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

Antiproliferative effects of masitinib and imatinib against canine oral fibrosarcoma in vitro

Background: Canine oral fibrosarcoma (COF) is one of the most common oral tumors in dogs and carries a guarded prognosis due to a lack of effective systemic therapeutic options. Mastinib and imatinib are two commonly used tyrosine kinase inhibitors (TKIs) in veterinary oncology but their potential efficacy against COF is uncharacterized. To begin investigating the rationale for use of these TKIs against COF, the present study tested for the presence TKI targets PDGFR-α, PDGFR-β, Kit, and VEGFR-2 and examined the in vitro effects on cell viability after TKI treatment alone or with doxorubicin. Immunohistochemistry for PDGFR-α, PDGFR-β, Kit, and VEGFR-2 was performed in 6 COF tumor biopsies. Presence of these same receptors within 2 COF cell lines was probed by reverse transcription-polymerase chain reaction and, for those with mRNA detected, confirmed via western blot. Effects on cell viability were assessed using an MTS assay after masitinib or imatinib treatment alone (0-100 μM), or in combination with doxorubicin (0-3000 nM doxorubicin). Anti-PDGFRB siRNA knockdown was performed and the effect on cell viability quantified. Results: Expression of the TKI targets evaluated was similar between the 2 COF cell lines and the 6 COF tumor biopsies: PDGFR-α and PDGFR-β were detected in neoplastic cells from most COF tumor biopsies (5/6 and 6/6, respectively) and were present in both COF cell lines; KIT and KDR were not detected in any sample. Masitinib and imatinib IC50 values ranged from 7.9–33.4 μM, depending on the specific TKI and cell line tested. The addition of doxorubicin resulted in synergistic cytotoxicity with both TKIs. Anti-PDGFRB siRNA transfection reduced PDGFR-β protein expression by 77 % and 67 % and reduced cell viability by 24 % (p < 0.0001) and 28 % (0 = 0.0003) in the two cell lines, respectively. Conclusions: These results provide rationale for further investigation into the use of TKIs, possibly in combination with doxorubicin, as treatment options for COF.Keywords: Masitinib, Oral fibrosarcoma, Imatinib, Platelet-derived growth factor receptor, Do

Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors.

Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103+CD39+ tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103+CD39+ CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103+CD39+ CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103+CD39+ CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies

Autocrine production of reproductive axis neuropeptides affects proliferation of canine osteosarcoma in vitro

Abstract Background Osteosarcoma strikes hundreds of people each year, of both advanced and younger ages, and is often terminal. Like many tumor types, these bone tumors will frequently undergo a neuroendocrine transition, utilizing autocrine and/or paracrine hormones as growth factors and/or promoters of angiogenesis to facilitate progression and metastasis. While many of these factors and their actions on tumor growth are characterized, some tumor-derived neuropeptides remain unexplored. Methods Using validated canine osteosarcoma cell lines in vitro, as well as cells derived from spontaneous tumors in dogs, we explored the autocrine production of two neuropeptides typically found in the hypothalamus, and most closely associated with reproduction: gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss-1). We evaluated gene expression and protein secretion of these hormones using quantitative RT-PCR and a sensitive radioimmunoassay, and explored changes in cell proliferation determined by MTS cell viability assays. Results Our current studies reveal that several canine osteosarcoma cell lines (COS, POS, HMPOS, D17, C4) synthesize and secrete GnRH and express the GnRH receptor, while COS and POS also express kiss1 and its cognate receptor. We have further found that GnRH and kisspeptin, exogenously applied to these tumor cells, exert significant effects on both gene expression and proliferation. Of particular interest, kisspeptin exposure stimulated GnRH secretion from COS, similarly to the functional relationship observed within the neuroendocrine reproductive axis. Additionally, GnRH and kisspeptin treatment both increased COS proliferation, which additionally manifested in increased expression of the bone remodeling ligand rankl within these cells. These effects were blocked by treatment with a specific GnRH receptor inhibitor. Both neuropeptides were found to increase expression of the specific serotonin (5HT) receptor htr2a, the activation of which has previously been associated with cellular proliferation, suggesting that production of these factors by osteosarcoma cells may act to sensitize tumors to circulating 5HT of local and/or enteric origin. Conclusions Here we report that kisspeptin and GnRH act as autocrine growth factors in canine osteosarcoma cells in vitro, modulating RANKL and serotonin receptor expression in a manner consistent with pro-proliferative effects. Pharmacological targeting of these hormones may represent new avenues of osteosarcoma treatment

A Preliminary Proteomic Investigation of Circulating Exosomes and Discovery of Biomarkers Associated with the Progression of Osteosarcoma in a Clinical Model of Spontaneous Disease

Circulating cancer exosomes are microvesicles which originate from malignant cells and other organs influenced by the disease and can be found in blood. The exosomal proteomic cargo can often be traced to the cells from which they originated, reflecting the physiological status of these cells. The similarities between cancer exosomes and the tumor cells they originate from exhibit the potential of these vesicles as an invaluable target for liquid biopsies. Exosomes were isolated from the serum of eight osteosarcoma-bearing dogs, five healthy dogs, and five dogs with traumatic fractures. We also characterized exosomes which were collected longitudinally from patients with osteosarcoma prior and 2 weeks after amputation, and eventually upon detection of lung metastasis. Exosomal proteins fraction were analyzed by label-free mass spectrometry proteomics and were validated with immunoblots of selected proteins. Ten exosomal proteins were found that collectively discriminate serum of osteosarcoma patients from serum healthy or fractured dogs with an accuracy of 85%. Additionally, serum from different disease stages could be distinguished with an accuracy of 77% based on exosomal proteomic composition. The most discriminating protein changes for both sample group comparisons were related to complement regulation, suggesting an immune evasion mechanism in early stages of osteosarcoma as well as in advanced disease