Immunotherapy of spontaneous mammary carcinoma with fusions of dendritic cells and mucin 1-positive carcinoma cells

Mice

Female C57BL/6 mice, 6 to 8 weeks old, were purchased from Taconic (Germantown, NY). The transgenic mice included: (i) MT mice expressing the polyomavirus middle-T oncogene driven by the mouse mammary tumour virus (MMTV) long-terminal repeat (LTR) that develop spontaneous mammary carcinomas,¹⁹ (ii) MUC1 transgenic mice (MUC1.Tg) expressing the human MUC1 antigen in a tissue-specific fashion similar to that in humans,²² and (iii) MMT mice that express the PyMT and the MUC1 antigen and develop spontaneous mammary carcinomas.¹⁵ The MT mice were generated by breeding the female wild-type C57BL/6 strain with male MT mice. The MMT mice were generated by crossing the female C57BL/6 strain of MUC1.Tg mice with male MT mice. All mice were congenic on the C57BL/6 background at N > 10. The mice were selected for the expression of the PyMT oncogene and/or MUC1 using the polymerase chain reaction (PCR).^22,23 Only female mice positive for either MT (MT mice) or MT/MUC1 double transgenes (MMT mice) were used for the experiments. The mice were maintained in microisolator cages under specific pathogen-free conditions.

Cell culture and fusion

Murine MC38 adenocarcinoma cells (C57BL/6) were stably transfected with a MUC1 cDNA (MC38/MUC1).²⁴ Cells were maintained in Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mml-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. DCs were obtained from bone marrow cultures of C57BL/6 mice.²⁵ The methods of DC generation and fusion with MC38 or MC38/MUC1 tumour cells in the presence of 50% polyethylene glycol have been described.²⁶ Briefly, DC and MC38/MUC1 cells were collected, washed twice in serum-free medium, and counted. DCs were mixed with MC38/MUC1 cells in a 10 : 1 ratio. The fusion process was carried out with 50% polyethylene glycol (PEG) in prewarmed Dulbecco's phosphate-buffered saline (PBS) without Ca²⁺ or Mg²⁺ at pH 7·4. After washing twice, the fused cells were plated in 24-well culture plates for 5 days. Then the cells were plated in six-well culture plates in complete RPMI 1640 medium supplemented with 20 ng/ml recombinant murine GM-CSF (Sigma Chemical Co., St. Louis, MO). By day 5 of culture, the unfused tumour cells had become firmly attached to the tissue culture flask, while the fused cells could be dislodged by gentle pipetting. The latter were then collected and analysed by flow cytometry for antigen expression.

PCR

The mice were examined for MUC1 and MT genes by PCR analysis. Ten-µg aliquots of tail and mammary tumour tissue were digested with proteinase K. DNA was extracted using the Dneasy™ Tissue Kit (QIAGEN, Valencia, CA). PCR was carried out in a total volume of 50 µl in Perkin-Elmer Gene Amp tubes (Perkin-Elmer, Norwalk, CT) with the following reagents: 5 µl 10 × PCR buffer including 15 mm MgCl₂; 0·02% formamide; 0·2 mm dNTPs; 100 nm 5′-CTTGCCAGCCATAGCACCAAG-3′ (bp 745–765) forward primer, and 100 nm 5′-CTCCACGTCGTGGACATTGATG-3′ (bp 1086–1065) reverse primer for the MUC1 gene; 100 nm 5′-AGTCACTGCTACTGCACCCAG-3′ (bp 282–302), and 100 nm 5′-CTCTCCTCAGTTCCTCGCTCC-3′ (bp 817–837) primers for the MT gene; 1·25 units of Taq polymerase; 2 µl of tail DNA (approximately 500 ng) and reagent quality H₂O. The amplification programme consisted of one cycle for 10 min at 94° and 40 cycles of 30 s each at 94°, 61° and 72°. The PCR product of each reaction was analysed by size fractionation in a 1% agarose gel. Amplification of MUC1-positive DNA resulted in a 500-bp fragment²² and that of MT-positive DNA in a 491-bp fragment.²³

Immunoblotting

Mammary carcinomas from MT or MMT mice were harvested, rapidly frozen in liquid nitrogen and stored at −80° until use. The samples were lysed in a solution containing 0·05 m sodium chloride, 0·02 m Tris, pH 7·4, 100 µg/ml leupeptin, 50 µg/ml aprotinin and 1% NP-40. Equivalent amounts of protein were separated in 5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were stained with Ponceau S, photographed, blocked in 5% non-fat dry milk in PBS + 0·05% Tween 20 and probed with anti-MUC1 mAb (DF-3) for 1 hr at 25°. Membranes were incubated with horseradish proxidase (HRP)-conjugated rabbit anti-mouse antibody (Amersham, Pharmacia Biotech, Piscataway, NJ) for 1 hr at 25°. The antigen–antibody complexes were visualized by Electro Chemiluminescence (ECL, Amersham).

Histological and immunohistochemical staining

Groups of MT or MMT mice were killed at various ages (from 3 weeks to 6 months). Mammary tissue or tumour was harvested and fixed in 2% paraformaldehyde. Sections (5 µm) were cut and stained with hematoxylin and eosin (H&E). For detecting MUC1 expression, sections were stained with anti-MUC1 mAb (DF3) for 30 min at room temperature and then subjected to indirect immunoperoxidase staining using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA).

Flow cytometry

DC, FC/MUC1 and MC38/MUC1 carcinoma cells were double stained with fluorescein-isothiocyanate (FITC)-conjugated-mAb HMPV (anti-MUC1, BD Pharmingen, San Diego, CA) for 30 min on ice. After washing twice with PBS, the phycoerythrin (PE)-conjugated mAb M5/114 (anti-MHC class II, BD Pharmingen) was added for another 30 min on ice. Splenocytes were purified by passage through nylon wool and stained with the following antibodies: anti-CD4 (H129·19), anti-CD8 (53-6·7), anti-NK1·1 (PK136), anti-αβ TCR (H57-597) and anti-γδ TCR (GL3) (BD Pharmingen) for 30 min on ice. The cells were washed with PBS and incubated with FITC-conjugated anti-rat, mouse and hamster immunoglobulin G (IgG) for an additional 30 min on ice. All the cells were washed, fixed, and analysed by FACScan (Becton Dickinson, Bedford, MA) with CellQuest analysis software (Becton Dickinson).

Vaccination

Groups of 4-week-old MT or MMT mice were vaccinated subcutaneously with 5 × 10⁵ FC/MUC1 cells, FC/MC38 cells or MC38/MUC1 tumour cells that had been exposed to 60-Gy ionizing radiation (Gammacell 1000 Atomic Energy of Canada, Ottawa, Canada). In another experiment, MMT mice were immunized at varying ages. The immunization was repeated four additional times at monthly intervals. The mice were followed for up to 6 months. From 6 weeks of age, the mice were palpated every other day to assess the presence of mammary tumours. A progressively growing mass was regarded as tumour and was measured by calipers in two perpendicular diameters. Tumour incidence was determined and recorded. Mice with tumours ≥ 2 cm were killed. The mice were cared for according to the Institutional Animal Care and Use Committee Guidelines.

Humoral immune responses

Microtitre plates were precoated overnight at 4° with 100 µl/well of MUC1 antigen (5 units/ml in PBS, pH 7·4). MUC1 antigen was purified from supernatant of ZR-75 human breast cancer cells using the method previously described.^27–30 Briefly, the supernatant obtained by culturing ZR-75 cells for 3 or 4 days in the serum-free medium was concentrated in a stirred ultrafiltration cell (Millipore, Billerica, MA) on a YM30 filter. The concentrate was centrifuged and applied to an agarose-wheat germ lectin column (Pharmacia, Peapack, NJ). The DF3/MUC1 antigen containing fractions was eluted with 1 m N-acetylgulcosamine. The elute was dialysed, concentrated, and then applied to Sephacryl S-300 (Pharmacia). The DF3/MUC1 antigen-containing fractions were monitored by immunoassay, concentrated and then incubated with mAb DF3-Sepharose 4B. The MUC1 antigen was eluted with 3 m MgCl_2. The MgCl₂ fraction was dialysed against water and then concentrated. Anti-mouse IgG at 100 µl/well (1 µg/ml in PBS, pH 7·4) was used to precoat the well as a control. Each well was washed three times with PBS/Tween (0·05% v/v Tween 20) and blocked with 120 µl/well 5% horse serum in PBS for 1 hr at room temperature. After washing, 4-fold dilutions of mouse sera were added to each well for 2 hr. The plates were washed and incubated with sheep anti-mouse IgG conjugated to horseradish peroxidase (Amersham, Piscataway, NJ). Antibody complexes were detected by development with o-phenylenediamine (Sigma) and measured in an enzyme-linked immunosorbent assay (ELISA) microplate autoreader EL310 (Bio-Rad, Hercules, CA)at an OD of 490 nm.

⁵¹Cr cytotoxicity assay

Splenocytes were isolated from MMT mice by Ficoll separation. The target cells include mammary carcinoma cells isolated from MMT or MT mice, MC38/MUC1, MC38 (the parent cells of MC38/MUC1), and YAC-1 cells. These target cells were prelabelled with ⁵¹Cr for 1 hr at 37° and added to wells of 96-well v-bottom plates with T cells (effector cells) for 5 hr at 37°. The supernatants were assayed for ⁵¹Cr release in a gamma counter and CTL activity was determined at the indicated effector:target (E:T) ratios. Percentage of specific ⁵¹Cr release was determined by the following equation:

Statistical analysis

Statistical significance was determined using Student's t-test.

Characterization of mammary carcinomas in MT and MMT mice

MMT mice have been generated by breeding MT mice with MUC1.Tg mice. Bitransgene-positive mice were selected using PCR. Mammary tumours first appeared in the female MMT mice at about 8 weeks of age. All mice developed mammary tumours by 15 weeks of age (range from 8 to 15 weeks) with a median of 12 weeks. The mammary tumours were multiple in nature with synchronous kinetics. The tumours progressed rapidly, and the mice became moribund at 16–18 weeks of age (Fig. 1a) when they were killed.

To establish the pattern of tumourigenesis, groups of MMT and MT mice were killed at varying time-points and their mammary tissue was subjected to histological examination. At least three stages of tumour development were observed. Mammary glands appeared normal until the 3rd week of age. Focal hyperplasia began to appear in the 4th week, which evolved into dysplasia and carcinoma in situ. Invasive tumours emerged at 8 to 9 weeks (Fig. 1b). A similar trend was observed in MT mice. These findings indicate that expression of PyMT resulted in transformation of the mammary epithelia and rapid production of mammary carcinomas in MT and MMT mice. Expression of MUC1 shortened the latency period to some extent, although no significant difference was found between the two groups. More importantly, multiple stages of tumour development, similar to those found in human cancers, were observed.

Expression of MUC1 on mammary carcinoma cells in MMT mice

To assess expression of MUC1 in the mammary carcinomas of MMT mice, western blotting, PCR and immunohistochemical staining techniques were used. Western blot showed the expression of MUC1 in spontaneous mammary carcinoma cells from MMT, but not from MT, mice at a level comparable to that found in MCF-7 human breast cancer cells (Fig. 2a). At the DNA level, the presence of MUC1 and PyMT was detected in tumour cells from MMT mice by PCR analysis. In contrast, only PyMT was detected in tumour cells from MT mice (Fig. 2b). Furthermore, we detected MUC1 expression on mammary tumour cells from MMT, but not MT, mice with immunohistochemical staining (Figs 2c and d). Whereas MUC1 expression was confined to the apical or lumenal surface of normal mammary epithelia, MUC1 was detectable over the entire surface of spontaneous mammary tumour cells. Taken together, these results indicate that MUC1 is overexpressed on mammary carcinoma cells in MMT mice.

Induction of anti-MUC1 immunity in MMT mice by immunization with FC/MUC1

MC38/MUC1 carcinoma cells were successfully fused with syngeneic DCs. The fused cells (FC/MUC1) expressed tumour-derived MUC1 and DC-derived MHC class II molecules (Table 1a). In contrast, fusion of DC with MC38 tumour cells (FC/MC38) resulted in the expression of MHC class II molecules but not MUC1 (Table 1a). The fusion efficiency was 21·16%, as determined by dual expression of MUC1 and MHC class II. To assess induction of anti-MUC1 humoral immune response, MT and MMT mice were immunized subcutaneously with FC/MUC1. The immunization was repeated four additional times at 4-week intervals. Irradiated MC38/MUC1 tumour cells, fusions of DCs and MC38 tumour cells (FC/MC38) or PBS injection were used as controls. The sera from immunized and control mice were collected 7 days after each immunization and analysed for the presence of anti-MUC1 antibody by ELISA assay. An anti-MUC1 humoral response was induced in MMT mice immunized with FC/MUC1, but not with irradiated MC38/MUC1, FC/MC38 or PBS injection (Fig. 3a). The anti-MUC1 antibody titre increased after the second immunization, peaked after the third and fourth immunizations, then decreased and was maintained at lower levels at 6 months of age (Fig. 3b). In contrast, there was no anti-MUC1 antibody detected in MMT mice injected with PBS (Fig. 3b). These results indicate that immunization with FC/MUC1 is associated with production of anti-MUC1 antibodies in MMT mice.

To assess the induction of CTL against MUC1-positive tumour cells, splenocytes from MMT mice immunized with FC/MUC1 were collected at multiple time-points. Table 1b shows the phenotypes of the splenocytes. The standard ⁵¹Cr release assay was used to determine the CTL activity against spontaneous mammary tumour cells from MMT mice (MMT tumour cells, MUC1-positive). CTL activity against MMT tumour cells (37%) was induced in splenocytes from MMT mice immunized with FC/MUC1 (Fig. 3c). In contrast, there was no apparent CTL activity induced in their littermates immunized with irradiated MC38/MUC1, FC/MC38 or PBS (Fig. 3c). The CTL activity against MUC1-positive targets was detectable at 20–38% lysis throughout the experiment (Fig. 3d). To assess the specificity of CTL activity, multiple targets were used. CTL activity against MC38/MUC1, MMT tumour and, to a lesser extent, MC38 cells was induced by FC/MUC1 immunization (Fig. 3e, right panel). This result is consistent with our previous findings that polyclonal CTLs are induced by the FC/MUC1 vaccination against MUC1 antigen as well as unknown tumour antigens expressed by MC38 and MC38/MUC1. In contrast, there was little, if any, CTL activity against MT and YAC-1 cells (Fig. 3e, right panel). As expected, no CTLs were induced in MMT mice immunized with irradiated MC38/MUC1 tumour cells (Fig. 3e, middle panel) or treated with PBS (Fig. 3e, left panel). Taken together, the results indicate that immunization with FC/MUC1 induces polyclonal and antigen-specific CTLs.

Inhibition of spontaneous mammary carcinomas in MMT mice by fusion cell immunization

To assess the efficacy of immunization with fusion cells in vivo, MMT mice were immunized with FC/MUC1 cells, FC/MC38 cells or irradiated MC38/MUC1 cells, or injected with PBS. Immunization was begun at 4 weeks of age. The mice received four additional immunizations at 4-week intervals (Fig. 4a). All MT mice treated with PBS, irradiated MC38/MUC1 cells or FC/MUC1 developed mammary tumours at 8–15 weeks and became moribund 4 weeks after tumour appearance (Fig. 4b, left panel). In contrast, immunization of MMT mice with FC/MUC1 significantly delayed the development of spontaneous mammary tumours (Fig. 4b, right panel). The specificity of these responses against MUC1 is supported by the finding that the immunization of MMT mice with FC/MC38 (MUC1-negative) or immunization of MT mice with FC/MUC1 gave no protection against the development of mammary carcinomas (Fig. 4b, left panel). These results indicate that the anti-MUC1 immune responses induced by the immunization with FC/MUC1 can block or delay the development of mammary tumours in a predisposed model.

Control of tumour progression with fusion cell immunization

One of the characteristics of MMT and MT mice is that they develop mammary tumours in multiple stages. We have shown that immunization of MMT mice in the early stage of tumourigenesis can block tumour development. Therefore, it would be of interest to determine whether immunization of MMT mice in the later stage of tumourigenesis can control the progression of spontaneous mammary tumour. To address this issue, MMT mice were divided into three groups according to age at initial immunization. The immunization was begun in the first group of mice at < 1 month of age, in the second group of mice at 1–2 months and in the third group of mice at > 2 months. The immunization was repeated four additional times at 4-week intervals. Immunization of MMT mice with FC/MUC1 at < 1, 1–2 and > 2 months of age rendered 33, 5 and 0% of mice free of tumours, respectively, up to 6 months (Fig. 5a). As expected, all the PBS-treated MMT mice developed mammary tumours and became moribund 4 weeks after tumour occurrence (Fig. 5b, left). However, immunization of MMT mice with FC/MUC1 in the early stages of tumour development rendered 33% mice free of the disease for at least 6 months (Fig. 5b, right), and delayed development of tumour in those in which mammary tumour developed. The percentage of tumour-free mice was statistically significant compared with 1–2 month, >2 months, and non-vaccine groups respectively (P < 0·05, P < 0·0001 and P < 0·0001). Invasive mammary carcinomas were found in samples from PBS-treated mice (Fig. 5c, upper and lower panels). In contrast, there was no tumour formation in MMT mice immunized with FC/MUC1 (Fig. 5d, upper and lower panels). These results indicate that the efficacy of immunization is dependent on the age at which the mice were immunized.