ovalbumin and Adenocarcinoma

Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8. CD4

Topics: Adenocarcinoma; Animals; B7-H1 Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; Epitopes, T-Lymphocyte; Glycoproteins; Humans; Immune Checkpoint Inhibitors; Immunotherapy; Melanoma; Mice; Ovalbumin; Programmed Cell Death 1 Receptor; Receptors, Antigen, T-Cell; Tumor Microenvironment; Vaccination

We report an integrated pipeline for efficient serum glycoprotein biomarker candidate discovery and qualification that may be used to facilitate cancer diagnosis and management. The discovery phase used semi-automated lectin magnetic bead array (LeMBA)-coupled tandem mass spectrometry with a dedicated data-housing and analysis pipeline; GlycoSelector (http://glycoselector.di.uq.edu.au). The qualification phase used lectin magnetic bead array-multiple reaction monitoring-mass spectrometry incorporating an interactive web-interface, Shiny mixOmics (http://mixomics-projects.di.uq.edu.au/Shiny), for univariate and multivariate statistical analysis. Relative quantitation was performed by referencing to a spiked-in glycoprotein, chicken ovalbumin. We applied this workflow to identify diagnostic biomarkers for esophageal adenocarcinoma (EAC), a life threatening malignancy with poor prognosis in the advanced setting. EAC develops from metaplastic condition Barrett's esophagus (BE). Currently diagnosis and monitoring of at-risk patients is through endoscopy and biopsy, which is expensive and requires hospital admission. Hence there is a clinical need for a noninvasive diagnostic biomarker of EAC. In total 89 patient samples from healthy controls, and patients with BE or EAC were screened in discovery and qualification stages. Of the 246 glycoforms measured in the qualification stage, 40 glycoforms (as measured by lectin affinity) qualified as candidate serum markers. The top candidate for distinguishing healthy from BE patients' group was Narcissus pseudonarcissus lectin (NPL)-reactive Apolipoprotein B-100 (p value = 0.0231; AUROC = 0.71); BE versus EAC, Aleuria aurantia lectin (AAL)-reactive complement component C9 (p value = 0.0001; AUROC = 0.85); healthy versus EAC, Erythroagglutinin Phaseolus vulgaris (EPHA)-reactive gelsolin (p value = 0.0014; AUROC = 0.80). A panel of 8 glycoforms showed an improved AUROC of 0.94 to discriminate EAC from BE. Two biomarker candidates were independently verified by lectin magnetic bead array-immunoblotting, confirming the validity of the relative quantitation approach. Thus, we have identified candidate biomarkers, which, following large-scale clinical evaluation, can be developed into diagnostic blood tests. A key feature of the pipeline is the potential for rapid translation of the candidate biomarkers to lectin-immunoassays.

Topics: Adenocarcinoma; Aged; Animals; Apolipoprotein B-100; Barrett Esophagus; Biomarkers, Tumor; Calibration; Case-Control Studies; Chickens; Complement C9; Diagnosis, Differential; Esophageal Neoplasms; Female; Gelsolin; Glycoproteins; Humans; Male; Middle Aged; Ovalbumin; Plant Lectins; Protein Array Analysis; Reference Standards; Tandem Mass Spectrometry

Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model.. Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8(+) T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively.. Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8(+) T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8(+) T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination.. Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy.

Topics: Adenocarcinoma; Animals; Antibody Specificity; Antimetabolites, Antineoplastic; B-Lymphocytes; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Combined Modality Therapy; Dendritic Cells; Deoxycytidine; Drug Screening Assays, Antitumor; Enzyme-Linked Immunosorbent Assay; Female; Gemcitabine; Immunity, Cellular; Immunity, Humoral; Immunosuppression Therapy; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Pancreatic Neoplasms; Peptide Fragments; Tumor Escape

Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin's action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.

Topics: Adenocarcinoma; Amino Acid Motifs; Amino Acid Sequence; Breast Neoplasms; Catalytic Domain; Cell Line, Tumor; Female; Humans; In Vitro Techniques; Models, Molecular; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Proteins; Ovalbumin; Proteasome Endopeptidase Complex; Protein Conformation; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Serpins; Transgenes; Ubiquitination

Vaccines based on immune stimulatory complexes (ISCOM) induce T-cell responses against tumor antigen (Ag). However, immune responses are impaired in pancreatic cancer patients. We investigated the efficacy of an ISCOM vaccine in a murine pancreatic carcinoma model. Panc02 cells expressing OVA as a model Ag were induced subcutaneously or orthotopically in the pancreas of C57BL/6 mice. Treatment consisted of an OVA containing ISCOM vaccine, either used alone or in combination with the TLR9 agonist CpG. The ISCOM vaccine effectively induced Ag-specific CTL capable of killing tumor cells. However, in mice with established tumors CTL induction by the vaccine was inefficient and did not affect tumor growth. Lack of efficacy correlated with increased numbers of Treg. Depletion of Treg with anti-CD25 mAb restored CTL induction and prolonged survival. Adding low-dose CpG to the ISCOM vaccine reduced Treg numbers, enhanced CTL responses and induced regression of pancreatic tumors in a CD8(+) T cell-dependent manner. Mice cured from the primary tumor mounted a memory T-cell response against wild-type Panc02 tumors, indicative of epitope spreading. Combining ISCOM vaccines with TLR agonists is a promising strategy for breaking tumor immune evasion and deserves further evaluation for the treatment of pancreatic carcinoma.

Topics: Adenocarcinoma; Animals; Antigen-Antibody Complex; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Immune Evasion; Immunization; Immunotherapy; Lymphatic Metastasis; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Ovalbumin; Pancreatic Neoplasms; Survival Rate; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Toll-Like Receptor 9; Tumor Cells, Cultured

Neoantigens derived from somatic mutations in tumors may provide a critical link between the adaptive immune system and cancer. Here, we describe a system to introduce exogenous antigens into genetically engineered mouse lung cancers to mimic tumor neoantigens. We show that endogenous T cells respond to and infiltrate tumors, significantly delaying malignant progression. Despite continued antigen expression, T cell infiltration does not persist and tumors ultimately escape immune attack. Transplantation of cell lines derived from these lung tumors or prophylactic vaccination against the autochthonous tumors, however, results in rapid tumor eradication or selection of tumors that lose antigen expression. These results provide insight into the dynamic nature of the immune response to naturally arising tumors.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adoptive Transfer; Animals; Antigen Presentation; Antigens, Neoplasm; Antigens, Surface; Apoptosis; Apoptosis Regulatory Proteins; B-Lymphocytes; Cancer Vaccines; CD3 Complex; CD8-Positive T-Lymphocytes; Cell Count; Cell Proliferation; Cytokines; Genetic Vectors; Graft Rejection; Histocompatibility Antigens Class I; Humans; Immunity, Cellular; Interleukin-7 Receptor alpha Subunit; Kaplan-Meier Estimate; Lentivirus; Lung; Lung Neoplasms; Lymph Nodes; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Oligopeptides; Ovalbumin; Peptide Fragments; Programmed Cell Death 1 Receptor; T-Lymphocytes; Time Factors; Transplantation, Isogeneic

Foxp3 is a central control element in the development and function of regulatory T cells (Treg), and mice expressing a diphtheria toxin (DT) receptor-enhanced green fluorescent protein fusion protein under the control of the foxp3 gene locus (DEREG mice) allow conditional and efficient depletion of Foxp3(+) Treg by DT injection. Herein, we use DEREG mice and a mouse model of carcinogenesis to show that conditional and effective Treg depletion can both protect mice from carcinogenesis by innate control, yet permanently eradicate a proportion of de novo-established tumors in mice in a largely CD8(+) T-cell- and IFN-γ-dependent manner. Tumors displayed a heterogeneous response to Treg depletion, and suppression of established tumors was accompanied by an increase in the tumor-infiltrating CD8(+) T-cell/B-cell ratio. Tumor rejection occurred in the absence of overt autoimmunity, suggesting that effective transient Treg depletion strategies may be therapeutic in at least a proportion of spontaneous tumors developing in the host.

Topics: Adenocarcinoma; Animals; B-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Division; Colonic Neoplasms; Diphtheria Toxin; Forkhead Transcription Factors; Genes, Reporter; Green Fluorescent Proteins; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Lymphocyte Depletion; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Ovalbumin; T-Lymphocytes, Regulatory

Heat shock proteins (HSP) convey both chaperoned propeptide and danger signal to dendritic cells (DC). However, few studies have compared the two activities. Using a murine inducible hsp70 secreted by cells distinct from those providing the tumor antigens, we showed that hsp70 exerts efficacious adjuvant effects toward DC cross-priming. Hsp70 induces DC maturation and phagocytosis of cellular debris both in vitro and in vivo, which are conducive to CTL response to chaperoned and nonchaperoned antigens. Whereas the ability of hsp70 to induce cross-presentation of chaperoned peptides is natural killer (NK) independent, the adjuvant activity requires NK cells at the site of DC-hsp70 interaction to induce CTL response and therapeutic effect against lung metastases. However, although bystander activity provides equal CTL induction, the best therapeutic efficacy rests on cell vaccine secreting hsp70 that combines chaperoned antigen and danger signal within the same cell.

Topics: Adenocarcinoma; Adjuvants, Immunologic; Animals; Antigen Presentation; Cell Communication; Colonic Neoplasms; Dendritic Cells; Female; HSP70 Heat-Shock Proteins; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; T-Lymphocytes, Cytotoxic

We hypothesized that ovarian tumors without oviductal involvement would not express the oviductal protein ovalbumin, the major protein found in the magnum of the hen's oviduct.. On the basis of gross visual examination, tissues samples were removed from hens determined to have ovarian tumors and were processed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Ovarian tumors and other peritoneal lesions were evaluated histologically. Paraffin sections of ovarian and oviductal tissue were deparaffinized and evaluated for the protein expression of ovalbumin, proliferating cell nuclear antigen (PCNA), and progesterone receptor (PR).. Hens with ovarian adenocarcinoma without (n = 10) or with (n = 6) oviductal involvement were positive for ovalbumin in the ovary. Ovary sections from normal hens (n = 9) were negative, and oviductal sections from normal hens (n = 3) were positive for ovalbumin. Expression of PCNA protein was abundant in all ovarian tumors (16 of 16). Oviductal epithelial cells strongly expressed PCNA protein. Expression of PR was observed in 9 of 14 ovarian tumors.. The presence of ovalbumin in ovarian tumors in the absence of any oviductal involvement suggests that ovarian tumors dedifferentiate during the disease process and thereby resemble serous-type ovarian tumors in women.

Topics: Adenocarcinoma; Animals; Chickens; Cystadenocarcinoma, Serous; Disease Models, Animal; Female; Humans; Ovalbumin; Ovarian Neoplasms; Oviducts; Paraffin Embedding; Proliferating Cell Nuclear Antigen; Receptors, Progesterone

This study was undertaken to test the effect of immunization against luteinizing hormone-releasing hormone (LHRH) fusion proteins on the development and progression of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Two LHRH fusion proteins, ovalbumin with seven LHRH peptides (OV-LHRH-7), and thioredoxin with seven LHRH peptides (TH-LHRH-7) were used in a cocktail vaccine. Two groups of male TRAMP mice were immunized with the cocktail. Primary immunizations were at either 4 or 8 weeks of age. LHRH immunized mice (n=19) were compared with castrated (n=19) and intact mice (n=18) for testosterone concentration, tumor weight, and lifespan. Immunization against LHRH in the TRAMP mice resulted in significant production of antibodies to LHRH compared with surgically castrated and intact control mice. Testicular weight was significantly reduced in the LHRH immunized groups compared with intact control mice. Serum testosterone was reduced (P<0.05) in the immunized mice compared with intact control mice and was not different from that of castrated mice (P>0.05). Tumor weight was variable and inconsistent throughout all treatment groups. Lifespan was not increased by immunization against LHRH or castration. Intact control mice (lived the longest (227+/-11 days), whereas immunized mice lived 206+/-11 days and castrated mice lived 213+/-13 days. Tumors from immunized TRAMP mice appeared more aggressive than tumors of castrated and intact mice, as demonstrated by 35% expression of gross lung tumors in the immunized mice whereas none were observed in the castrated or intact TRAMP mice. Prostate cancer is initially dependent upon androgens for growth and development, but cells have the ability to escape androgen dependence and progress to an androgen independent state, which was evident in this study. The TRAMP mouse model immunized against LHRH may have utility in future studies and treatments of the androgen independent prostate cancer.

Topics: Adenocarcinoma; Animals; Disease Models, Animal; Disease Progression; Gonadotropin-Releasing Hormone; Immunization; Male; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Orchiectomy; Organ Size; Ovalbumin; Prostatic Neoplasms; Recombinant Fusion Proteins; Testis; Testosterone; Thioredoxins

Most murine lung tumors are composed of differentiated epithelial cells. We have reported previously that surfactant protein (SP)-D is expressed in urethane-induced tumors. Serum levels of SP-D are increased in patients with interstitial lung disease and acute respiratory distress syndrome and in rats with acute lung injury but have not been measured in mice. In this study, we sought to determine whether SP-D could be detected in murine serum and discovered that it was increased in mice bearing lung tumors. Serum SP-D concentration was 5.0 +/- 0.2 ng/ml in normal C57BL/6 mice, essentially absent in SP-D nulls, and 63.6 +/- 9.0 ng/ml in SP-D-overexpressing mice. SP-D in serum was verified by immunoblotting. Serum SP-D was increased in mice bearing tumors induced by three different protocols, and the SP-D level correlated with tumor volume. However, in mice with a single adenoma or a few adenomas, SP-D levels were usually within the normal range. SP-D was expressed by the tumor cells, and there was also a field effect whereby type II cells near the tumor expressed more SP-D than type II cells in the remainder of the lung. Serum SP-D was also increased by lung inflammation. In airway inflammation induced by aerosolized ovalbumin in sensitized BALB/c mice, the serum levels were elevated after challenge. In conclusion, serum SP-D concentration is increased in mice bearing lung tumors and generally reflects the tumor burden but is also elevated during lung inflammation.

Topics: Adenocarcinoma; Animals; Carcinogens; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, ras; Lung Neoplasms; Male; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Transgenic; Ovalbumin; Pulmonary Surfactant-Associated Protein D; Rats; Transforming Growth Factor beta; Urethane

Trp, Phe, and Tyr ethyl esters and their dipeptides with Gly at the C-terminals inhibited ovalbumin (OVA) permeation through Caco-2 monolayers. The inhibitory activity of Trp ethyl ester was the highest at near the concentration of 10(-6) M. It was suggested that Trp ethyl ester inhibited transcellular permeation of OVA.

Topics: Adenocarcinoma; Amino Acids, Aromatic; Biological Transport; Cell Line, Tumor; Colonic Neoplasms; Esters; Humans; Intestinal Mucosa; Kinetics; Ovalbumin; Peptides

A combined method has been developed for selective cytolysis in vitro as well as in vivo using a photosensitizer haematoporphyrin-protein conjugate as the targeting molecule and low-power He-Ne laser (632.8 nm) irradiation in order to activate the sensitizer to its excited, toxic triplet energy state. The specificity of the procedure was demonstrated in vitro by purging a mixed cell population from one component, and in vivo in an animal (nude mice) xenograft tumour model, where human cancer cells were destroyed by the immunotargeting method using monoclonal-antibody-haematoporphyrin (mAb-HP) conjugate (a-PNAr-I mAbs, which bind to the cell surface antigens of gastric cancer cells) and soft laser irradiation. The cell destruction was dependent on the doses of both mAb-HP and He-Ne laser light energy, and occurred only in target cell populations.

Topics: Adenocarcinoma; Animals; Hematoporphyrin Photoradiation; Humans; Hybridomas; Immunotoxins; Lasers; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Ovalbumin; Receptors, Mitogen; Stomach Neoplasms

Uridine diphosphogalactose:glycoprotein galactosyltransferases were examined in human lung adenocarcinoma and squamous cell carcinoma. The galactosyltransferase activities in tissue homogenates from both carcinomas were higher than in adjacent normal control with asialoagalactofetuin as a substrate. This activity in adenocarcinoma (27 cases) was two times higher than that in squamous cell carcinoma (19 cases) with statistical significance (p less than 0.001). Using Triton-solubilized enzymes from a particulate fraction, similar differences in the activity were observed with ovalbumin, asialoagalactofetuin, and its beta-eliminated derivative as acceptors but not with bovine submaxillary mucin. These observations mean that the higher activity of galactosyltransferase(s) in lung carcinomas (especially in adenocarcinoma) is mainly responsible for galactosylation of carbohydrate chains in N-glycoside-type but not O-glycoside-type glycoproteins.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Galactosyltransferases; Gangliosides; Glycoproteins; Humans; Lung Neoplasms; Mucins; Ovalbumin; Substrate Specificity