpaired Student’s t test) control versus all three therapy groups.
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f Tumor volume was calculated by the formula: v = ab2/2, where a is the longest diameter and b is the shortest diameter of the tumor. P < 0.0001 control versus all three therapy groups and C225 versus C225 plus gemcitabine therapy group.
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- Table 2
Immunohistochemical analysis of tumor cell proliferation (anti-PCNA) and apoptosis (TUNEL) in tumors from animals treated for 11, 18, and 30 days
Therapy % PCNA/Total cellsa Median (range) Pb % TUNEL/Total cellsa Median (range) Pb Control 11 days 92 (87–98) 1.4 (1–7) 18 days 94 (79–98) 0.9 (0–3) 30 days 72 (31–94) 1.1 (0.5–2) Gemcitabine 11 days 73 (51–80) 0.01 10 (3–13) 0.006 18 days 69 (31–75) 0.01 15 (8–21) 0.005 30 days 56 (37–67) NSc 30 (18–39) 0.005 C225 11 days 26 (9–85) 0.04 8 (2–21) NS 18 days 34 (26–52) 0.04d 17 (7–18) 0.004d 30 days 30 (14–41) 0.02d 39 (23–41) 0.004 C225+ gemcitabine 11 days 19 (13–30) 0.000001 7 (5–14) 0.02 18 days 7 (3–23) 0.000001 27 (21–43) 0.009 30 days 4 (0–6) 0.004 39 (36–68) 0.009d -
a PCNA- or TUNEL-positive cells were counted in 10 microscopic fields per tumor section at ×100 (0.159 mm2). The total number of cells/field and the number of PCNA- or TUNEL-positive cells in that field were determined to calculate the median percentage in each tumor section.
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b Control versus the therapy shown.
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c NS, not significant.
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d For C225 versus C225 plus gemcitabine, P < 0.008 (unpaired Student’s t test).
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- Table 3
In vitro inhibition of VEGF and IL-8 protein production by C225a
Protein C225 0 μg/ml 1.25 μg/ml 2.5 μg/ml 5.0 μg/ml VEGF (pg/ml) 909 ± 141 486 ± 11b 499 ± 75 455 ± 15b IL-8 (pg/ml) 326 ± 11 251 ± 32b 274 ± 78b 255 ± 32b -
a L3.6pl cells were cultured in the presence or absence of C225 for 48 h in 5% serum containing medium. VEGF and IL-8 were measured by ELISA. Values are the mean ± SD of triplicate cultures and have been normalized to account for cell number.
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b For 5 μg/ml C225 versus control, P < 0.05 (unpaired Student’s t test).
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- Table 4
Immunohistochemical analysis in tumors after 11, 18, and 30 days of therapy with C225 and gemcitabine
Therapy VEGFa expression Average (SD) IL-8a expression Average (SD) MVDb Median (range) % apoptotic EC/Total ECc Median (range) Control 11 days 172 (28) 170 (28) 37 (30–54) 0 (0–12) 18 days 220 (26) 191 (17) 73 (53–90) 0 (0–5) 30 days 151 (20) 202 (32) 56 (24–180) 0 (0–6) Gemcitabine 11 days 162 (21)d 168 (21)d 38 (31–64)d 0 (0–15)d 18 days 150 (21)d 210 (35)d 69 (51–72)d 0 (0–5)d 30 days 165 (33)d 204 (48)d 78 (60–89)d 10 (7–26)e C225 11 days 97 (13)e 100 (13)f 27 (15–34)e 19 (0–40)e 18 days 98 (13)e 94 (14)f 16 (14–44)f 69 (60–81)f 30 days 110 (2)e 113 (20)f 19 (16–22)e 63 (60–67)f C225+ gemcitabine 11 days 109 (9)e 110 (9)e 22 (20–40)e 21 (0–43)e 18 days 107 (12)e 109 (6)e 14 (11–18)f 62 (27–67)f 30 days 114 (29)e 111 (32)e 9 (4–22)e 68 (61–100)f -
a VEGF and IL-8 cytoplasmic staining was evaluated by computer-assisted image analysis using 10 microscopic fields/tumor section at ×200 (0.033 mm2) and is expressed as the ratio of tumor expression to normal pancreatic epithelium expression multiplied by 100.
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b The number of CD31-positive vessels was evaluated in 10 microscopic fields per tumor section at ×100 (0.159 mm2) using the Optimas 6.0 computer-assisted image analysis software.
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c The percentage of TUNEL-positive endothelial cells was determined in 10 microscopic fields/tumor section at ×400 (0.011 mm2) by evaluating the ratio between the number of TUNEL-positive endothelial cells/field and the total number of endothelial cells/field.
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d No significant difference compared with the control at the same time point.
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e P < 0.05 compared with the control at the same time point.
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f P < 0.002 compared with the control at the same time point.
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Volume 6, Issue 5
