Exosomal proteomic signatures correlate with drug resistance and carboplatin treatment outcome in a spontaneous model of canine osteosarcoma.

BACKGROUND: Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. METHODS: In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. RESULTS: Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of > 300 days, and dogs with < 100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient's exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. CONCLUSIONS: The findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

Exosomes from Osteosarcoma and normal osteoblast differ in proteomic cargo and immunomodulatory effects on T cells.

BACKGROUND: Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant-derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells. RESULTS: Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF-ß, α fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree. CONCLUSIONS: Osteosarcoma-derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast-derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype.

Mutations in PIK3CA sensitize breast cancer cells to physiologic levels of aspirin.

A review of the literature finds that women diagnosed with breast cancer, who were on an aspirin regimen, experienced a decreased risk of distant metastases and death. Several recent studies have reported an improvement in overall survival in colorectal cancer patients who harbored mutations in the oncogene PIK3CA and received a daily aspirin regimen. Breast cancer patients on a daily aspirin regimen experienced decreased risk of distant metastases and death. PIK3CA is the most frequently mutated oncogene in breast cancer, occurring in up to 45 % of all breast cancers. In order to determine if mutations in PIK3CA sensitized breast cancers to aspirin treatment, we employed the use of isogenic cellular clones of the non-tumorigenic, breast epithelial cell line MCF-10A that harbored mutations in either PIK3CA or KRAS or both. We report that mutations in both PIK3CA and KRAS are required for the greatest aspirin sensitivity in breast cancer, and that the GSK3ß protein was hyperphosphorylated in aspirin-treated double knockin cells, but not in other clones/treatments. A more modest effect was observed with single mutant PIK3CA, but not KRAS alone. These observations were further confirmed in a panel of breast cancer cell lines. Our findings provide the first evidence that mutations in PIK3CA sensitize breast cancer cells to aspirin.

Caloric restriction maintains OX40 agonist-mediated tumor immunity and CD4 T cell priming during aging.

Immune responses wane during aging, posing challenges to the potential effectiveness of cancer immunotherapies. We previously demonstrated that in the context of a promising immunotherapeutic, OX40 agonist (αOX40), older animals exhibited impaired anti-tumor immune responses and diminished CD4 T cell effector differentiation. In this study, we hypothesized that tumor immune responses could be maintained during aging through caloric restriction (CR) or dietary supplementation with resveratrol (RES), a CR mimetic. Mice were placed on either a calorically restricted diet or a RES-formulated diet starting between 4 and 6 months of age and continued until mice reached 12 months of age. Tumor immune responses were assessed after challenging with either sarcoma or breast tumor cells followed by αOX40 treatment. Our results show that CR, but not RES, maintained OX40-mediated anti-tumor immunity. In addition, CR fully sustained antigen-specific CD4 T cell priming in aged hosts (12 months old), whereas tumor-specific CD8 T cell priming was not fully maintained compared to young reference animals (2 months old). Thus, CR appears to maintain immunological fitness of the CD4 T cell priming environment during aging, which is critical for optimal OX40-mediated responses.

Interleukin-2 alters distribution of CD144 (VE-cadherin) in endothelial cells.

BACKGROUND: High-dose IL-2 (HDIL2) is approved for the treatment of metastatic melanoma and renal cell carcinoma, but its use is limited in part by toxicity related to the development of vascular leak syndrome (VLS). Therefore, an understanding of the mechanisms that underlie the initiation and progression of HDIL2-induced increases in endothelial cell (EC) permeability leading to VLS are of clinical importance. METHODS: We established a novel ex vivo approach utilizing primary human pulmonary microvascular ECs to evaluate EC barrier dysfunction in response to IL-2. RESULTS: Complementary in vitro studies using exogenous IL-2 and ex vivo studies using serum from patients treated with IL-2 demonstrate that HDIL2 induces VLS through CD144 (vascular endothelial (VE)-cadherin) redistribution. CONCLUSIONS: These findings provide new insight into how IL-2 induces VLS and identifies VE-cadherin as a potential target for preventing IL-2-related VLS.

Potential Promises and Perils of Human Biological Treatments for Immunotherapy in Veterinary Oncology.

The emergence of immunotherapy for the treatment of human cancers has heralded a new era in oncology, one that is making its way into the veterinary clinic. As the immune system of many animal species commonly seen by veterinarians is similar to humans, there is great hope for the translation of human therapies into veterinary oncology. The simplest approach for veterinarians would be to adopt existing reagents that have been developed for human medicine, due to the potential of reduced cost and the time it takes to develop a new drug. However, this strategy may not always prove to be effective and safe with regard to certain drug platforms. Here, we review current therapeutic strategies that could exploit human reagents in veterinary medicine and also those therapies which may prove detrimental when human-specific biological molecules are used in veterinary oncology. In keeping with a One Health framework, we also discuss the potential use of single-domain antibodies (sdAbs) derived from camelid species (also known as Nanobodies™) for therapies targeting multiple veterinary animal patients without the need for species-specific reformulation. Such reagents would not only benefit the health of our veterinary species but could also guide human medicine by studying the effects of outbred animals that develop spontaneous tumors, a more relevant model of human diseases compared to traditional laboratory rodent models.

OX40 engagement stabilizes Mxd4 and Mnt protein levels in antigen-stimulated T cells leading to an increase in cell survival.

OX40 engagement on activated T cells leads to increased proliferation, expansion and survival of Ag-specific T cells. Direct ex vivo examination of Ag-stimulated murine T cells show that the Myc antagonists, Mxd4 and Mnt, are transiently upregulated and translocated to the nucleus following OX40 engagement and may be involved in suppressing cell death. Both Mxd4 and Mnt are upregulated following OX40 stimulation through increased protein stability and we identify a critical phosphorylation site in Mxd4 that controls Mxd4 stability. The upregulation of Mxd4 and Mnt contributes to OX40-mediated T-cell survival because siRNA knockdown of Mxd4 and Mnt led to increased cell death. We hypothesize the upregulation of c-Myc following OX40 engagement drives T-cell proliferation and that upregulation of Mxd4 and Mnt suppresses Myc-dependent cell death. Thus, Mxd4 and Mnt upregulation following OX40 engagement most likely increases T-cell survival.

Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins.

A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.

OX40-enhanced tumor rejection and effector T cell differentiation decreases with age.

OX40 agonists have potent immunotherapeutic effects against a variety of murine tumors, yet it is unclear the role that age-related immune senescence plays on their efficacy. We found that middle-aged and elderly tumor-bearing mice (12 and 20 mo old, respectively) treated with anti-OX40 were less responsive compared with young mice 6 mo or less of age. Decreased tumor-free survival was observed in both male and female mice, and was not due to changes in the surface expression of OX40 on T cells in older animals. Enumeration of cytokine-producing effector T cells in tumor-bearing mice revealed a significant decline in these cells in the older mice treated with anti-OX40 compared with their younger counterparts. The decrease of this critical T cell population in middle-aged mice was not a result of inherent T cell deficiencies, but was revealed to be T cell extrinsic. Finally, combining IL-12, an innate cytokine, with anti-OX40 boosted levels of differentiated effector T cells in the older anti-OX40-treated mice and partially restored the defective antitumor responses in the middle-aged mice. Our data show that the anti-OX40-enhancement of tumor immunity and effector T cell numbers is decreased in middle-aged mice and was partially reversed by coadministration of the proinflammatory cytokine IL-12.

Cutting Edge: OX40 agonists can drive regulatory T cell expansion if the cytokine milieu is right.

We report that OX40 stimulation drives all lineages of CD4 T cell development, including regulatory T cells (Tregs), and the plasticity of the response is dependant on local cytokines. In TGF-beta1-treated cultures, an OX40 agonist increased IFN-gamma and IL-4 production and diverted T cells from the Treg lineage. However, cytokine blockade in the context of OX40 stimulation promoted enhanced Treg accumulation. This observation was evident in naive mice, as OX40 engagement enhanced Treg proliferation and accumulation in vivo. Lastly, OX40 agonist administration influenced experimental autoimmune encephalomyelitis disease severity in opposing directions, depending on the timing of administration. Given during Ag priming, the OX40 agonist drove Treg expansion and inhibited disease, whereas given later it enhanced T cell effector cytokine production in the CNS and exacerbated disease. Hence, OX40 signaling can augment the accumulation of all CD4 T cell lineages; however, its accentuation of immune responses may have vastly different biologic outcomes depending upon the local cytokine milieu.

Fasting reduces the incidence of vincristine-associated adverse events in dogs.

Fasting has been shown to decrease chemotherapy-associated adverse events (AEs), in part through insulin-like growth factor (IGF-1) reduction, and may induce a protective effect on normal cells during chemotherapy treatment in mice and people. The purpose of this study was to evaluate the effect of fasting on constitutional, bone marrow and gastrointestinal (GI) AEs, and serum glucose, IGF-1 and insulin levels in dogs receiving vincristine. The study was a prospective, crossover clinical trial in tumour-bearing dogs. Dogs were randomized to be fasted for 24 to 28 hours prior to and 6 hours following their first or second vincristine treatment, and fed normally for the alternate dose. A significant reduction in nausea, anorexia, lethargy and serum insulin was observed when dogs were fasted; however, no significant differences were found in other GI symptoms, neutrophil count, serum glucose or IGF-1. Fasting prior to vincristine therapy is a safe and effective treatment modality that helped mitigate constitutional and GI AEs in tumour-bearing dogs.

The role of OX40-mediated co-stimulation in T-cell activation and survival.

The extent of T-cell activation, proliferation, and survival that follows T-cell receptor (TCR) ligation is controlled by several factors, including the strength of TCR stimulation, the availability of prosurvival cytokines, and the presence or absence of co-stimulatory signals. In addition to engagement of the CD28 co-stimulatory receptor by its natural ligands, B7.1 (CD80) and B7.2 (CD86), recent work has begun to elucidate the mechanisms by which signaling through the OX40 (CD134) co-stimulatory receptor, a member of the tumor necrosis factor receptor (TNFR) superfamily, affects T-cell responses. Importantly, OX40 ligation has been shown to augment CD4 and CD8 T-cell clonal expansion, effector differentiation, survival, and in some cases, abrogate the suppressive activity of regulatory FoxP3+CD25+CD4+ T cells. In this review, we focus on the mechanisms regulating OX40 expression on activated T cells as well as the role of OX40-mediated co-stimulation in boosting T-cell clonal expansion, effector differentiation, and survival.

The effect of aging on OX40 agonist-mediated cancer immunotherapy.

Agents that enhance T cell co-stimulatory signaling have emerged as promising cancer immunotherapies. Our laboratory has been evaluating the TNF receptor co-stimulatory molecule, OX40, which has the capacity to augment critical aspects of T cell function and induce tumor regression in animal models. Effective stimulation of OX40 expressing T cells was accomplished with agonist antibodies to OX40 that were eventually translated into a clinical trial for cancer patients. A recent attempt to assess the affect of immune senescence on OX40 therapy, revealed a dramatic loss of efficacy of the agonist therapy in older tumor-bearing mice. The deficiency in OX40-enhanced anti-tumor responses in older mice correlated with a decrease in the number of differentiated effector T cells. Further investigation suggests that the underlying age-related decline in the agonist OX40-mediated T cell responses was not inherent to the T cells themselves, but related to the host environment. Thus, effective use of immunotherapies based on T cell co-stimulatory molecules may require additional modifications, such as immune stimulants to increase innate immunity, to address age-related defects that reside outside of the T cell and within the host environment.

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.

Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines.

In patients with malignancy, the major barrier to achieving complete response is emergence of resistance to current chemotherapeutic agents. One of the major mechanisms by which tumour cells become resistant to therapies is by altering cellular drug targets through mutations and/or deletions. Resistance by this mechanism is achieved more easily if the drug has limited cellular targets and/or processes. We hypothesized that as Pseudomonas aeruginosa exotoxin T (ExoT) targets six proteins that are required for cancer cell survival and proliferation, it is highly unlikely for cancer cells to develop resistance to this toxin. We assessed ExoT's cytotoxicity against multiple invasive and highly resistant tumour cell lines in order to evaluate its potential as a chemotherapeutic agent. Our data demonstrated that ExoT induced potent cytotoxicity in all tumour cell lines that we examined. Collectively, our data highlighted the potential of ExoT as a possible chemotherapeutic candidate for the treatment of cancer.

NK cells and CD8+ T cells cooperate to improve therapeutic responses in melanoma treated with interleukin-2 (IL-2) and CTLA-4 blockade.

BACKGROUND: Melanoma is one of the few types of cancer with an increasing annual incidence. While a number of immunotherapies for melanoma have been associated with significant clinical benefit, including high-dose IL-2 and cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade, clinical response to either of these single agents has been limited to 11-20% of treated patients. Therefore, in this study, we sought to test the hypothesis that the combination of IL-2 and CTLA-4 blockade could mediate a more profound therapeutic response. METHODS: Here, B6 mice were challenged with poorly immunogenic B16 melanoma on day 0, and treated with CTLA-4 blocking antibody (100 µg/mouse) on days 3, 6, and 9, and IL-2 (100,000 units) twice daily on days 4-8, or both. RESULTS: A highly significant synergistic effect that delayed tumor growth and prolonged survival was demonstrated with the combination immunotherapy compared to either monotherapy alone. The therapeutic effect of combination immunotherapy was dependent on both CD8+ T and NK cells and co-depletion of these subsets (but not either one alone) abrogated the therapeutic effect. CTLA-4 blockade increased immune cell infiltration (including CD8+ T cells and NK cells) in the tumor and IL-2 reduced the proportion of highly differentiated/exhausted tumor-infiltrating NK cells. CONCLUSIONS: These results have implications for the design of clinical trials in patients with metastatic melanoma and provide new insights into how the immune system may be mediating anti-tumor activity with combination IL-2 and CTLA-4 blockade in melanoma.

Current status of granulocyte-macrophage colony-stimulating factor in the immunotherapy of melanoma.

In 2012, it was estimated that 9180 people in the United States would die from melanoma and that more than 76,000 new cases would be diagnosed. Surgical resection is effective for early-stage melanoma, but outcomes are poor for patients with advanced disease. Expression of tumor-associated antigens by melanoma cells makes the disease a promising candidate for immunotherapy. The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) has a variety of effects on the immune system including activation of T cells and maturation of dendritic cells, as well as an ability to promote humoral and cell-mediated responses. Given its immunobiology, there has been interest in strategies incorporating GM-CSF in the treatment of melanoma. Preclinical studies with GM-CSF have suggested that it has antitumor activity against melanoma and can enhance the activity of anti-melanoma vaccines. Numerous clinical studies have evaluated recombinant GM-CSF as a monotherapy, as adjuvant with or without cancer vaccines, or in combination with chemotherapy. Although there have been suggestions of clinical benefit in some studies, results have been inconsistent. More recently, novel approaches incorporating GM-CSF in the treatment of melanoma have been evaluated. These have included oncolytic immunotherapy with the GM-CSF-expressing engineered herpes simplex virus talimogene laherparepvec and administration of GM-CSF in combination with ipilimumab, both of which have improved patient outcomes in phase 3 studies. This review describes the diverse body of preclinical and clinical evidence regarding use of GM-CSF in the treatment of melanoma.

Early-onset age-related changes in dendritic cell subsets can impair antigen-specific T helper 1 (Th1) CD4 T cell priming.

Decline in CD4 T cell immune responses is associated with aging. Although a number of immunological defects have been identified in elderly mice (>18 months old), a key early-onset immune defect at middle age could be a driver or contributor to defective CD4 T cell responses. Our studies demonstrate that age-related alterations in DC subsets within the priming environment of middle-aged mice (12 months old) correlate with and can directly contribute to decreases in antigen-specific CD4 T cell Th1 differentiation, which measured by T-bet and IFN-γ expression, was decreased significantly in T cells following VSV infection or s.c. immunization with a protein antigen in the context of immune stimulation via OX40. The deficient Th1 phenotype, observed following protein antigen challenge, was found to be the result of an age-related decrease in an inflammatory DC subset (CD11b+ Gr-1/Ly6C+) in the dLN that corresponded with T cell dysfunction. In the virus model, we observed significant changes in two DC subsets: mDCs and pDCs. Thus, different, early age-related changes in the DC profile in the priming environment can significantly contribute to impaired Th1 differentiation, depending on the type of immunological challenge.

Inhibition of p70 S6 kinase (S6K1) activity by A77 1726 and its effect on cell proliferation and cell cycle progress.

Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor-mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 µM, respectively. Exogenous uridine partially blocked A77 1726-induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726-induced cell cycle arrest in the S phase.