Rodriguez-Ruiz, M.E. (María Esperanza)
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- Cancer immunotherapy with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies requires Batf3-dependent dendritic cells(American Association for Cancer Research, 2016) Jure-Kunkel, M. (María); Sancho, D. (David); Sanchez-Paulete, A.R. (Alfonso R.); Rodriguez-Ruiz, M.E. (María Esperanza); Cueto, F.J. (Francisco J.); Quetglas, J.I. (José Ignacio); Morales-Kastresana, A. (Aizea); Aznar, M.A. (María Ángela); Azpilicueta, A. (Arantza); Melero, I. (Ignacio); Martinez-Lopez, M. (María); Labiano, S. (Sara)Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory mAbs targeting PD-1 or CD137. Using Batf3−/− mice, which are defective for cross-presentation of cell-associated antigens, we show that BATF3-dependent dendritic cells (DC) are essential for the response to therapy with anti-CD137 or anti–PD-1 mAbs. Batf3−/− mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFLT3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti–CD137- and anti–PD-1–mediated immunostimulation in tumor therapy against B16-ovalbumin–derived melanomas, whereas this function was lost in Batf3−/− mice. These experiments show that cross-priming of tumor antigens by FLT3L- and BATF3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects.
- Immune mechanisms mediating abscopal effects in radioimmunotherapy(Elsevier, 2019) Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Leaman, O. (Olwen); Aristu-Mendioroz, J.J. (José Javier); Montero, Á. (Ángel); Calvo, F.M. (Felipe Manuel); Melero, I. (Ignacio); Conde, A.J. (Antonio José); Lopez-Campos, J.L. (José Luis)Radiotherapy of cancer has been traditionally considered as a local therapy without noticeable effects outside the irradiated fields. However, ionizing radiation exerts multiple biological effects on both malignant and stromal cells that account for a complex spectrum of mechanisms beyond simple termination of cancer cells. In the era of immunotherapy, interest in radiation-induced inflammation and cell death has considerably risen, since these mechanisms lead to profound changes in the systemic immune response against cancer antigens. Immuno- therapies such as immunomodulatory monoclonal antibodies (anti-PD-1, anti-CTLA-4, anti-CD137, anti-OX40, anti-CD40, anti-TGFβ), TLR-agonists, and adoptive T-cell therapy have been synergistically combined with radio- therapy in mouse models. Importantly, radiation and immunotherapy combinations do not only act against the irradiated tumor but also against distant non-irradiated metastases (abscopal effects). A series of clinical trials are exploring the beneficial effects of radioimmunotherapy combinations. The concepts of crosspriming of tumor neoantigens and immunogenic cell death are key elements underlying this combination efficacy. Proinflamatory changes in the vasculature of the irradiated lesions and in the cellular composition of the leukocyte infiltrates in the tumor microenvironment contribute to raise or dampen cancer immunogenicity. It should be stressed that not all effects of radiotherapy favor antitumor immunity as there are counterbalancing mechanisms such as TGFβ, and VEGFs that inhibit the efficacy of the antitumor immune response, hence offering additional therapeu- tic targets to suppress. All in all, radiotherapy and immunotherapy are compatible and often synergistic approaches against cancer that jointly target irradiated and non-irradiated malignant lesions in the same patient.
- Thymidylate synthase polymorphisms in genomic DNA as clinical outcome predictors in a European population of advanced non-small cell lung cancer patients receiving pemetrexed(BioMed Central, 2014) Gil-Bazo, I. (Ignacio); Patiño-García, A. (Ana); Arevalo, E. (Estefanía); Rodriguez-Ruiz, M.E. (María Esperanza); Castañón-Álvarez, E. (Eduardo); Santisteban, M. (Marta); Rolfo, C. (Christian); Zubiri, L. (Leire); Lopez, I. (Inés); Salgado, J. (Josefa); Martin, P. (Patricia); Collado, V. (Víctor)BACKGROUND: We studied whether thymidylate synthase (TS) genotype has an independent prognostic/predictive impact on a European population of advanced non-small cell lung cancer (NSCLC) patients receiving pemetrexed. METHODS: Twenty-five patients treated with pemetrexed-based regimens were included. Genomic DNA was isolated prior to treatment. The variable number of tandem repeat (VNTR) polymorphisms, the G > C single nucleotide polymorphisms (SNP) and the TS 6-bp insertion/deletion (6/6) in the 3' untranslated region (UTR) polymorphisms were analyzed and correlated with overall response rate (ORR), progression-free survival (PFS), overall-survival (OS) and toxicity. RESULTS: The genotype +6/+6 predicted a higher ORR among active/former smokers compared to +6/-6 genotype (100% vs. 50%; p = 0.085). Overall, the 3R/3R genotype predicted a higher ORR (100%) over the rest VNTR polymorphisms (p = 0.055). The presence of 3R/3R genotype significantly correlated with a superior ORR in patients without EGFR activating mutations (100%) compared to 2R/2R, 2R/3R and 3R/4R genotype (77.8%, 33.3% and 0% respectively; p = 0.017). After a median follow-up of 21 months, a trend towards a better PFS, although not significant, was found among subjects showing 3R/3R polymorphisms (p = 0.089). A significantly superior OS was found in patients showing 3R/3R genotype rather than other VNTR polymorphisms (p = 0.019). No significant correlation with the toxicity was observed. CONCLUSION: In our series, 3R/3R polymorphism correlated with a superior OS. Also, this polymorphism, when associated to wild type EGFR, was related to a higher ORR to pemetrexed. Toxicity was not significantly correlated with a specific TS genotype.
- Nivolumab and urelumab enhance antitumor activity of human T lymphocytes engrafted in Rag2-/-IL2Rγnull immunodeficient mice(AACR, 2015) Mazzolini, G. (Guillermo); Jure-Kunkel, M. (María); Alfaro, C. (Carlos); Azpilikueta, A. (Arantza); Perez-Gracia, J.L. (Jose Luis); Martin-Algarra, S. (Salvador); Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Hidalgo, M. (Manuel); Morales-Kastresana, A. (Aizea); Sarno, F. (Francesca); Oñate, C. (Carmen); Melero, I. (Ignacio); Labiano, S. (Sara); Schalper, K.A. (Kurt A.); Korman, A.J. (Alan J.); Fernandez-Sanmamed, M. (Miguel)A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.
- CXCR1 and CXCR2 chemokine receptor agonists produced by tumors induce neutrophil extracellular traps that interfere with immune cytotoxicity(Elsevier, 2020) Berraondo, P. (Pedro); Cirella, A. (Assunta); Etxeberria, I. (Iñaki); Alfaro, C. (Carlos); Azpilikueta, A. (Arantza); Miguéliz-Basterra, I. (Itziar); Perez-Gracia, J.L. (Jose Luis); Rouzaut, A. (Ana); Gato-Cañas, M. (María); Rodriguez-Ruiz, M.E. (María Esperanza); Pizzol, M. (Maria) de; Carleton, M. (Michael); Garasa, S. (Saray); Otano, I. (Itziar); Ponz-Sarvise, M. (Mariano); Andueza, M.P. (Maria P.); Nieto, C.P. (Celia P.); Ochoa, M.C. (María Carmen); Allegretti, M. (Marcello); Mellado, M. (Mario); Melero, I. (Ignacio); Andrea, C.E. (Carlos Eduardo) de; Resano, L. (Leyre); Schalper, K.A. (Kurt A.); Fernandez-Sanmamed, M. (Miguel); Teijeira, A. (Álvaro)Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
- Intratumoral BO-112 in combination with radiotherapy synergizes to achieve CD8 T-cell-mediated local tumor control(2023) Escuin-Ordinas, H. (Helena); Perez-Gracia, J.L. (Jose Luis); Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Arbea-Moreno, L. (Leire); Garate-Soraluze, E. (Eneko); Serrano-Mendioroz, I. (Irantzu); Quintero, M. (Marisol); Melero, I. (Ignacio); Diaz-Pascual, V. (Victor); Barrio-Alonso, C. (Celia); Álvarez, M. (Maite); Sánchez-Mateos, P. (Paloma); Fernandez-Sanmamed, M. (Miguel)BackgroundRadioimmunotherapy combines irradiation of tumor lesions with immunotherapy to achieve local and abscopal control of cancer. Most immunotherapy agents are given systemically, but strategies for delivering immunotherapy locally are under clinical scrutiny to maximize efficacy and avoid toxicity. Local immunotherapy, by injecting various pathogen-associated molecular patterns, has shown efficacy both preclinically and clinically. BO-112 is a viral mimetic based on nanoplexed double-stranded RNA (poly I:C) which exerts immune-mediated antitumor effects in mice and humans on intratumoral delivery. BO-112 and focal irradiation were used to make the proof-of-concept for local immunotherapy plus radiation therapy combinations.MethodsMurine transplantable tumor cell lines (TS/A, MC38 and B16-OVA) were used to show increased immunogenic features under irradiation, as well as in bilateral tumor models in which only one of the lesions was irradiated or/and injected with BO-112. Flow cytometry and multiplex tissue immunofluorescence were used to determine the effects on antitumor immunity. Depletions of immune cell populations and knockout mice for the IFNAR and BATF-3 genes were used to delineate the immune system requirements for efficacy.ResultsIn cultures of TS/A breast cancer cells, the combination of irradiation and BO-112 showed more prominent features of immunogenic tumor cell death in terms of calreticulin exposure. Injection of BO-112 into the tumor lesion receiving radiation achieved excellent control of the treated tumor and modest delays in contralateral tumor progression. Local effects were associated with more prominent infiltrates of antitumor cytotoxic tumor lymphocytes (CTLs). Importantly, local irradiation plus BO-112 in one of the tumor lesions that enhanced the therapeutic effects of radiotherapy on distant irradiated lesions that were not injected with BO-112. Hence, this beneficial effect of local irradiation plus BO-112 on a tumor lesion enhanced the therapeutic response to radiotherapy on distant non-injected lesions.ConclusionThis study demonstrates that local BO-112 immunotherapy and focal irradiation may act in synergy to achieve local tumor control. Irradiation plus BO-112 in one of the tumor lesions enhanced the therapeutic effects on distant irradiated lesions that were not injected with BO-112, suggesting strategies to treat oligometastatic patients with lesions susceptible to radiotherapy and with at least one tumor accessible for repeated BO-112 intratumoral injections.
- PSA reactivity in extracellular microvesicles to commercial immunoassays(2023) Alegre-Martinez, E. (Estibaliz); Perez-Gracia, J.L. (Jose Luis); Rodriguez-Ruiz, M.E. (María Esperanza); Gonzalez-Hernandez, A. (Alvaro); Sandúa-Condado, A. (Amaia); Gurpide, A. (Alfonso); Fernandez-Sanmamed, M. (Miguel); Ancizu-Marckert, J. (Javier)Aims: Characterization of PSA in extracellular microvesicles (EVs) and its reactivity to commercial methods. Materials and methods: EVs derived from serum of 47 prostate cancer (PCa) patients, 27 benign prostatic hyperplasia (BPH) patients and 42 healthy controls were analyzed. EVs isolation and quantification of PSA immunoreactive to total (ev-T-PSA) or free (ev-F-PSA) PSA immunoassays, were performed using commercial assays. PSA in CD81+ or CD63+ EVs was determined directly in serum by an immunocapture-ELISA (IC-ELISA). Results: Ev-T-PSA immunoreactive to Elecsys assay was detected in all samples. Median T-PSA ev/srm ratio was 2.20 % (Q1-Q3: 0.80-4.00 %), although in some samples this ratio reached 59 %. T-PSA ev/srm ratio was higher in those samples with serum T-PSA below 4 µg/L than in those exceeding that cut-off (p < 0.001). T-PSA ev/srm ratio was lower in PCa patients compared to healthy controls and BPH patients (p < 0.001). Elecsys immunoassays detected higher concentrations of ev-T-PSA and ev-F-PSA than Immulite (p < 0.001). PSA was detected by IC-ELISA more intensely in CD81+ EVs than in CD63+ EVs, and ev-T-PSA correlated with PSA+ CD63+ (p < 0.001) but not with PSA+ CD81+. Conclusion: EVs-bound PSA is another form of circulating PSA whose measurement could be easily performed in clinical laboratories by automated immunoassays.
- Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8(Wiley, 2021) Sainz, C. (Cristina); Lozano, M.D. (María Dolores); Perez-Gracia, J.L. (Jose Luis); Rodriguez-Ruiz, M.E. (María Esperanza); Eguren-Santamaría, I. (Iñaki); Ortiz-de-Solorzano, C. (Carlos); Garasa, S. (Saray); Villalba-Esparza, M. (María); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Abengozar-Muela, M. (Marta); Echeveste, J.I. (José I.); Andrea, C.E. (Carlos Eduardo) de; Schalper, K.A. (Kurt A.); Fernandez-Sanmamed, M. (Miguel); Sánchez-Gregorio, S. (Sandra); Teijeira, A. (Álvaro); Ariz, M. (Mikel)Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8+ T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8+ T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8+ T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8+ tumour-infiltrating lymphocytes.
- Intercellular adhesion molecule-1 and vascular cell adhesion molecule are induced by ionizing radiation on lymphatic endothelium(Elsevier, 2016) Etxeberria, I. (Iñaki); Rouzaut, A. (Ana); Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Aristu-Mendioroz, J.J. (José Javier); Barbes-Fernandez, B. (Benigno); Garasa, S. (Saray); Yanguas, A. (Alba); Halin, C. (Cornelia); Melero, I. (Ignacio); Solórzano-Rendón, J.L. (José Luis); Teijeira, A. (Álvaro)Purpose/Objectives The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Materials/Methods Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGFβ in this process anti-TGFβ blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGFβ and /or anti-ICAM1 blocking mAb. Results We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGFβ. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Conclusion Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy.
- Combined immunotherapy encompassing intratumoral poly-ICLC, dendritic-cell vaccination and radiotherapy in advanced cancer patients(Elsevier, 2018) Berraondo, P. (Pedro); Gil-Bazo, I. (Ignacio); Salazar, A.M. (A. M.); Alfaro, C. (Carlos); Perez-Gracia, J.L. (Jose Luis); Martin-Algarra, S. (Salvador); Rodriguez, I. (Inmaculada); Rodriguez-Ruiz, M.E. (María Esperanza); Lopez-Diaz-de-Cerio, A. (Ascensión); Barbes-Fernandez, B. (Benigno); Oñate, C. (Carmen); Inoges, S. (Susana); Gurpide, A. (Alfonso); Ponz-Sarvise, M. (Mariano); Melero, I. (Ignacio); Benito, A. (Amparo); Perez, G. (Guiomar); Andrea, C.E. (Carlos Eduardo) de; Resano, L. (Leyre); Fernandez-Sanmamed, M. (Miguel)Background: Combination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methods: In this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24 h with poly-ICLC (Hiltonol), TNF-a and IFN-a. On days þ8 and þ10 of each cycle, patients received intratumoral image-guided 0.25 mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600 mg/m2 was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre- and posttreatment PBMC from patients achieving durable stable disease (SD) were studied by IFNc ELISPOT-assays responding to tumor-lysate loaded DC and by TCRb sequencing. Results: Combined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABRþ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-b and IFN-a mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1b concentrations, especially in patients presenting SD. IFNc-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. Conclusions: This radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy.