12 research outputs found
Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts
BACKGROUND: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. RESULTS: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. CONCLUSIONS: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis
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A Multiplex Biomarker Approach for the Diagnosis of Transitional Cell Carcinoma from Canine Urine
Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.Keywords: Liquid chromatography, Biomarkers, Tandem mass spectrometry, Canine, Transitional cell carcinom
Correction: Cell death induced by cytotoxic CD8+ T cells is immunogenic and primes caspase-3–dependent spread immunity against endogenous tumor antigens
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BrachaShayVetMedMultiplexBiomarkerApproach_Figures1-5.zip
Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.Keywords: Transitional cell carcinoma, Liquid chromatography, Tandem mass spectrometry, Canine, BiomarkersKeywords: Transitional cell carcinoma, Liquid chromatography, Tandem mass spectrometry, Canine, Biomarker
NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8+ T cell responses capable of curing multi-focal cancer
Background High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8+ T cell and natural killer cell stimulation compared with IL-2.Methods Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray.Results We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8+ T cell responses and increased trafficking of these T cells to both irradiated and distant, unirradiated, tumors.Conclusions Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors
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BrachaShayVetMedMultiplexBiomarkerApproach.pdf
Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.Keywords: Canine, Biomarkers, Tandem mass spectrometry, Liquid chromatography, Transitional cell carcinomaKeywords: Canine, Biomarkers, Tandem mass spectrometry, Liquid chromatography, Transitional cell carcinom
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Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts
Background: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis
despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for
development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a
cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration,
adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two
validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system
to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally,
we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semiquantitative
immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition
between the OSA and the normal osteoblast cells.
Results: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with
good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene
expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell
lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three
proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed
at much higher levels on the surface of the OSA than the normal osteoblast cell lines.
Conclusions: The results of the present study identified numerous differences, and similarities, in the SEP of canine
OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the
subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be
applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized
differences between samples. While this study yielded data that may prove useful for OSA researchers and
clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a
more thorough SEP analysis.Keywords: Mass spectrometry, Biotinylation, Dog, Proteomics, Osteosarcom
Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice
NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8
BACKGROUND: High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8
METHODS: Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray.
RESULTS: We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8
CONCLUSIONS: Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors