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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 09, 2008 |
| Title |
Immunity to Grb10, a signal transduction molecule, inhibits the growth of breast cancer in mice. |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
This study describes the application of a unique strategy to identify breast cancer antigens (TAA). In a mouse model, the strategy led to the identification of growth factor receptor-bound protein10 (Grb10) as a newly identified TAA. Grb10 is signal transduction molecule associated with multiple transmembrane tyrosine-kinase receptors. It was discovered by comparing microarrays of cellular breast cancer vaccines highly enriched for cells that induced breast cancer immunity in tumor-bearing mice, with non enriched vaccines.
The vaccines were prepared by transferring a cDNA expression library derived from SB5b cells, a breast cancer cell line (C3H/He origin (H-2k), into LM mouse fibroblasts (H-2k). As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide, and is expressed, the vaccine could be prepared from microgram amounts of tumor tissue. Relatively few cells in the transduced cell-population, however, incorporated cDNA-fragments that included genes specifying TAA. (The vast majority specified normal cellular constituents.) A unique strategy was employed, therefore, to enrich the vaccine for immunotherapeutic cells. Twenty genes were over-represented in the enriched vaccines. One, the gene for Grb10, was approximately 100 fold over-represented. To determine if Grb10 in the enriched vaccine was partly responsible for its therapeutic benefits, the gene was transferred into the fibroblast cell line, which was then used as a vaccine. Mice with established breast cancer treated solely by immunization with the modified fibroblasts developed robust immunity to the breast cancer cells, which, in some instances, was sufficient to result in tumor-rejection.
Keywords: cell type comparisons, genetic modifications, transfected cells, fibroblasts, immunity
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| Overall design |
Experimental animals, tumor cell lines and monoclonal antibodies (mAbs). Pathogen-free C3H/He female mice (H-2k) between 10 to 14 weeks old were from the Jackson Laboratory (Bar Harbor, ME). They were maintained according to NIH Guidelines for the Care and Use of Laboratory Animals. SB5b cells were a breast cancer cell line established from an adenocarcinoma that arose spontaneously in the mammary gland of a C3H/He mouse in our animal colony. LM fibroblasts, of C3H/He mouse origin, were from the American Type Culture Collection (ATCC). B16F1 cells, a melanoma cell line of C57BL/6 mouse origin (H-2b), were from the ATCC. Each of the cell types was maintained at 370 in a humidified 7% CO2/air atmosphere in DMEM (Gibco BRL, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum (FBS) and antibiotics (Gibco BRL) (growth medium). mAbs for CD4+, CD8+ and NK1.1 cells and fluoroscein-conjugated mAbs for H-2Kb class I-determinants were from B-D Pharmingen (San Jose, CA).
Modification of LM fibroblasts to secrete IL-2. The fibroblasts were modified to secrete IL-2 before transfection (LM-IL-2 cells) as a means of augmenting their non-specific immunogenic properties, as described previously (30).
Modification of the cytokine-secreting fibroblasts to express H-2Kb-class I-determinants, allogeneic in C3H/He mice. Allogeneic class I-determinants are strong immune adjuvants (32,33). To stimulate up take of the vaccine by dendritic cells of the tumor-bearing host, and to ensure rejection, the fibroblasts (H-2k) were modified to express H-2Kb-determinants, allogeneic in C3H/He mice (LM-IL-2Kb cells), as described previously (34, 35).
Preparation of cDNA libraries from SB5b breast cancer cells. Guanidine isothiocyanate was used to recover total RNA from SB5b cells or from B16F1 melanoma cells. The mRNA, derived from approximately 1 x 107 cells, was isolated using an mRNA isolation system (Promega, Madison, WI). The cDNA-expression libraries were constructed with a Lambda Zap vector using a cDNA library kit (Stratgene, La Jolla, CA). cDNAs greater than 0.5 kb were selected by size fractionation via gel filtration and directionally cloned into a pBK-CMV vector with an EcoRI restriction site at the 5’ end and an XhoI site at the 3’ end. The expression libraries yielded approximately 4 x 105 PFU/μg cDNA with an individual cDNA insert. The size distribution of the cDNA transduced into the modified fibroblasts was 0.5-7.0 kb.
Preparation of the cDNA-based cellular vaccines. LM-IL-2Kb cells were transduced with a cDNA library from SB5b cells, or, for use as a specificity control, with a cDNA library from B16F1 cells, using Lipfectamine 2000 (Invitrogen, Calsbad, CA) to aid cDNA uptake. In brief, 30 μg of cDNA from either of the cell types were mixed with 3 μg pcDNA6/Bla (Invitrogen), a plasmid specifying a gene conferring resistance to blasticidin, used for selection. Afterward, the cDNA/pcDNA6/Bla mixture was added to Lipfectamine 2000 and then to 2.0 x 107 LM-IL-2Kb cells divided into four 100 mm plastic cell culture dishes 24 hours previously. After incubation for 18 hours, the cells were divided into sixteen 100 mm dishes, and incubated for 14 days in fresh growth medium containing 5 ug/ml blasticidin and 500 ug/ml G418. The surviving blasticin/G418-resistant cells (at least 2 x 106 colonies) were pooled and maintained as cell lines for use in the experiments (LM-IL-2Kb/cSB5b and LM-IL-2Kb/B16F1 cells respectively). For use as a control, the same procedure was followed except that the fibroblasts were transduced with pcDNA/Bla alone (LM-IL-2Kb cells).
Mouse interferon gamma (IFN-γ) ELISPOT IFN-γ assays. Mouse ELISPOT IFN-γ assays were used to determine the number of responding T cells in mice immunized with the transduced fibroblasts. The spots were counted by computer-assisted image analysis (ImmunoSpot Series 2 analyzer: Cellular Technology Limited, Cleveland, OH).
51Cr-release cytotoxicity assays. Mononuclear cells from the spleens of C3H/He mice immunized with the cDNA-transduced cells, or with cells transduced with a plasmid encoding the gene specifying Grb10, were isolated by Ficoll-Hypaque density gradient centrifugation. After washing, the cells were co cultured at 370 with mitomycin C-treated (45 min, 50 μg/ml) SB5b cells for 5 days (ratio of spleen cells:SB5b cells = 30:1). Afterward, the population that failed to adhere to the plastic cell culture flasks was collected and used as effector cells for the cytotoxicity determinations. Spleen cell mediated cytotoxicity was determined in a standard 51Cr-release assay, using 51Cr-labeled SB5b cells as targets in the reaction.
The percent specific cytolysis was calculated as:
Experimental 51Cr release - Spontaneous 51Cr release
---------------------------------------------------------------X 100
Maximum 51Cr release - Spontaneous 51Cr release
The spontaneous release of 51Cr was less than 15% of the total release in each instance.
Enrichment of the vaccine for cells that induce immunity to (SB5b) mammary carcinoma cells.
The vaccine was prepared by transfer of a cDNA library derived from SB5b cells into the modified mouse fibroblasts. Since only a small proportion of the transduced cell population was expected to have incorporated cDNAs that included genes specifying tumor antigens (TAA), a unique strategy was used to enrich the vaccine for TAA-positive cells, as described previously (30). In brief, aliquots of the suspension of transduced cells were added to each of ten wells of a 96 well plate. Each pool contained a starting inoculum of 1 x 103 cells. Wells containing higher numbers of TAA-positive cells were detected by comparing the response of C3H/He mice to immunization with cells from the individual pools, as determined by both ELISPOT IFN-γ and 51Cr-release cytotoxicity assays. To obtain a sufficient number of cells for immunization, cells from the individual pools were allowed to increase to approximately 5 x 107, through periodic transfers to larger culture plates and eventually cell culture flasks. An aliquot of each of the expanded cell populations was maintained frozen/viable (for later recovery). The remaining portion was used for immunization. Frozen cells derived from the pool that stimulated immunity to the breast cancer cells to the greatest extent (immunohigh), and, for use as a control, from the pool that induced immunity to SB5b cells to the least extent (immunolow), were recovered, reestablished in culture and subjected to additional rounds of positive or negative immune selection (30). (As an additional control, one pool was not subjected to either positive or negative selection (master pool)). After five rounds of selection, microarrays were used to compare the gene expression profiles of cells in the immunohigh and immunolow pools.
Microarrays of cellular vaccines enriched for transduced fibroblasts that induced immunity to SB5b cells.
cRNA microarrays were used to the compare the gene expression profiles of transduced fibroblasts from the immunohigh and immunolow pools, as described previously (36).
RT-PCR of Grb10, a candidate gene specifying a breast cancer antigen, identified by comparing microarrays of the enriched and non enriched vaccines.
Grb10 was highly over represented in cells from immunohigh pools. RT-PCR was used to determine if the gene was expressed. Approximately 6 x 106 cells from the immunohigh pool in monolayer culture were disrupted and homogenized. 1 volume of 70% ethanol was added before the extracts were loaded onto RNeasy mini columns. RT-PCR was performed on RNA eluted from the column with a one-step RT-PCR kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. 1 ug RNA was mixed with buffer containing 1.25 mM MgCl2, 40uM dNTPs, 0.6 μM of each forward and backward primers and 2 μl of a mixture containing reverse transcriptase and Taq polymerase.
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| Contributor(s) |
O-Sullivan I, Chopra A, Carr J, Kim T, Cohen EP |
| Citation(s) |
18381455 |
| Submission date |
Jan 07, 2008 |
| Last update date |
Mar 19, 2012 |
| Contact name |
Michael Falduto |
| E-mail(s) |
mfalduto@genusbiosystems.com
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| Phone |
847-291-9602
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| Organization name |
GenUs BioSystems, Inc.
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| Street address |
1808 Janke, Unit M
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| City |
Northbrook |
| State/province |
IL |
| ZIP/Postal code |
60062 |
| Country |
USA |
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| Platforms (1) |
| GPL2894 |
GE Healthcare/Amersham Biosciences CodeLink™ UniSet Mouse 20K I Bioarray |
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| Samples (6)
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| GSM254990 |
Breast cancer cell antigen Grb10_non-selected vaccine_rep1 |
| GSM254993 |
Breast cancer cell antigen Grb10_non-selected vaccine_rep2 |
| GSM254996 |
Breast cancer cell antigen Grb10_immuno high vaccine_rep1 |
| GSM255000 |
Breast cancer cell antigen Grb10_immuno high vaccine_rep2 |
| GSM255002 |
Breast cancer cell antigen Grb10_immuno low vaccine_rep1 |
| GSM255005 |
Breast cancer cell antigen Grb10_immuno low vaccine_rep2 |
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| Relations |
| BioProject |
PRJNA108273 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE10095_RAW.tar |
4.5 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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