Antibodies targeted to TRAIL receptor-2 and ErbB-2 synergize in vivo and induce an antitumor immune response

Abstract

Despite the development of human epidermal growth factor receptor-2 (ErbB-2/HER2)-targeted therapies, there remains an unmet medical need for breast cancer patients with ErbB-2 overexpression. We investigated the therapeutic activity of an agonist mAb to mouse tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 (DR5) against ErbB2-driven breast cancer. Established tumors in BALB/c transgenic mice expressing a constitutively active ErbB-2/neuT were treated with anti-DR5 mAb and/or anti-ErbB-2 mAb and monitored for tumor progression. Treatment with anti-DR5 or anti-ErbB2 mAb as single agents significantly delayed tumor growth, although all tumors eventually progressed. Remarkably, treatment with a combination of anti-DR5 and anti-ErbB-2 mAbs induced complete response in a majority of mice. In vivo blockade of CD11b⁺ cells, but not natural killer cell depletion, significantly abrogated the early antitumor response. Notably, depletion of CD8⁺ T cells provoked primary and secondary tumor relapse, revealing the induction of antitumor immunity by the combination treatment. Combined therapy with anti-DR5 and anti-ErbB-2 mAbs further significantly suppressed the growth of advanced spontaneous tumors in ErbB-2/neuT transgenic mice, even when treatment was delayed until tumors were palpable. We thus demonstrated that the combination of anti-DR5 and anti-ErbB2 mAbs might be an effective form of treatment for ErbB-2-overexpressing breast cancer.

Human epidermal growth factor receptor-2 (ErbB-2/HER2) is overexpressed in 20–30% of patients with breast cancer and is associated with a poor prognosis (1). The use of trastuzumab (Herceptin; Genentech), a mAb that blocks the activity of ErbB-2, in combination with chemotherapy has been associated with an increased median survival in patients with advanced ErbB-2-positive breast cancer (2–4). In addition to its inhibitory effect on ErbB-2 signaling, trastuzumab mediates its therapeutic effect through the recruitment of immune cells such as monocytes and natural killer (NK) cells (1, 5). Accordingly, trastuzumab-like mAb unable to bind Fc receptors has less therapeutic activity (6), and complete or partial remission induced by trastuzumab correlates with higher antibody-dependent cell cytotoxicity (7–9).

Although the development of trastuzumab constitute a major advance in the treatment of ErbB-2-overexpressing breast cancer, there remains an unmet medical need for new therapies. Proapoptotic receptor agonists (PARAs), including recombinant Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mAbs, have now emerged as promising anticancer agents (10, 11). PARAs exploit the enhanced susceptibility of cancer cells versus nontransformed cells to TRAIL-mediated apoptosis. Activation of TRAIL-R1 (DR4) or TRAIL-R2 (DR5) triggers the formation of a death-inducing signaling complex (DISC) that activates caspase-8. In some cells (type I), the activation of the ensuing caspase cascade is sufficient to trigger apoptosis, whereas in other cells (type II) mitochondrial release of proapoptotic factors via the cleavage of Bid by caspase-8 is required. Overexpression of antiapoptotic Bcl-2 family proteins can protect tumor cells from DR4- or DR5-mediated apoptosis. At the DISC level, overexpression of the caspase-8 inhibitor cellular FLICE-like inhibitory protein (cFLIP) further contributes to TRAIL resistance (11–15).

The potential for agonistic anti-DR4 or anti-DR5 mAbs to be incorporated in the treatment regimen of ErbB-2-overexpressing breast cancer is currently unknown. Here, we investigated the therapeutic activity of agonistic anti-DR5 mAb against spontaneous mammary carcinomas arising in BALB/c transgenic mice expressing a constitutively active rat ErbB-2 transgene (ErbB-2/neuT) under the mouse mammary tumor virus (MMTV) promoter (16–18). We provide evidence that administration of agonistic anti-DR5 mAb can synergize in vivo with anti-ErbB2 mAb therapy for the treatment of spontaneous ErbB-2-driven breast cancer.

Results

Primary ErbB-2/neuT Tumor Cells Are Sensitive to Anti-DR5 and Anti-ErbB-2 mAbs.

Primary ErbB-2/neuT tumor cells consistently expressed DR5 (Fig. 1A). Plate-bound anti-DR5 mAb (clone MD5-1) (Fig. 1B) or MD5-1 cross-linked by Fc receptor-expressing P815 cells (Fig. 1C) induced caspase-dependent apoptosis of ErbB-2/neuT tumor cells. To determine whether MD5-1 activated the intrinsic and/or extrinsic apoptotic pathways, we retrovirally gene-modified ErbB-2/neuT tumor cells to overexpress Bcl-2 or the caspase-8 inhibitor CrmA. CrmA overexpression, but not Bcl-2 overexpression, inhibited MD5-1-mediated apoptosis, suggesting that ErbB-2/neuT tumor cells do not rely on the mitochondrial pathway for DR5-mediated apoptosis (Fig. 1A).

Fig. 1.

Tumor cells derived from ErbB-2/neuT transgenic mice are sensitive to anti-DR5 and anti-ErbB-2 mAbs. (A) ErbB-2/neuT tumor cell lines were analyzed by flow cytometry for cell surface expression of rat (r)ErbB-2/neuT and DR5. (B and C) H2N100 cells were retrovirally gene-modified to express Bcl-2, CrmA, or control plasmid and cultured for 6 h on protein A-coated plates (B) or cocultured with Fc receptor-expressing P815 cells (C), with increasing concentrations of anti-DR5 mAb (clone MD5-1). The percentage of apoptotic cells was determined by flow cytometry using allophycocyanin-conjugated Annexin-V. Means ± standard errors of three independent experiments are shown (*, P < 0.05 by Student's t test). (D) H2N100 cells were treated for 24 h with 10 μg/ml control mouse IgG2a or anti-ErbB-2 mAb (clone 7.16.4), and immunoblotting was performed. (E) Real-time PCR analysis of VEGF expression relative to GAPDH expression (means of triplicates ± standard errors; *, P < 0.05 by Student's t test).

We next assessed whether ErbB-2/neuT tumor cells were sensitive to anti-ErbB-2 mAb treatment in vitro. Anti-ErbB-2 mAb (clone 7.16.4) down-regulated AKT activity, Bcl-2, Bcl-X_L, and cFLIP protein levels (Fig. 1D) and decreased vascular endothelial growth factor (VEGF) transcription (Fig. 1E). We assessed whether anti-DR5 and anti-ErbB-2 mAbs could enhance the antitumor activity of either mAb. Anti-DR5 and anti-ErbB-2 mAbs were added at increasing concentrations to primary ErbB-2/neuT tumor cell cultures. As shown in Fig. 2, combined treatment with anti-DR5 and anti-ErbB-2 mAbs significantly enhanced the antiproliferative effect of either mAb.

Fig. 2.

Combined anti-DR5 and anti-ErbB-2 mAbs enhanced the antitumor activity of either mAb in vitro. ErbB-2/neuT tumor cells were cultured for 4 days on protein A-coated plates with increasing concentrations of anti-DR5 (MD5-1) and anti-ErbB-2 (7.16.4) mAb clones (or control Ig). Tritiated thymidine was added for the last 16 h of culture, and radioactivity was measured with a β-counter. Means ± standard errors of triplicates are shown. One of two representative experiments is shown (*, P < 0.05 compared with control-treated cells; **, P < 0.05 compared with 7.16.4- or MD5-1-treated cells).

In Vivo Antitumor Synergy Between Anti-DR5 and Anti-ErbB-2 mAbs.

To test the in vivo activity of anti-DR5 and anti-ErbB-2 mAbs, we first transplanted ErbB-2/neuT tumors into ErbB-2/neuT transgenic mice. When tumors reached an average size of 9 mm², mice were treated with anti-DR5 and/or anti-ErbB-2 mAb as indicated. Although single therapy with anti-DR5 or anti-ErbB2 mAb significantly delayed tumor growth, all tumors eventually progressed. Remarkably, combined therapy with anti-DR5 and anti-ErbB-2 mAbs induced a rapid and sustained tumor regression (Fig. 3A). Of a total of 60 mice treated with a combination of MD5-1 and 7.16.4 mAbs, 38 (63.3%) achieved a complete response.

Fig. 3.

In vivo antitumor synergy between anti-DR5 and anti-ErbB-2 mAbs. (A) A total of 2 × 10⁵ ErbB-2/neuT tumor cells were injected s.c. into transgenic mice. When tumors reached an average size of 9 mm², mice were treated with anti-DR5 (MD5-1) and/or anti-erbB2 (7.16.4) (or control Ig). Mean tumor size ± standard errors are shown. One of six representative experiments is shown. (B) The same as A, except that mice treated with MD5-1 and 7.16.4 mAbs that remained tumor-free after cessation of treatment were injected with depleting anti-CD8 mAb or control Ig twice weekly from day 40. Mean tumor size ± standard errors of 10 mice per group from two independent experiments are shown. (C) The same as B. Some mice were additionally challenged s.c. on the opposite flank with 2 × 10⁵ cells parental tumor cells. Mean tumor size ± standard errors of five mice per group are shown. (D) The same as A, except that SCID mice were used. Mean tumor size ± standard errors of five mice per group are shown. (E) The same as D, except that some groups of mice were additionally injected twice weekly with 300 μg of anti-CD11b (5C6) mAb. Mean tumor size ± standard errors of six mice per group from two independent experiments are shown. *, P < 0.05 compared with single treatment or control-treated by Mann–Whitney test.

We investigated whether combined MD5-1 and anti-ErbB-2 mAb therapy induced adaptive immunity in ErbB-2/neuT transgenic mice. Mice with established tumors treated with the combination that remained tumor-free after cessation of treatment were injected with depleting anti-CD8 mAb or control Ig twice per week from day 40. As shown in Fig. 3, mice depleted of CD8⁺ cells had relapsing primary tumors and developed a secondary tumor at a greater rate then control-treated mice. These data support the development of an adaptive antitumor immune response after therapy. In addition, although the initial antitumor effect of all treatments was essentially independent of adaptive immunity, total tumor suppression was not achieved in severe-combined immunodeficient (SCID) mice (Fig. 3D). In vivo blockade of CD11b⁺ cells partially inhibited the early therapeutic response (Fig. 3E), whereas injection of the NK cell-depleting anti-asialoGM1 antibody had no effect (data not shown). Collectively, these findings suggest the rapid suppression of tumor size mediated by anti-DR5 and anti-ErbB-2 mAbs involved immune and nonimmune mechanisms. TUNEL staining of tumor sections revealed an enhanced level of apoptosis in tumors treated with the combination (Fig. 4A). We thus investigated whether anti-ErbB-2 mAb could directly synergize with MD5-1. In vitro treatment with anti-ErbB-2 mAb failed to increase the sensitivity of ErbB-2/neuT tumor cells to MD5-1 [supporting information (SI) Fig. S1]. Thus, although the two mAbs were strongly synergistic in vivo, we did not detect any direct synergistic activity in vitro. An important distinguishing feature of in vivo tumors is the cross-talk between malignant and stromal cells. We thus investigated whether MD5-1 and anti-ErbB-2 mAbs disrupted tumor–stroma interactions (19). We observed that whereas anti-ErbB-2 mAb directly inhibited VEGF transcription (Fig. 1E), it did not affect hypoxia-inducible factor 1α (HIF-1α) expression in vivo (Fig. 4B; consistent with other studies) (20). In contrast, MD5-1 significantly decreased HIF-1α expression in ErbB-2/neuT tumors (Fig. 4B). Thus, administration of MD5-1 and anti-ErbB-2 mAbs may disrupt tumor–stroma interactions in ways not achievable with single-mAb therapy.

Fig. 4.

TUNEL and HIF-1α expression after treatment. ErbB-2/neuT tumor cells were injected s.c. into transgenic mice, and when tumors reached an average size of 9 mm², mice were treated with anti-DR5 (MD5-1) and/or anti-erbB2 (7.16.4) (or control Ig). Tumors were surgically removed 24 h after treatment and fixed. (A) TUNEL staining was performed. Representative sections of each treatment group are shown. (B) HIF-1α protein expression was detected by immunohistochemistry. Representative sections of each treatment group are shown. (Scale bars: 250 microns.)

Combined Anti-DR5 and Anti-ErbB-2 mAb Therapy Prolonged the Latency of Spontaneous Mammary Carcinomas.

We investigated whether anti-DR5 mAb could be effectively combined with anti-ErbB-2 mAb for the treatment of advanced spontaneous tumors. Whole-mount staining of mammary glands of ErbB-2/neuT transgenic mice at 80 days of age confirmed the presence of multiple lesions (Fig. 5A). When treatment was initiated at 80 days of age, the combination therapy significantly prolonged the latency of spontaneous tumors compared with single treatment (Fig. 5B). We investigated whether tumors that developed despite treatment had acquired resistance to MD5-1 or anti-ErbB-2 mAb. When tumor cells were isolated from MD5-1 and/or anti-ErbB2 mAb-treated mice and exposed to MD5-1 or anti-ErbB-2 mAb in vitro, they remained sensitive to either antibody (Fig. 5 C–E). Our data suggests that whereas the combination prolonged the latency of spontaneous tumors, therapy eventually fails despite tumor cells remaining sensitive in vitro. Most remarkably, the enhanced therapeutic effect of combining MD5-1 and anti-ErbB-2 mAb was significant even when treatment was delayed until tumors were palpable (i.e., treatment initiated at 87 days of age; Fig. 5F).

Fig. 5.

Combined anti-DR5 and anti-ErbB-2 mAb therapy prolonged the latency of spontaneous mammary carcinomas. (A) Whole-mount staining of inguinal mammary glands of WT and ErbB-2/NeuT (Tg) transgenic mice at 80 days of age. (B) ErbB-2/neuT transgenic mice were treated starting at 80 days of age with anti-DR5 (MD5-1) and/or anti-erbB2 (7.16.4) (or control Ig). Mean tumor multiplicity ± standard errors of two independent experiments are shown. (C) Tumor cell lines generated from spontaneous tumors treated with MD5-1 (H2N126) or treated with combined MD5-1 and 7.16.4 mAbs (H2N61) were cultured for 4 days on protein A-coated plates with 10 μg/ml control mIgG2a or 7.16.4 mAb. Tritiated thymidine was added for the last 16 h of culture, and radioactivity was measured with a β-counter. Means ± standard errors of triplicates are shown (*, P < 0.05 compared with control by Student's t test). (D) H2N61 tumor cells were cultured for 4 days on protein A-coated plates with increasing concentrations of MD5-1. Tritiated thymidine was added for the last 16 h of culture, and radioactivity was measured with a β-counter. Mean ± standard errors of triplicates are shown (*, P < 0.05 compared with control by Student's t test). (E) H2N126 tumor cells were cultured for 20 h on protein A-coated plated with 2.5 μg/ml control Ig or MD5-1 mAb. A representative experiment of two is shown. (F) The same as B, except that mice were treated starting at 87 days of age (in B and F, *, P < 0.05 compared with single treatment by Mann–Whitney test).

Discussion

Therapeutic strategies aimed at inducing a proimmunogenic form of cancer cell death may have the potential to significantly benefit cancer patients (21, 22). We investigated the therapeutic activity of anti-DR5 mAb against spontaneously arising mammary tumors in ErbB-2/neuT transgenic mice. We hypothesized that combined therapy with anti-DR5 and anti-ErbB2 mAbs might induce synergistic antitumor activity. Our studies provide evidence that anti-DR5 mAb can greatly improve anti-ErbB2 mAb therapy by triggering extensive tumor cell death and CD8-dependent antitumor immune response.

Consistent with studies showing that DR5 is expressed at high levels on human breast cancer cells (23–25), tumor cells derived from ErbB-2/neuT transgenic mice expressed high levels of DR5. In vitro, agonistic anti-DR5 mAb (clone MD5-1) induced caspase-dependent apoptosis of ErbB-2/neuT-driven breast cancer. Interestingly, Bcl-2 overexpression failed to protect tumor cells against MD5-1, suggesting that ErbB-2-driven cancer cells do not rely on the intrinsic mitochondrial pathway for DR5-mediated apoptosis.

In vitro, anti-DR5 and anti-ErbB-2 mAbs significantly enhanced the antiproliferative effect induced by either mAb. Remarkably, treatment of established tumors with a combination of anti-DR5 and anti-ErbB-2 mAbs induced complete response in a majority of mice. The combination of MD5-1 and anti-ErbB-2 mAbs induced greater tumor cell apoptosis as early as 24 h after treatment. Blocking CD11b⁺ cell infiltration partially inhibited the early antitumor effect of the combination treatment, whereas NK cell depletion had no effect. Previous experiments in another model of mouse mammary carcinoma have shown that MD5-1 activity completely depended on a combination of CD11b⁺ cell infiltration and NK cells, and that FcγR expression was essential (26). We now need to create FcγR-deficient erbB2 transgenic mice and explore the role of FcR in single- and dual-mAb therapy, as well as examine which CD11b⁺ cells (neutrophils and/or monocytes/macrophages) and CD11b⁻ innate cells are potentially suppressing tumor growth. Interestingly, the two mAbs did not show direct synergistic activity in vitro, although they induced potent synergistic activity in vivo. One possible explanation involves interactions between tumor cells and stromal cells that may have been disrupted by the combination treatment in ways not achieved with either mAb. In support of this hypothesis, we observed that treatment with anti-ErbB-2 mAb decreased VEGF transcription without affecting HIF-1α expression in vivo, whereas MD5-1 significantly decreased HIF-1α expression in vivo.

An important finding of our study is the observation that tumor-bearing mice treated with MD5-1 and anti-ErbB2 mAbs that were subsequently depleted of CD8⁺ cells had relapsing primary tumors and developed a secondary tumor at a greater rate then control-treated mice. This result suggests that combined therapy with MD5-1 and anti-ErbB-2 mAbs can break immune tolerance in ErbB-2/neuT transgenic mice. Others have shown that adaptive antitumor immune responses can be generated in ErbB-2/neuT transgenic mice (27–29). We demonstrated that adaptive antitumor immunity is also achievable with the administration of anti-DR5 and anti-ErbB-2 mAbs. Interestingly, trastuzumab has been shown to enhance the cytotoxicity of ErbB-2-specific cytotoxic T lymphocytes (CTLs). Through enhanced endocytic degradation of ErbB-2, trastuzumab was shown to enhance MHC class I presentation of ErbB-2 epitopes, resulting in a higher susceptibility of ErbB-2 tumors to lysis by CTLs (30, 31). Taken together, these data suggest that the combined administration of anti-DR5 mAb and trastuzumab may synergize in the generation of antitumor adaptive immune responses.

Remarkably, the combination therapy with MD5-1 and anti-ErbB-2 mAbs significantly delayed the development of advanced established spontaneous tumors in ErbB-2/neuT transgenic mice. Notably, when tumor cells were isolated from MD5-1 and/or anti-ErbB2 mAb-treated mice and exposed to MD5-1 or anti-ErbB-2 mAb in vitro, they remained sensitive to either antibody. Our data thus suggest that in vivo resistance to therapy is not the result of acquired intrinsic resistance by tumor cells. A better understanding of the failure mechanism might improve the development of mAb-based cancer therapies. Although we did not observe any toxicity of the single or combination mAbs in our study and MD5-1 can be administered safely to BALB/c mice at very high doses (26, 32), cholangiocyte toxicity and bile duct occlusion is observed in C57BL/6 mice treated with high doses of MD5-1, and hepatotoxicity with increased serum alanine aminotransferase, aspartate aminotransferase, and bilirubin was reported in a few patients when treated with higher doses (20 mg/kg) of lexatumumab (human anti-human DR5) (33). It is too early to know whether hepato- or bile duct toxicity might limit anti-DR5-based therapeutic approaches in humans, but care needs to be taken when progressing to combination therapies in humans where new sensitivities to the TRAIL–DR5 pathway may manifest.

In conclusion, despite the significant improvement in quality of care afforded by ErbB-2 targeted therapies, there remains an unmet medical need for breast cancer patients with ErbB-2 overexpression. We have provided preclinical evidence that agonistic anti-DR5 mAb can induce potent antitumor effect against primary ErbB-2-driven breast tumors in mice. Notably, anti-DR5 mAb therapy was effective against ErbB-2/neuT tumor cells overexpressing antiapoptotic Bcl-2. Moreover, we have demonstrated that combining ErbB2 blockade and TRAIL receptor activation induces potent synergistic antitumor effect in vivo and could be an effective form of therapy combining targeted tumor cell death and systemic antitumor immune response.

Materials and Methods

Mice.

BALB/c MMTV-ErbB-2/neuT mice, BALB/c SCID mice, and WT BALB/c mice were bred and maintained at the Peter MacCallum Cancer Centre.

Cell Lines.

4T1.2, NIH 3T3, and P815 cells have been described (19). ErbB-2/neuT tumor lines were generated as described (34). Briefly, lobular breast carcinomas were excised from 20- to 23-week-old female BALB/c MMTV-ErbB-2/neuT transgenic mice and digested with DNase I and collagenase. ErbB2/neuT expression was confirmed by flow cytometry using purified anti-rat ErbB-2/neuT mAb (10 μg/ml clone 7.16.4) and phycoerythrin (PE)-conjugated anti-mouse Ig F(ab)₂ fragment (Chemicon). DR5 expression was assessed by using biotinylated anti-DR5 mAb (clone MD5-1; BD Bioscience) and PE-conjugated streptavidin (BD Bioscience).

Retroviral Gene Transfer.

Control and ErbB-2/neuT plasmids were a generous gift from William J. Muller (McGill University, Montreal). Plasmids for mouse Bcl-2, CrmA, or control have been described (35). Phoenix-Eco 293 packaging cells were used to generate gene-modified tumor cells.

Antibodies.

Anti-rat ErbB-2/neuT mAb (clone 7.16.4) hybridoma was generously provided by Mark I. Greene, University of Pennsylvania (Philadelphia). MD5-1, 7.16.4, anti-CD8 mAb (clone 53–6.7), anti-CD11b mAb (clone 5C9), control IgG (clone UC8–1B9), and control IgG (clone MAC4) were produced at the Peter MacCallum Cancer Centre as described (26). Rabbit anti–asialo-GM1 antibody was purchased from Wako Chemicals and injected according to the manufacturer's instructions.

In Vitro Apoptosis and Proliferation Assays.

MD5-1 was diluted in 100 μl of PBS and incubated overnight at 4°C on Reacti-Bind 96-well protein A-coated plates (Pierce Biotechnology). The next day, wells were washed with PBS, and 10⁵ tumor cells were added per well in complete media. At the indicated time points, cells were trypsinized and analyzed by flow cytometry for annexin-V binding (BD Bioscience). In some experiments, the pan-caspase inhibitor Zvad-fmk (MP Biomedicals) was added at a final concentration of 100 μM. Tumor cells and P815 cells were cocultured at a ratio of 5:1 with 10 μg/ml control IgG or 7.16.4. For in vitro proliferation, 10⁴ tumor cells per well were cultured in 96-well protein A-coated plates in complete media for 4 days. For the last 16 h, 10 μCi per well of tritiated thymidine was added. Cells were harvested by using a cell harvester and radioactivity measured by using a Chameleon β-counter (Hidex).

Immunoblotting and Intracellular Staining.

The following antibodies were used: hamster anti-mouse Bcl-2 (clone 3F11, at 1:2,000; BD Biosciences), rabbit anti-FLIP (clone H-202, at 1:500; Santa Cruz), anti-Bcl-XL (clone H5, at 1:500; Santa Cruz), anti-B actin (at 1:2,000), rabbit anti-pS6 (2215 at 1:2,000; Cell Signaling Technology), anti-CrmA (clone A71–1, at 1:500; BD Biosciences), anti-p53 (at 1:1,000), anti-Bax (clone 6A7, at 1:1,000; BD Biosciences), anti-hamster IgG-HRP (mixture of clones G70-204 and G94-56, at 1:4,000; BD Biosciences), anti-rabbit IgG-HRP (at 1:2,000; Dako), and anti-mouse IgG-HRP (at 1:2,000; DAKO).

Real-Time PCR.

RNA from cells was extracted by using the RNeasy Midi Kit (Qiagen). Oligonucleotides and Taqman probe mix (Gene Expression Assays) specific for mouse VEGF (Mm00437306_m1) and mouse GAPDH (Mm99999915_g1) were purchased from Applied Biosystems. PCRs were performed by using the 7500 Fast Real-Time PCR System (Applied Biosystems). VEGF levels were quantified as relative expression to endogenous GAPDH levels by using 7500 Fast System SDS Software (Applied Biosystems).

In Vivo Treatment.

Tumor cells (2 × 10⁵) were injected s.c. into 4-week-old ErbB-2/neuT transgenic mice or SCID mice. Mice were treated with three i.p. injections of 100 μg of MD5-1 every 4 days and/or 100 μg of 7.16.4 twice a week. Tumor size was measured with a caliper as the product of two perpendicular diameters. In some mice, 100 μg of anti-CD8 (clone 53-6.7), anti-asialoGM1, 300 μg of anti-CD11b (clone 5C6), or control Ig (clone MAC4) were injected i.p. twice a week. CD8⁺ T cell and NK cell depletion was confirmed by flow cytometry. MMTV-ErbB-2/neuT transgenic mice were injected starting at 80 or 87 days of age with four i.p. injections of 100 μg of MD5-1 and/or 100 μg of 7.16.4 twice weekly. Development of mammary tumors was monitored by palpation of mammary glands.

TUNEL and HIF-1α Immunohistochemistry.

Transplanted tumors were surgically removed 24 h after treatment, fixed in 10% formalin, embedded in paraffin, and cut into 4-μm sections. Apoptosis was assessed by using an ApopTag peroxidase in situ apoptosis detection kit (Chemicon) per the manufacturer's instructions. For HIF-1α immunohistochemistry, sections were dewaxed and rehydrated, and anti-HIF1α antibody (Novus Biologicals; NB100-123) diluted 1:200 in antibody diluent (Dako; S0809) was added for 30 min at room temperature. Secondary EnVision+ antibody (Dako; K4007) was added for 30 min. Diaminobenzidine substrate (Dako; K3468) was added for 10 min. Sections were then counterstained with hematoxylin.

Whole-Mount Mammary Gland Staining.

Inguinal mammary fat pads were scraped from the skin of WT or ErbB-2/neuT transgenic mice, spread on clear glass slides, and immersed in Canoy's fixative overnight. The mammary fat pads were then rehydrated, stained in Carmine red solution overnight, and dehydrated in increasing concentrations of alcohol. Digital pictures were taken with a Nikon D80 digital camera.