Induction of Antitumor Immunity by Dendritic Cells Loaded with Membrane-Translocating Mucin 1 Peptide Antigen

Tr a n s l a t i o n a l O n c o l o g y

Volume 4 Number 1

February 2011

pp. 1–8 1

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Induction of Antitumor Immunity by Dendritic Cells Loaded with MembraneTranslocating Mucin 1 Peptide Antigen1

Saho Kobukai*, Gert-Jan Kremers†, Jared G. Cobb*, Richard Baheza*, Jingping Xie*, Alex Kuley*, Meiying Zhu* and Wellington Pham*,‡ *Institute of Imaging Science, Vanderbilt University, Nashville, TN, USA; †Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA; ‡ Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA

Abstract To investigate the role of enhanced antigen presentation in dendritic cell (DC)–based immunotherapy. Here, we describe the development of a cell-penetrating mucin 1 (MUC1) antigen and its immunotherapeutic potential against tumors. After animal groups received two immunizations of MUC1-MPA11P–pulsed DCs, we observed a marked tumor regression compared with the mice treated with DCs alone or DCs pulsed with MUC1 peptide. We confirmed the migration and homing of DCs in the popliteal lymph node using magnetic resonance imaging during the study. In summary, enhanced antigen uptake using an MPA11P delivery molecule improves cell therapy. Translational Oncology (2011) 4, 1–8

Introduction Dendritic cells (DCs) play important roles as antigen-presenting cells in innate and adaptive immunity, therefore they have become a primary target for the development of immunotherapy against cancers [1,2]. Several studies have demonstrated the role of DCs in the induction of antigen-specific immune responses against bacteria, viruses, and allergens [3]. Furthermore, a DC regimen is capable of inducing specific antitumor immune responses in mouse models [4,5] and humans [6]. In these studies, DCs were isolated and pulsed with exogenous tumorspecific antigens. Afterward, the antigen-loaded DCs were transferred to the hosts as cancer vaccines to enhance the immune responses against tumor targets. To date, DC-based therapy has been used in clinical trials for the potential treatment of a wide variety of cancers [7–12]. One of the most frequently tested tumor antigens in DC-based clinical trials is mucin1 (MUC1). MUC1 is a large transmembrane glycoprotein secreted on the apical surface of epithelial cells of mammary, colon, and salivary tissues [13]. The extracellular domain of MUC1 is composed of a repeating 20–amino acid sequence (GVTSAPDTRPAPGSTAPPAH)n, which is heavily O-glycosylated [14]. The tandem repeated peptide contains several serine and threonine moieties where glycosylation occurs. In neoplasic tissues, MUC1 is underglycosylated, loses its polarity, and is overexpressed in most human epithelial cell adenocarcinomas, including more than 90% of human breast [15–18], colon [19,20], and pancreatic cancers [19–23]. Thus, it became an optimal target for therapy and imaging [24,25]. Although MUC1-specific antibodies and/or cytolytic T lymphocytes have been observed in some

patients, there is no consistent clinical response that demonstrates that lymphocyte proliferation leads to the inhibition of cancer in patients [26–29]. Moreover, a recent review of DC vaccines in more than 1000 patients indicated that the overall response rate was just 8.9% [30]. Thus, the efficacy of DC vaccines still requires extensive research effort for further treatment optimization. Particularly, the process of incubating tumor peptide antigens with isolated DCs in vitro must be examined carefully because the uptake of individual tumor antigens differs widely from one type to another. In addition, it is unclear if there is a marked difference in the uptake of tumor antigens between bona fide versus isolated DCs. In a recent study, however, our group reported that the latter did not uptake the fluorescence probe unless the myristoylated polyarginine 11-mer peptide (MPA11P) delivery vehicle was used [31]. In line with this approach, a number of other works also focused on the development of a reliable technique to deliver the antigens inside DCs using penetratin [32] or Tat peptide [33]. In this study, we demonstrated the benefit of enhanced antigen delivery in improving cell therapy.

Address all correspondence to: Wellington Pham, PhD, Vanderbilt University, Institute of Imaging Science, 1161 21st Ave S, AA. 1105 MCN, Nashville, TN 37232-2310. E-mail: [email protected] 1 This work was supported by grants from National Institute on Aging (AG038325) and American Cancer Society–Institutional Research Grant (IRG-58-009-50). Received 4 June 2010; Revised 14 October 2010; Accepted 18 October 2010 Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1944-7124/11/$25.00 DOI 10.1593/tlo.10166

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Materials and Methods

Reagents and Cell Lines Murine breast cancer cell lines (mammary epithelial tumor cell line) C57MG or the MUC1-transfected C57MG were generously provided by Dr Sandra Gendler of the Mayo Clinic, Scottsdale, AZ. These cell lines were cultured and maintained in Dulbecco modified Eagle medium (Mediatech, Manassas, VA) in the presence of 10% fetal calf serum (FCS; Invitrogen, Carlsbad, CA), penicillin-streptomycin antibiotics (Mediatech), and 10 μg/ml insulin (Sigma-Aldrich, St Louis, MO) at 37°C and 5% CO2 incubator. Chicken anti-EEA1 and Alexa 488– labeled secondary antibodies were purchased from Invitrogen. Mouse anti-MUC1 and Alexa 647–labeled secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

MUC1.Tg Mice A colony of MUC1 transgenic (MUC1.Tg) mice was maintained by crossing MUC1.Tg mice obtained from Sandra Gendler (Mayo Clinic) with a wild-type C57BL/6 strain (Jackson Laboratory, Bar Harbor, ME). The mice were genotyped by a standard polymerase chain reaction using DNA isolated from tail tips with the following primers: forward, 5′-CTTGCCAGCCATAGCACCAAG-3′; reverse, 5′-CTCCACGTCGTGGACATTGATG-3′. After polymerase chain reaction amplification, the DNA product of each reaction was analyzed by size fractionation through a 1% agarose gel. The size of the DNA product from MUC1-positive mice corresponded with a 500-bp fragment. MUC1 transgenic mice were maintained as hemizygous animals. Animal experiments were carried out in accordance with the guidelines provided by Vanderbilt University’s Institutional Animal Care and Use Committee.

Synthetic MUC1 Peptides The 30-mer antigen (APDTRPAPGSTAPPAHGVTSAPDTRPAPGS) with the most antigenic epitope recognized by anti–mucin mAb and cytotoxic T cells and its counterpart that is covalently linked to the delivery molecule, MPA11P (MPA11P (C14-(R)11)-APDTRPAPGSTAPPAHGVTSAPDTRPAPGS), were synthesized using conventional fluoren-9-ylmethoxy carbonyl (Fmoc) chemistry [34,35]. After synthesis, the products were purified to 99% purity, as determined by high-performance liquid chromatography and characterized by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. For the 30-mer antigen and the MPA11P-antigen, the calculated molecular weights (MH+) 2837.87 and 4739.89 were found to be 2837.08 and 4739.40, respectively.

Bone Marrow–Derived Dendritic Cells DCs were isolated from the bone marrow of C57BL/6 mice as described in the past [31]. Briefly, bone marrow precursors were flushed out with RPMI from the femurs and tibias of mice and were subsequently processed into a single-cell suspension using a 70-μm mesh strainer. Erythrocytes were lysed with 0.83% ammonium chloride with 2 minutes of incubation at room temperature. The cells were centrifuged for 5 minutes at 300g and washed twice in RPMI. After washing, the cells were cultured in RPMI 1640 supplemented with 10% FCS, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 1000 U/ml recombinant granulocyte– macrophage colony-stimulating factor, and 250 U/ml recombinant interleukin 4 (both from PeproTech, Rocky Hill, NJ). On days 3 and 6 of the culture, nonadherent granulocytes and the B and T cells were

Translational Oncology Vol. 4, No. 1, 2011 gently removed by suction from half of the media, after which fresh media with cytokines were added. The released immature, nonadherent, loosely adherent cells were collected on day 8, with typical morphologic features of immature DCs. Cells in the day 8 culture were used as immature DCs. For most of the experiment, the immature DCs were pulsed without any antigen, with the MUC1 peptide, or with MUC1-MPA11P for 24 hours, and they were washed before experimental use.

T-cell Purification T cells were purified from spleen and lymph nodes (LNs) of MUC1. Tg mice with nylon wool (Wako Chemicals, Richmond, VA) column. Briefly, spleen and LNs were removed from mice at the end of the therapy and subsequently processed into a single-cell suspension using a 70-mm mesh strainer. The cell suspension was incubated in the column filled with the sterile nylon wool and RPMI medium at 37°C for 1 hour to adhere and trap B and other non–T cells to the nylon wool. Then, T cells were collected from run-through medium of the column and were used for the proliferation assay. The purity of the T cell was confirmed as greater than 95% by staining with a cell surface expression of CD3 molecule with PE-conjugated anti–CD3e mAb (eBiosciences, San Diego, CA) by FACS analysis.

T-cell Proliferation Assay Carboxyfluorescein succinimidyl ester (CFSE) was used for the detection of cell proliferation by FACS analysis. Purified T cells from MUC1.Tg mice were used to determine the frequency of proliferating cells against subsequent antigen presentation of MUC1 peptide by DCs. For staining T cells with CFSE (Invitrogen), T cells at 1 × 106 cells/ml were incubated with 0.5 μM CFSE for 10 minutes at 37°C and 5% CO2. Staining was quenched by the addition of an excess volume of ice-cold cell culture medium to the cell suspension for 5 minutes. Day 8 cultures of DCs were pulsed with no antigen, with MUC1 peptide antigen or MUC1-MPA11P. After the extensive washing of DCs or T cells, those two populations were cocultured at a 1:10 (DC/T) ratio for 5 days. The whole cells were further labeled with mAbs for lineage markers, PE-conjugated anti-CD3e, antigenpresenting cell–conjugated anti-CD4, and Cy7-conjugated anti-CD8a (eBiosciences) for FACS analysis. Values from T cells incubated alone without any DCs’ presence were referred as a background, whereas T cells with mitogenic concanavalin A (ConA; Sigma-Aldrich) or anti-CD3e activating antibody (eBiosciences) were referred as a spontaneous positive proliferation. Unstained cells were included in all experiments and were applied for compensation setting of the flow cytometer. The CD3+CD4+ or CD3+CD8+ T-cell proliferation was respectively determined by gating on the lineage marker–positive subsets and CFSE staining levels.

Immunotherapy One million C57MG.MUC1 or C57MG (control) cells in 100-μl serum-free Dulbecco modified Eagle medium were injected subcutaneously into the mammary fat pad as described previously [36]. At 8 to 10 days after the inoculation, small palpable tumors of approximately 0.4 cm were confirmed at the injection site, and the immunotherapy course was commenced as day 0. Tumor-bearing mice were immunized subcutaneously in the footpads with PBS or 1 × 106 unpulsed immature DCs or were prepulsed with 40 μM MUC1 peptides or 40 μM MUC1-MPA11P (n = 6, each group) at days 0 and 10 of the therapy course. At the conclusion of therapy on day 30, the

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mice were killed, after which LN, spleen, serum, and tumor were cryopreserved to test the therapy profile as described below.

Pad software (GraphPad Software, La Jolla, CA). P values are two-tailed; differences with a P value < .05 were considered statistically significant.

Tumor Measurement

Results

Tumor volumes were measured with a high-resolution ultrasound system (770 High-Resolution Imaging System; Visual Sonics, Toronto, Ontario, Canada). The tumors were digitally sliced into 0.5-mm ultrasound images, which were applied to the three-dimensional volumetric calculation by manually selecting a tumor area on each image in the imaging system. During the course of therapy, tumor size was carefully monitored and measured at 10-day intervals, with the methods mentioned above taking place from days 0 to 30. Once the tumor volume reached 10% of body weight or larger than 2 cm, the mouse was withdrawn from the therapy study and killed.

ELISA The amount of cytokines in the blood sera was determined with interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) ELISA kit (eBiosciences). Briefly, 96-well microtiter plates were coated with capture Ab, either anti–IFN-γ (AN-18) or anti–TNF-α (TN3.19). Sample sera were incubated in the coated plate in triplicate serial dilutions. Biotin-conjugated Abs, either anti–IFN-γ (R4-6A2) or anti–TNF-α (rabbit polyclonal), were used for detection. Finally, measurements of the cytokines were determined with pretitrated avidin-HRP and tetramethylbenzidine (TBS) substrate solution and detected on a spectrophotometer (Spectramax M5; Molecular Devices, Sunnyvale, CA) at 450 nm. As a standard for each cytokine amounts, mouse recombinant IFN-γ or TNF-α was used, and the value was referred for standard line.

Immunofluorescence and Immunohistochemistry To determine the internalization of the MUC1 antigen inside DCs, fixed and permeabilized DCs were stained for MUC1 and EEA1 using anti-MUC1 antibody (1:500) and anti-EEA1 antibody (1:500). After incubation with the corresponding Alexa-conjugated secondary antibody (Alexa 647 for MUC1 and Alexa 488 for EEA1), slides were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Dual-channel confocal microscopy was performed using a Zeiss LSM510 (Carl Zeiss, Thornwood, NY ). For the detection of apoptotic cells in the tumor after therapy, we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using a commercial kit (Millipore, Billerica, MA). Briefly, tumors were cryopreserved and sectioned for immunohistochemistry on termination of the therapy. Five-micrometer sections were fixed in cold acetone for 10 minutes and incubated with 2.5% FCS containing PBS for 30 minutes to block the nonspecific binding of the primary Abs to the sample. The sections were washed three times with PBS, then sealed with mounting medium (Vector Laboratories, Burlingame, CA). Finally, the section was analyzed with a light microscope (Carl Zeiss MicroImaging, Inc, Thornwood, NY). For the quantitative evaluation of apoptotic cells in the tumor slides representative images of each tumor slide were digitized and imported into Adobe Photoshop (version CS3; Adobe Systems, San Jose, CA) in TIFF file format. The area of diaminobenzidine staining was determined using the Color Range tool by selecting for the brown color of positive cells (RGB = 131/75/50; range = 100). Pixel area was captured using the Record Measurements analysis function and calculated in Microsoft Excel (Microsoft Corp, Redmond, WA).

Statistical analysis. The experimental data are reported as mean ± SD. We compared the test groups using a paired Student’s t-test using Graph-

Uptake of MUC1 Antigens To confirm the tumor antigens were taken up by DCs, MUC1 antigen and its counterpart MUC1-MPA11P derivative were incubated with immature DCs overnight. Then, the internalized antigens were detected using confocal microscopy after staining the Triton X–treated cells with anti-MUC1 and anti–endosomes antibodies. Figure 1 shows that both MUC1 and MUC1-MPA11P were taken up inside DCs; albeit, the concentration of the former seems to be inferior to the latter (n = 5). This is not surprising because, according to the previous study, the MPA11P delivery module could mediate the transfer of exogenous materials inside DCs within 15 minutes of incubation [31].

Both MUC1- and MUC1-MPA11P–Laden DCs Induce Remarkable CD8+ T-cell Proliferation Because the hallmark of DC therapy is the immune response manifested by the proliferation of T cells, before the therapy started, we were interested to learn whether T cells from MUC1 Tg mice could be induced to proliferate on exposure to the MUC1 antigen–laden DCs. Purified T cells from MUC1.Tg mice were isolated and assessed for frequency of proliferating cells against subsequent antigen presentation of MUC1 peptides by DCs. The T cells were first incubated with CFSE and then they were cocultured with DCs that were pulsed with no antigen, with MUC1, or with MUC1-MPA11P peptide antigens. T-cell proliferation was assessed 5 days after stimulation by flow cytometric analysis. Both MUC1- and MUC1-MPA11P–laden DCs induced CD8+ T cells with similar strength (Figure 2). In contrast, no proliferation was observed for CD4+ T cells.

Delayed Tumor Growth in Mice Immunized with MUC1-MPA11-P–Pulsed DC We generated a mouse model of tumor tolerance by injection of the C57MG-MUC1 tumor cells (kindly provided by Dr. Sandra Gendler) in the fat pad of the MUC1 Tg mice. The animals were then divided into four treatment groups comprised of untreated animals and animals immunized with DCs alone, DCs pulsed with MUC1 peptide, or MUC1-MPA11P. The therapy commenced when the tumor size became palpable or approximately 3 to 6 mm3. The mice were immunized in the footpads with the doses of DCs as described. During the 30-day course of therapy with two identical treatment doses administered 10 days apart (Figure 3A), we observed that DCs pulsed with MUC1 peptide or MUC1-MPA11P showed a marked delay in tumor growth by day 30, whereas tumor growth demonstrated a significantly rapid pace among untreated mice or mice immunized with DCs alone (Figure 3B). Furthermore, mice immunized with MUC1-MPA11P–pulsed DCs exhibited a marked suppression of tumor growth compared with other treatment groups. These results suggest that enhanced antigen delivery into DCs elicits significant antitumor immune responses against the MUC1-expressing tumor in MUC1.Tg mice.

Enhanced Antigen Delivery Induced a Marked Increase in Cytokine Level during Therapy To assess the effects of the vaccine on the production of cytokines, presumably from T cells, blood from each mouse were evaluated for the

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Figure 1. Confocal immunocytochemistry analysis the internalization of MUC1 antigens. Untreated DCs (A, B, C) and DCs treated with MUC1 antigen (D, E, F) or MUC1-MPA11P (G, H, I) were fixed, permeablized, and stained with anti-MUC1 and anti-EEA1 antibodies followed by fluorescently labeled secondary antibodies. MUC1 is pseudocolored in blue; EEA1, in green.

cytokine profiles associated with in vivo antitumor response. When the therapy was completed on day 30, sera were analyzed for elevated serum cytokine levels, particularly TNF-α and IFN-γ. A substantial difference in cytokine levels was observed between the untreated and treated groups. After the therapy, the level of cytokine production from the former was significantly lower compared with the latter (Figure 4). Notably, the levels of TNF-α and IFN-γ in the serum of mice treated with MUC1-MPA11P–pulsed DCs was higher compared with those in mice treated with the MUC1-pulsed DCs.

Reduced Tumor Growth Is Associated with Increased Apoptotic Death To corroborate the retarded tumor growth with cell death, tumors from each treatment group were dissected and subjected to immunohistochemistry to determine the presence of apoptotic cell death using TUNEL assay. The degree of cell death in the tumor section was significant when there was enhanced antigen delivery into DCs (Figure 5). Under similar conditions, the tumor sections from mice treated with MUC1-pulsed DCs failed to stain to the same degree. The ratio of apoptotic cells in tumor treated with MUC1-pulsed DCs versus MUC1MPA11P–pulsed DCs was 1:1.72.

Discussion DCs are central to the priming and development of antigen-specific T-cell immunity, which is necessary to elicit effective T-cell responses to tumors. In this regard, the notion of using tumor antigen-pulsed DCs with high specificity as tumor vaccines has been embraced as both an ideal strategy and a burgeoning area of study in several ongoing clinical trials. Despite significant advancements in DC-based therapy, the approach remains challenging, especially with regard to developing a simple yet robust method for activating the immune system against cancer in the context of using tumor antigen–activated DCs. In this work, we test the concept of enhanced antigen delivery in DC therapy using the membrane-translating delivery system developed in our laboratory [31,34]. This effort was pursued to answer one of the fundamental questions in DC therapy such as what would be the outcome of therapy if a technique was available to guarantee antigen delivery? Toward that goal, we developed the mouse model of tumor tolerance reported previously [37] and tested the therapy using MUC1 tumor antigen. Before therapy, we confirmed that DCs could uptake the MUC1 antigens developed in our laboratory. Figure 1 shows that both MUC1 and MUC1-MPA11P were taken up inside DCs after an overnight incubation. However, the concentration of MUC1-MPA11P was apparently higher than that of MUC1. By combining this observation with the

Translational Oncology Vol. 4, No. 1, 2011 one made in the past [31], we hope that future therapy would not require overnight incubation of the antigen with DC using a more efficient means of delivering the antigen. The 30-day in vivo therapy (Figure 3A, timeline) conducted on tumor-bearing mice showed a significant regression in tumor growth among mice treated with MUC1-MPA11P–pulsed DCs compared with other treatment groups, thus indicating the potential contribution of MPA11P in inducing potent DCs. Altogether, these data suggest that the appropriate delivery of antigens into DCs can induce potent antitumor immunity. During therapy, the selected animals were chosen for magnetic resonance imaging to confirm the effective migration of DCs from the injection site to the lymph nodes. Toward that approach, before adaptively transferring the cells to the treated mice, DCs were pulsed briefly with iron nanoparticles using the previously described protocol [38]. Twenty-four hours after injection, magnetic resonance scans detected the decreased signal intensity in the popliteal lymph nodes. This phenomenon is attributed to the homing of the iron nanoparticle-laden DCs (data not shown). As shown in Figure 2, MUC1.Tg-derived CD8+ T cells incubated with DCs pulsed with either MUC1 peptide or MUC1-MPA11P

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showed proliferation. Interestingly, no effect was noted on the proliferation of CD4+ T cells in either group. The levels of CD8+ T-cell proliferations, led by the MUC1-MPA11P–pulsed DCs, were not superior to those of MUC1 peptide–pulsed DCs. Nevertheless, the in vivo therapy study showed a marked slowdown in tumor growth among the mice treated with MUC1-MPA11P–pulsed DCs compared with those treated with MUC1 peptide–pulsed DCs. This observation suggests the possibility that the number of CD8+ T cells proliferated by MUC1MPA11P–pulsed DCs might have a higher number of activated/functional cytotoxic T cells than those proliferated by MUC1 peptide–pulsed DCs. Further, we cannot ignore the complexity of the factors engaged in the in vivo environment, which promoted a slightly higher and persistent antitumor immune response in the presence of MUC1-MPA11P– pulsed DCs. In fact, immunohistochemical data indicated that the level of tumor cell death in the MUC1-MPA11P treatment group is apparently more obvious than in the group of mice treated with MUC1-pulsed DCs (Figure 5). Semiquantitative analysis of the signal associated with apoptotic cells showed that MPA11P-MUC1–pulsed DCs induced apoptosis nearly two-fold more than MUC1-pulsed DCs counterparts.

Figure 2. Induction of T-cell proliferation by MUC1 peptide or MUC1-MPA11P–pulsed DCs. MUC1.Tg mouse-derived purified T cells were labeled with 0.5 μM CFSE, and T-cell proliferation was determined by flow cytometry. DCs were pulsed overnight with no antigen, with 40 μM of MUC1 antigen, or with 40 μM MUC1-MPA11P. CFSE-labeled T cells and DCs were cocultured at a 1:10 ratio for 5 days. CD3+CD4+ or CD3+ CD8+T-cell populations were gated and analyzed for the intensity of the CFSE by flow cytometry. For positive control, the CFSE–T cells were pulsed with ConA; for negative control, the CFSE–T cells were cultured alone without the addition of cells or reagents for 5 days (not shown).

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Figure 3. Induction of antitumor immunity with DC-based vaccines. (A) Timeline of the therapy. (B) DC-based therapy. Four groups of age- and sex-matched MUC1 Tg mice (n = 6, each) were inoculated with 1 × 106 C57MG-MUC1 cells subcutaneously in the fat pad area. Eight days later, the untreated mice (♦) or mice challenged with DCs ( ), DCs loaded with MUC1 tumor peptide antigens ( ), or DCs loaded with MUC1MPA11P ( ) vaccine regiments. The mice were treated with similar doses on day 10. (B) The tumor growth was monitored, measured, and compared with the original size (day 0) using high-resolution ultrasound. Each point represents a mean of six measurements. We observed a modest reduced tumor growth but with significant difference in the groups of mice treated with DC + MUC1-MPA11P compared with those treated with DC + MUC1. *P < .05 in B (DC + MUC1-MPA11P vs DC + MUC1).

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It is worthwhile mentioning that although we observed a modest delay in tumor growth within the group of animals treated with MUC1-MPA11P–pulsed DCs compared with those treated with the MUC1-pulsed DCs, that observation is credible. Here, we used high-resolution ultrasound to measure tumor size, a method that is much more precise than the caliper technique. Exact assessment of

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the tumor volume is crucial in this study because tumor volume is considered an indicator of therapeutic outcome. During the course of this study, we confirmed an error made while using the manual caliper approach to the volumetric measurement of tumors. The error became evident when caliper-determined volumetric measurement data began to exhibit overestimates at larger volumes as the size of

Figure 4. Analysis of the cytokine profiles in the serum of mice after a 30-day therapy by ELISA. Each filled circle corresponds to data from one mouse. Vertical lines indicate mean value. Statistical analysis was performed using a paired Student’s t-test (*P = .0001). Cytokines were induced by DC vaccines and shown as their concentrations (pg/ml). High levels of TNF-α and IFN-γ were observed in mice treated with MUC1-MPA11P–pulsed DCs.

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Figure 5. The TUNEL assay was performed on the tumors that were harvested from the animals at the end of the 30-day therapy. Cryosections of the tumors were stained for DNA fragmentation associated with apoptosis. The sections were processed with diaminobenzidine (DAB) and counterstained with methyl green. Tumor sections from the MUC1-MPA11P treatment group revealed higher levels of apoptotic nuclei compared with those from the MUC1 treatment group. Magnification, ×40.

the tumor increased compared with those done using the ultrasound system (data not shown). Using the caliper technique, a tumor can be measured externally; however, this method cannot measure tumors that grow underneath the tissue. Furthermore, the various shapes of tumors are not always ideal for applying the mathematical formulas.

Conclusions In conclusion, we demonstrated the implication of enhanced antigen delivery and imaging in DC-based therapy. These findings provide a new avenue that might facilitate the implementation of protocols in DC preparation for cancer immunotherapy and cancer vaccine development. Notably, the MPA11P represents a promising delivery module for carrying tumor antigens inside DCs. Finally, we envision a broad application of our findings across many different types of cancers given that MUC1 is overexpressed in various epithelial malignancies. Thus, it is a suitable candidate for broadly applicable vaccine therapies.

Acknowledgments The authors thank Joseph Roland for quantitative analysis of the immunohistochemistry data.

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