Suchergebnisse

Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. ; This work was supported by grants from Fondo de Investigación Sanitaria [PI11/00298] [PS09/00181]; from the Spanish Ministry of Economy and Competitivity (MINECO) [SAF2012-32293], and from the Regional Government of Madrid [grant number S2010/BMD-2326 INMUNOTHERCAN]; EG-C was a recipient from a fellowship from Fundación La Caixa and received the Immunotools IT-special-Award 2014; SL-C was a recipient from a fellowship from the Spanish Ministry of Education, partially supported by CSIC [grant 201020E086 (to MV-G)] and received the Immunotools IT-BOX-139 award 2012; GR-C was a recipient of a JAE-predoc fellowship from CSIC; RC-C was supported by a BBSRC studentship; MR was funded by a postdoctoral fellowship from the Spanish Cancer Foundation (AECC). ; Peer reviewed ; Peer Reviewed

Abstract Background Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. Methods Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. Results Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. Conclusions These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques. ; This work has been supported by Grants from Madrid Regional Government [IMMUNOTHERCAN-CM (S2010/BMD-2326)] and the Spanish Ministry of Economy [SAF2015-69169-R (MINEICO/FEDER) and MAT2017-84959-C2-1-R.; the Network of Excellence for Research in Exosomes, Rediex (MINEICO/FEDER); the Consejería de Economía y Empleo del Principado de Asturias (Plan de Ciencia, Tecnología e Innovación 2013-2017), under the Grant GRUPIN14-022. SL-C was a recipient of a FPU fellowship (MECD), and a travel fellowship from the Spanish Group of Extracellular Vesicles (GEIVEX); CC-S was a recipient of a master's fellowship from "Postgrado de la Fundación Ramón Areces-UAM"

Background: Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. Methods: Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. Results: Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. Conclusions: These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques ; This work has been supported by Grants from Madrid Regional Government [IMMUNOTHERCAN-CM (S2010/BMD-2326)] and the Spanish Ministry of Economy [SAF2015-69169-R (MINEICO/FEDER) and MAT2017-84959-C2-1-R.; the Network of Excellence for Research in Exosomes, Rediex (MINEICO/FEDER); the Consejería de Economía y Empleo del Principado de Asturias (Plan de Ciencia, Tecnología e Innovación 2013-2017), under the Grant GRUPIN14-022. SL-C was a recipient of a FPU fellowship (MECD), and a travel fellowship from the Spanish Group of Extracellular Vesicles (GEIVEX); CC-S was a recipient of a master's fellowship from "Postgrado de la Fundación Ramón Areces-UAM".

This work has been supported by Grants from Madrid Regional Government [IMMUNOTHERCAN-CM (S2010/BMD-2326)] and the Spanish Ministry of Economy [SAF2015-69169-R (MINEICO/FEDER) and MAT2017-84959-C2-1-R.; the Network of Excellence for Research in Exosomes, Rediex (MINEICO/FEDER); the Consejería de Economía y Empleo del Principado de Asturias (Plan de Ciencia, Tecnología e Innovación 2013-2017), under the Grant GRUPIN14-022. SL-C was a recipient of a FPU fellowship (MECD), and a travel fellowship from the Spanish Group of Extracellular Vesicles (GEIVEX); CC-S was a recipient of a master's fellowship from "Postgrado de la Fundación Ramón Areces-UAM".

Immunotherapy, via intra-vesical instillations of BCG, is the therapy of choice for patients with high-risk non-muscle invasive bladder cancer. The subsequent recruitment of lymphocytes and myeloid cells, as well as the release of cytokines and chemokines, is believed to induce a local immune response that eliminates these tumors, but the detailed mechanisms of action of this therapy are not well understood. Here, we have studied the phenotype and function of the responding lymphocyte populations as well as the spectrum of cytokines and chemokines produced in an in vitro model of human peripheral blood mononuclear cells (PBMCs) co-cultured with BCG. Natural killer (NK) cell activation was a prominent feature of this immune response and we have studied the expansion of this lymphocyte population in detail. We show that, after BCG stimulation, CD56dim NK cells proliferate, upregulate CD56, but maintain the expression of CD16 and the ability to mediate ADCC. CD56bright NK cells also contribute to this expansion by increasing CD16 and KIR expression. These unconventional CD56bright cells efficiently degranulated against bladder cancer cells and the expansion of this population required the release of soluble factors by other immune cells in the context of BCG. Consistent with these in vitro data, a small, but significant increase in the intensity of CD16 expression was noted in peripheral blood CD56bright cells from bladder cancer patients undergoing BCG therapy, that was not observed in patients treated with mitomycin-C instillations. These observations suggest that activation of NK cells may be an important component of the anti-tumoral immune response triggered by BCG therapy in bladder cancer. ; This work was supported by grants from Madrid Regional Government "INMUNOTHERCAN"[S2010/BMD-2326 (LMP, MVG)]; the Spanish Ministries of Economy and Health [SAF-2012–32293, SAF2015–69169-R(MVG) and SAF2014–58752-R (HTR)]; EMGC and SLC are recipients of Fellowships from La Caixa and Spanish Ministry of Education (FPU),respectively. ; Peer reviewed

Exosomes are cell-secreted nanovesicles (40–200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×105 exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition. ; Funding from projects CTQ2013-47396-R, SAF2012-32293, SAF2014-58752-R, BFU2014-55478-R (Spanish Ministry of Economy and Competitivity), FC15-GRUPIN14-022 (Regional Government of Asturias) and S2010/BMD-2326-INMUNOTHERCAN (Regional Government of Madrid) is acknowledged. M. Oliveira-Rodríguez thanks FICYT for her pre-doctoral grant. SLC is a recipient of an FPU fellowship from the Spanish Ministry of Education.

© 2019 by the authors. ; [Background]: Intra-vesical instillation of Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is an effective therapy for high-grade non-muscle invasive bladder cancer (NMIBC), which provokes a local immune response resulting in 70% of patients free of relapse after three years. Because non-responder patients usually have a bad prognosis, the early identification of treatment failure is crucial. We hypothesized that, if an effective immune response was taking place in the bladder, soluble factors would be released to the urine many days after BCG instillations. ; [Methods]: An extensive panel of cytokines and chemokines released into the urine seven days after every BCG instillation was screened in a cohort of NMIBC patients over three years. ; [Results]: The determinations of the urinary concentrations of cytokines, chemokines, and creatinine showed that increasing concentrations of C-X-C motif chemokine 10 (CXCL10) also known as interferon-inducible protein 10 (IP10) could be detected during the six-week induction cycle of BCG-treated patients released into the urine by CD14+ cells. In vitro, CXCL10 facilitated the recruitment of effector immune cells after the BCG-mediated upregulation of CXCR3 in both T- and natural killer (NK)-cells. Conclusions: The high concentrations of chemokine detected one week after the encounter with mycobacteria suggest that the CXCL10 axis might be related to the intensity of the immune anti-tumor response. ; This research was funded by the Madrid Regional Government (S2010/BMD-2326 IMMUNOTHERCAN-CM (B2017/BMD-3733)/FEDER), and the Spanish Ministries of Economy and Health (SAF2015-69169-R, RTC-2017-6379-1, RTI2018-093569-B-I00 (MINECO/FEDER)); E.M.G.C. and S.L.C. were the recipients of fellowships from the La Caixa and Spanish Ministry of Education (FPU), respectively; C.S. was an Erasmus-visiting student from Sorbonne University. The APC was partially funded by CSIC. ; Peer reviewed