Prevention of gastrointestinal tumors based on adenomatous polyposis coli gene mutation by dendritic cell vaccine

Abstract

Here we describe the effect of immunization with dendritic cells loaded with syngeneic tumor cells (DC/Ts) by polyethylene glycol treatment, on tumor development in adenomatous polyposis coli (APC) gene mutant mouse models, APC1309 and APC(Min-/+), in which adenomatous polyps of the gastrointestinal tracts develop with a high incidence. Treatment with DC/Ts prevented the development of gastrointestinal tumors, and coadministration of DC/Ts and IL-12 caused a further reduction in tumor incidence. Splenocytes from APC1309 mice treated with DC/Ts and IL-12 showed no cytotoxic activity toward the tumor cells, but serum antibody specific to them was detected. IgG from the treated mice exhibited cytotoxic activity against the tumor cells in vitro. Predominance of Th2 cell response over Th1 response was also suggested by ELISPOT assays in the treated mice. Depletion in vivo of CD4(+) T cells, not CD8(+) T cells, by the intraperitoneal administration of corresponding mAb's decreased the antitumor effect of DC/T inoculation. Immunofluorescence microscopic studies showed that Ig was attached to tumor cells in mice treated with DC/Ts and IL-12. These findings indicate that DC/T vaccination prevents tumor development through APC gene mutation and that its preventive effects are mediated by humoral antitumor immunity.

Figures

Figure 1

FACS analysis and fluorescence microscopy of DCs and tumor cells simply mixed or treated with PEG. DCs stained with FITC-conjugated anti-CD80 and tumor T cells stained with the red-fluorescent dye PKH26GL were mixed, treated with PEG or not, and incubated overnight as described in the text. A mixture of DCs and tumor cells that had not been treated with PEG served as control. After overnight incubation, the cells were collected, analyzed by FACS, and examined under a fluorescence microscope. FACS analysis results: (A) DCs stained with FITC-conjugated anti-CD80; (B) tumor cells stained with PKH26GL; (C) mixture of DCs and tumor cells; (D) PEG-treated DCs and tumor cells. Fluorescence microscopy: (E) DCs stained with FITC-conjugated anti-CD80; (F) tumor cells stained with PKH26GL; (G) mixture of DCs and tumor cells; (H) PEG-treated DCs and tumor cells. Six independent experiments were performed with similar results. A typical experiment is shown. FACS analysis of DCs and DC/Ts: DCs (blue solid line) and DC/T (red solid line) were stained with FITC-conjugated mAb’s against (I) H-2Kb (J) I-Ab (K) CD80, and (L) CD86. Cells stained with FITC-conjugated isotype-matched control antibody are indicated by the dotted lines (green, DCs; orange, DC/Ts).

Figure 2

Macroscopic view of the upper ilei of APC1309 mice. The entire gastrointestinal tracts of 10-week-old mice were processed as described in the text. The upper ilei were excised and are shown here. The tumors are identified as black dots on the mucosa. The arrow indicates a stenotic change of the intestine by tumor growth in an untreated mouse. (A) Upper ileum from an untreated mouse (B) from a mouse treated with DC/Ts, and (C) from a mouse treated with DC/T + IL-12.

Figure 3

Effect of treatment with DC/Ts on the development of gastrointestinal tumors in APC1309 and APCMin_/+ mice. (A) Ten APC1309 mice were sacrificed at the start of treatment (6 weeks of age). Other groups of APC1309 mice that were untreated or treated with IL-12, with a mixture of PEG-treated DCs and PEG-treated tumor T cells (DC-PEG + T-PEG), with DC/Ts alone, or with DC/T + IL-12, were sacrificed at 10 weeks of age. The gastrointestinal tracts were processed as described in the text. The tumors in the entire gastrointestinal tracts were counted under the microscope. Each column represents mean ± SD (error bar) of the number of tumors. *P < 0.05; **P < 0.001. (B) Six APCMin_/+ mice were sacrificed at the start of treatment (6 weeks of age). Other groups of seven APCMin_/+ mice, which were untreated or treated with IL-12, DC/Ts, or DC/T + IL-12, were sacrificed at 10 weeks of age. The tumors in the entire gastrointestinal tracts were counted as described for A. Each column represents mean ± SD (error bar) of the number of tumors. Figures in parentheses show the number of mice examined. *P < 0.01; **P < 0.001.

Figure 4

Reactivity of serum with tumor T cells. (A-D) Reactivity of serum from mouse treated with DC/T + IL-12 against the cells derived from various carcinoma cell lines. Tumor T cells and other irrelevant tumor cells (2 ∞ 105) were incubated with 50 ∝l of the serum at 4_C for 30 minutes, washed extensively, and then incubated with 2 ∝l of FITC-conjugated rat anti-mouse Ig antibody. Fluorescence histograms shown by the cells were obtained with FACS: (A) tumor T; (B) MC38 colon carcinoma; (C) B16 melanoma; (D) Hepa1-6 hepatocellular carcinoma. Thick line, negative control; dotted line, serum from untreated mouse; thin line, serum from mouse treated with DC/T + IL-12. (E) Decrease of serum reactivity by incubation with tumor T cells. Serum from a mouse treated with DC/T + IL-12 was diluted 100-fold with PBS, incubated with tumor T cells (2 ∞ 105) at 4_C for 1 hour and centrifuged. Tumor T cells were incubated with 50 ∝l of the supernatant (dashed line), 100-fold diluted serum (dotted line), or untreated mouse serum (thick line) at 4_C for 30 minutes, washed extensively, incubated with 2 ∝l of FITC-conjugated rat anti-mouse Ig antibody, and submitted for flow-cytometric analysis using FACS. Three independent experiments were performed with similar results. A typical experiment is shown. (F) Serum dilution and reactivity with tumor T cells. Tumor T cells (2 ∞ 105) were incubated with 50 ∝l of the diluted serum at 4_C for 30 minutes, washed extensively, and then incubated with 2 ∝l of FITC-conjugated rat anti-mouse Ig antibody. Fluorescence histograms shown by the cells were obtained using FACS, and the median fluorescence intensity was measured. All determinations were carried out in triplicate. Squares and triangles, sera from untreated mice; diamonds and circles, sera from mice treated with DC/T + IL-12.

Figure 5

Analysis of Ig subclass of antibody to tumor T cells. Tumor T cells were incubated, first with 30-fold diluted sera from mice treated with DC/T + IL-12 as described in the legend for Figure 4, and then with rat anti-mouse antibody specific for IgG1, IgG2a, IgM, or IgA as described in the text. (A) Serum from untreated mouse; (B) serum from mouse treated with DC/T + IL-12.

Figure 6

Relationship between the number of gastrointestinal tumors and the median fluorescence intensity shown by the tumor cells incubated with serum. Gastrointestinal tumors were counted as described in the legend for Figure 3A. The median fluorescent intensities shown by tumor cells incubated with sera and then with FITC-conjugated rat anti-mouse Ig antibody were determined as described in the legend for Figure 4. Each symbol represents the number of gastrointestinal tumors and the median fluorescence intensity for individual mice. Filled squares, untreated mice; open circles, mice treated with DC/T; filled circles, mice treated with DC/T + IL-12 (correlation coefficient: _0.535; P < 0.05).

Figure 7

Effect of administration of anti-CD4 mAb’s or anti-CD8 mAb’s on the antitumor effect induced by DC/Ts. Anti-mouse CD4 mAb’s or anti-mouse CD8 mAb’s were administered at a dose of 0.5 mg/mouse intraperitoneally, 1 day before and 2 days after each of the DC/T inoculations; these mice were treated with IL-12 and sacrificed at 10 weeks of age as described in the text. The tumors in the gastrointestinal tracts were counted as described in the legend for Figure 3. Each column represents mean ± SD (error bar) of the number of tumors (n = 3). *P < 0.01; **P < 0.001.

Figure 8

Phase-contrast microscopy of tumor cells cultured with IgG purified from mice treated with DC/T + IL-12. Tumor T cells were cultured for 48 hours in the presence of 0.02 mg/ml IgG purified from untreated mice (A) and mice treated with DC/T + IL-12 (B). Many tumor cells were detached from the culture plate in the presence of IgG from mice treated with DC/T + IL-12.

Figure 9

Cytotoxicity shown by IgG purified from sera of mice treated with DC/Ts or DC/T + IL-12. Tumor T cells (2 ∞ 105 cells) were cultured for 48 hours in the medium containing various concentrations of IgG purified from sera of mice untreated or treated with DC/Ts or DC/T + IL-12. After removal of the medium, the cells that adhered to the plate were detached by trypsinization and counted. All determinations were carried out in triplicate. Three independent experiments were performed with similar results. A typical experiment is shown.

Figure 10

Immunofluorescence microscopy of gastrointestinal tumors. Tumors of APC1309 mice were analyzed with FITC-conjugated rat anti-mouse Ig antibody. Note the abundant Ig on tumor cells in the tumor tissue of a mouse treated with DC/T + IL-12. Original magnification: ∞40. (A) Tumor tissue of untreated mouse; (B) tumor tissue of mouse treated with DC/T + IL-12. Arrows indicate Ig bound to tumor cells under the immunofluorescence microscope.