Mining the pre-diagnostic antibody repertoire of TgMMTV-neu mice to identify autoantibodies useful for the early detection of human breast cancer

BACKGROUND: The use of autoantibodies for the early detection of breast cancer has generated much interest as antibodies can be readily assayed in serum when antigen levels are low. Ideally, diagnostic autoantibodies would be identified in individuals who harbored pre-invasive disease/high risk lesions leading to malignancy. Prospectively collected human serum samples from these individuals are rare and not often available for biomarker discovery. We questioned whether transgenic animals could be used to identify cancer-associated autoantibodies present at the earliest stages of the malignant transformation of breast cancer. METHODS: We collected sera from transgenic mice (TgMMTV-neu) from the time of birth to death by spontaneous mammary tumors. Using sera from a time point prior to the development of tumor, i.e. “pre-diagnostic”, we probed cDNA libraries derived from syngeneic tumors to identify proteins recognized by IgG antibodies. Once antigens were identified, selected proteins were evaluated via protein arrays, for autoantibody responses using plasma from women obtained prior to the development of breast cancer and matched controls. The ability of the antigens to discriminate cases from controls was assessed using receiver-operating-characteristic curve analyses and estimates of the area under the curve. RESULTS: We identified 6 autoantibodies that were present in mice prior to the development of mammary cancer: Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35. In rodent validation cohorts, detecting both IgM and IgG antibody responses against a subset of the identified proteins could discriminate pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. IgG and IgM autoantibodies, specific for a subset of the identified antigens, could discriminate the samples of women who eventually developed breast cancer from case-matched controls who did not develop disease. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if plasma samples were collected greater than 5 months prior to a breast cancer diagnosis (AUC 0.68; CI 0.565-0.787, p = 0.0025). CONCLUSION: Genetically engineered mouse models of cancer may provide a facile discovery tool for identifying autoantibodies useful for human cancer diagnostics

Preservation of tumor-host immune interactions with luciferase-tagged imaging in a murine model of ovarian cancer

BACKGROUND: Ovarian cancer is immunogenic and residual tumor volume after surgery is known to be prognostic. Ovarian cancer often follows a recurring-remitting course and microscopic disease states may present ideal opportunities for immune therapies. We sought to establish the immune profile of a murine model of ovarian cancer that allows in vivo tumor imaging and the quantitation of microscopic disease. RESULTS AND DISCUSSION: Baseline imaging and weight measurements were taken within 1 and 2 weeks after intraperitoneal tumor injection, respectively. Significantly higher photons per second from baseline imaging were first observed 5 weeks after tumor cell injection (p < 0.05) and continued to be significant through 8 weeks after injection (p < 0.01), whereas a significant increase in weight above baseline was not observed until day 56 (p < 0.0001). Expression of luc2 in ID8 cells did not alter the cellular immune microenvironment of the tumor. FOXP3+ T cells were more likely to be detected in the intraepithelial compartment and CD4+ T cells in the stroma as compared to CD3+ T cells, which were found equally in stroma and intraepithelial compartments. CONCLUSIONS: Use of an intraperitoneal tumor expressing a codon-optimized firefly luciferase in an immunocompetent mouse model allows tumor quantitation in vivo and detection of microscopic tumor burdens. Expression of this foreign protein does not significantly effect tumor engraftment or the immune microenvironment of the ID8 cells in vivo and may allow novel immunotherapies to be assessed in a murine model for their translational potential to ovarian cancers in remission or minimal disease after primary cytoreductive surgery or chemotherapy. METHODS: Mouse ovarian surface epithelial cells from C57BL6 mice transformed after serial passage in vitro were transduced with a lentiviral vector expressing a codon optimized firefly luciferase (luc2). Cell lines were selected and luc2 expression functionally confirmed in vitro. Cell lines were intraperitoneally (IP) implanted in albino C57BL/6/BrdCrHsd-Tyrc mice and albino B6(Cg)-Tyrc-2 J/J mice for serial imaging. D-luciferin substrate was injected IP and tumors were serially imaged in vivo using a Xenogen IVIS. Tumor take, weights, and luminescent intensities were measured. Immunohistochemistry was performed on tumors and assessed for immune infiltrates in stromal and intraepithelial compartments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40425-015-0060-6) contains supplementary material, which is available to authorized users

Abstract 2415: Evaluation of a DNA vaccine and adjuvant construct for producing anti tumor immunity targeting IGFBP2

Abstract DNA based vaccines offer the advantage of stability, low cost, and longer duration of antigen exposure than protein based vaccines. We sought to evaluate strategies to augment the anti tumor potency. Using three immunogenic peptides from the ovarian cancer antigen, human insulin-like growth factor binding protein-2 (IGFBP2) we created a DNA epitope construct that encodes these three peptides in sequence and vaccinated mice with the epitope vaccine or an admixture of the peptides or plasmids expressing either the full length human or mouse genes. Mice received three vaccinations separated by two weeks followed fourteen days later by a lethal injection of syngeneic MMC tumor cells known to express IGFBP2. The DNA epitope vaccine produced a greater level of protection than any of the other constructs proving it to be the best formulation for inducing protective anti tumor responses. Full length human and mouse IGFBP2 DNA constructs induced a similar level of protection as the peptide based vaccine. This IGFBP2 epitope DNA vaccine was then tested in conjunction with plasmids encoding murine GM-CSF and IL-12 either alone or in combination in an identical vaccination strategy. Each genetic adjuvant either alone or in combination improved the level of protection induced by the IGFBP2 DNA epitope vaccine though no difference in tumor protection was seen between adjuvants used either alone or together. Evaluation of the inoculation site revealed a persistence of CD11c+ dendritic cells and CD3+ T cells fourteen days after final vaccination predominantly in mice who received plasmids containing the cytokines versus those who received soluble protein. These data support the use of epitope based DNA vaccines in conjunction with genetic adjuvants for the production of anti tumor efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2415.</jats:p

Abstract 1806: TLR2 agonist PSK activates NK cells and enhances the anti-tumor effect of HER2-targeted monoclonal antibody therapy

Abstract Purpose: The therapeutic effect of trastuzumab, the anti-HER2 monoclonal antibody (mAb), is dependent on functional NK cells which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). Novel agents that enhance NK cell function could potentially improve the therapeutic effect of trastuzumab. We recently identified polysaccharide krestin (PSK), a natural product extracted from medicinal mushroom, as a novel TLR2 agonist. The current study was undertaken to evaluate the effect of PSK on human NK cells and the potential of PSK to enhance HER2-targeted mAb therapy. Experimental Design: Human PBMC were stimulated with PSK to evaluate the effect of PSK on NK cell activation and IFN-g secretion. K 562 was used as a target to measure the lytic function of NK cells. Effect of PSK on ADCC was also measured using trastuzumab-coated SKBR3 and MDA-MB-231 breast cancer cells. Finally, in vivo experiment in neu transgenic mice was carried out to determine the potential of PSK to augmented HER2-targeted mAb therapy. Results: PSK upregulated the expression of activation markers, CD25 and CD69, on NK cells, and stimulated IFN-g production by NK cells. The cytolytic potential of NK cells, as shown by CD107a degranulation and lysis of K562, was also augmented by PSK treatment. PSK enhanced trastuzumab-mediated ADCC in lysing both SKBR3 and MDA-MB-231 breast cancer cells. In vivo treatment in neu transgenic mice showed that oral administration of PSK significantly potentiated the anti-tumor effect of anti-rat neu/HER2 mAb therapy. Conclusion: These results demonstrated that natural product PSK activates human NK cells and potentiates trastuzumab-mediated ADCC. Concurrent treatment of PSK and trastuzumab may be a novel way to augment the anti-tumor effect of trastuzumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2011-1806</jats:p

The role of TLR2 in the immunostimulatory effect of Polysaccharide krestin (PSK) (41.13)

Abstract Polysaccharide krestin (PSK) is a mushroom extract that has long been used in Asia as an anti-cancer drug, although the mechanism of action is unclear. Using neu-transgenic (neu-tg) mice, a model of human HER2 positive breast cancer, we investigated the anti-tumor effect of PSK and its potential mechanisms. We found that oral administration of PSK significantly inhibited the growth of both implanted and spontaneous breast tumors in these mice. In vitro PSK treatment stimulated splenocytes proliferation and secretion of TNFalpha. PSK activated dendritic cells (DC) from neu-tg mice as shown by increased expression of CD86, CD80, and MHCII. The expression of toll-like receptor 2 (TLR2) on DC was also increased. To study the potential activating effect of PSK on TLRs, we used HEK293 cells transfected with different TLRs and incubated them with PSK (0.5 to 1500ug/ml, 16 hour). PSK activated NFkB in HEK cells transfected with TLR2 in a dose-dependent manner, but had no effect on HEK transfected with other TLRs (4, 7, or 8). To further investigate the role of TLR2 in mediating the immunostimulatory effect of PSK, we used splenocytes from TLR2 knockout (k/o) mice and WT mice and treated them with PSK (25 to 400ug/ml, 72 hour). PSK stimulated the secretion of TNFalpha in splenocytes from WT mice dose-dependently but not in splenocytes from k/o mice. These results suggest that TLR2 is critical in mediating the immunostimulatory effect of PSK.</jats:p

Abstract 750: Identification of tumor antigens in transgenic mouse model as vaccine targets for the prevention of human breast cancer

Abstract The development of a vaccine to prevent breast cancer would be facilitated if tumor antigens were identified which were associated with the initiation of the malignant transformation. Transgenic mouse models have been proven to demonstrate a similar antigenic repertoire as that seen in breast cancer patients, therefore, we propose to use these models to identify tumor antigens as vaccine targets for breast cancer prevention. We have chosen two mouse models: TgMMTV-neu and TgC3(1)-Tag for study. TgMMTV-neu mouse has a genotype similar to luminal breast cancer and the TgC3(1)-Tag model reflects a basal phenotype of breast cancer. We identified potential vaccine candidate antigens in one of two ways; (1) determining which proteins expressed by the cancer are immunogenic very early in the malignant process by screening pre-diagnostic sera for immunogenicity against tumor cDNA libraries, and (2) injecting parental animals with a syngeneic tumor cell line resulting in “tumor rejection” which we have shown previously is mediated by T cells. To date, we have identified nine pre-diagnostic tumor antigens in TgMMTV-neu mouse; Rpl5, TNFaip3/A20, Pdhx, Otud6b, Stk39, Zfp238, Dnajc10, Lgals8 and Vps35. Moreover, we have identified four rejection antigens in TgC3(1)-Tag mouse; Ddx21, Sfrs11, Edh1 and Tdg. We have initiated vaccination experiments to examine the anti-tumor effect of vaccination with plasmids encoding these antigens. We found that vaccination targeting Ddx21, Dnajc10, and Pdhx mediated tumor growth inhibition in implant models compared with a control group (p&amp;lt;0.01). Our preliminary studies also demonstrate that several of the proteins are immunogenic in humans and we have identified putative promiscuous class II epitopes suitable for screening human PBMC for T cells specific to these proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2011-750</jats:p

Abstract 747: Tumor vaccine adjuvant effect of TLR7 agonist Imiquimod is limited by inducing immusuppressive cells

Abstract Toll-like receptor (TLR) agonists are immune stimulators and have been studied as potential vaccine adjuvants with great interest. We evaluated the adjuvant effects of the TLR7 agonist imiquimod as compared to GM-CSF, which is widely used as a vaccine adjuvant in clinical trials. We found that both topical imiquimod and intradermal GM-CSF effectively induced the accumulation and activation of dentritic cells in draining lymph nodes and equally enhanced OVA specific CD4+ and CD8+ T cell responses in an OVA-tg mouse model. We further evaluated the efficacy of tumor prevention of imiquimod and GM-CSF in a neu-transgenic (neu-tg) mouse model of human breast cancer which over-expresses both neu and IGFBP2 tumor associated antigens. The neu-tg mice were immunized with a vaccine composed of three immunogenic IGFBP2 peptides prior to being inoculated with syngeneic MMC tumor cells. The tumor growth was significantly inhibited in the groups which received GM-CSF as an adjuvant, but not in imiquimod-treated groups. We questioned whether imiquimod induces immune suppression in response to tumor associated antigens and assessed the frequencies of myeloid-derived suppressive cells (MDSC) and T regulatory cells (Treg) in the splenocytes after three immunizations. We found that both CD11c+GR1+ MDSC and CD4+FOXP3+Treg cells were significantly increased in imiquimod-treated mice, but did not increase in GM-CSF-treated group. Serum levels of IL-10 were also significantly increased in imiquimod-treated, but not in GM-CSF-treated group. IFN-gamma secretion in response to IGFBP2 antigen, on the contrary, was remarkably enhanced in the GM-CSF-treated mice, but not in the imiquimod-treated mice as tested by an IFN-gamma ELISPOT assay. Thus, the TLR7 agonist imiquimod, as a vaccine adjuvant did not achieve a protective effect on breast cancer growth. The limited anti-tumor effect observed is associated with elevated levels of immunosuppressive MDSC and Treg cells. These results may have significant implications in the future development of imiquimod as a vaccine adjuvant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 747. doi:10.1158/1538-7445.AM2011-747</jats:p

Abstract 2401: IGF-IR peptide vaccine inhibits tumor growth via IFN-g-dependent biologic remodeling of the tumor

Abstract The insulin-like growth factor (IGF) pathway plays an important role in breast cancer growth and metastasis. The majority of breast tumors overexpress the IGF-I receptor (IGF-IR). Increased IGF-IR signaling in breast cancer is correlated with a poor prognosis and is associated with disease that is resistant to chemotherapy, radiotherapy, and biologic therapy. Thus, therapeutically targeting the IGF-IR signaling pathway is a promising approach to treat breast cancer. We have determined that the IGF-IR is immunogenic in breast cancer. Furthermore, vaccination with MHCII IGF-IR-specific peptides p384-398, p575-589, p951-965, and p1122-1136 induces a CD4+ T cell-dependent anti-tumor immune response in Neu-transgenic mice. Preliminary data suggests that IGF-IR specific immunization results in both decreased proliferation and increased apoptosis of tumor cells. Antigen specific CD4+ T cells secrete IFN-g and we have demonstrated that the inhibition of tumor growth with the IGF-IR vaccine is dependent on IFN-g. We questioned the mechanism whereby IFN-g-secreting CD4+ T cells targeting IGF-IR could inhibit tumor growth. Studies suggest that tumor-associated IGF-IR activation is decreased in tumors from vaccinated mice, with concomitant upregulation in SOCS-3, which has been shown to be modulated by IFN-g. In addition, IFN-g stimulation of the tumor cell line derived from the Neu-transgenic mouse decreased IGF-IR activation and induced SOCS-3 expression. Thus, active immunization targeting IGF-IR may induce both immunologic and biologic effects resulting in inhibition of breast cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2401.</jats:p