RESUMO
A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias/tratamento farmacológico , Proclorperazina/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Biópsia , Cetuximab/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Xenoenxertos , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Trastuzumab/farmacologiaRESUMO
Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.
Assuntos
Marcação de Genes/métodos , Genoma Humano/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Reparo Gênico Alvo-Dirigido/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Animais , Antígenos CD34/metabolismo , DNA Complementar/genética , Endonucleases/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Sangue Fetal/transplante , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Mutação/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapiaRESUMO
Inflammation is a critical phase in the healing of skin wounds. Excessive inflammation and inflammatory macrophages are known to cause impaired wound closure and outcome. This prompted us to test the role of IL-23 in IL-17 expression and in modulating wound inflammation and macrophage polarization. Full-thickness wounds (4 × 6 mm) were created on the dorsal surface of multiple genetically modified mouse models. Obese diabetic mouse wounds were treated with anti-IL-17A, anti-IL-23, or isotype-matched antibodies. We found IL-23- but not IL-12-deficient mice displayed significantly reduced IL-17 expression in wounds. This was rescued by delivery of recombinant IL-23. IL-23- and IL-17-deficient mice showed a significant increase in noninflammatory macrophages. Obese diabetic mice treated with anti-IL-17A and anti-IL-23p19 blocking antibodies had significantly improved wound reepithelialization. Similarly, IL-17-/- obese mice had accelerated wound closure, resulting in reduced iNOS expression and inflammatory macrophages while maintaining prohealing CD206 and lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1)-expressing macrophages. This study highlights the importance of the IL-17 pathway in wound closure offering new possibilities of therapeutic intervention in chronic wounds.-Lee, J., Rodero, M. P., Patel, J., Moi, D., Mazzieri, R., Khosrotehrani, K. Interleukin-23 regulates interleukin-17 expression in wounds, and its inhibition accelerates diabetic wound healing through the alteration of macrophage polarization.
Assuntos
Pé Diabético/imunologia , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Cicatrização , Animais , Feminino , Interleucina-17/genética , Interleucina-23/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Brain metastases are the most prevalent of intracranial malignancies. They are associated with a very poor prognosis and near 100% mortality. This has been the case for decades, largely because we lack effective therapeutics to augment surgery and radiotherapy. Notwithstanding improvements in the precision and efficacy of these life-prolonging treatments, with no reliable options for adjunct systemic therapy, brain recurrences are virtually inevitable. The factors limiting intracranial efficacy of existing agents are both physiological and molecular in nature. For example, heterogeneous permeability, abnormal perfusion and high interstitial pressure oppose the conventional convective delivery of circulating drugs, thus new delivery strategies are needed to achieve uniform drug uptake at therapeutic concentrations. Brain metastases are also highly adapted to their microenvironment, with complex cross-talk between the tumor, the stroma and the neural compartments driving speciation and drug resistance. New strategies must account for resistance mechanisms that are frequently engaged in this milieu, such as HER3 and other receptor tyrosine kinases that become induced and activated in the brain microenvironment. Here, we discuss molecular and physiological factors that contribute to the recalcitrance of these tumors, and review emerging therapeutic strategies, including agents targeting the PI3K axis, immunotherapies, nanomedicines and MRI-guided focused ultrasound for externally controlling drug delivery.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/terapia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antineoplásicos/farmacologia , Encéfalo/cirurgia , Neoplasias Encefálicas/imunologia , Quimiorradioterapia Adjuvante , Sistemas de Liberação de Medicamentos , Humanos , Imunoterapia , Terapia de Alvo Molecular , Nanomedicina , Nanopartículas , Resultado do Tratamento , Microambiente TumoralRESUMO
Despite significant advances, most current in vivo models fail to fully recapitulate the biological processes that occur in humans. Here we aimed to develop an advanced humanized model with features of an organ bone by providing different bone tissue cellular compartments including preosteoblasts, mesenchymal stem/stromal (MSCs), endothelial and hematopoietic cells in an engineered microenvironment. The bone compartment was generated by culturing the human MSCs, umbilical vein endothelial cells with gelatin methacryloyl hydrogels in the center of a melt-electrospun polycaprolactone tubular scaffolds, which were seeded with human preosteoblasts. The tissue engineered bone (TEB) was subcutaneously implanted into the NSG mice and formed a morphologically and functionally organ bone. Mice were further humanized through the tail vein injection of human cord blood derived CD34+ cells, which then populated in the mouse bone marrow, spleen and humanized TEB (hTEB). 11 weeks after CD34+ transplantation, metastatic breast cancer cells (MDA-MB-231BO) were orthotopically injected. Cancer cell injection resulted in the formation of a primary tumor and metastasis to the hTEB and mouse organs. Less frequent metastasis and lower tumor burden were observed in hematochimeric mice, suggesting an immune-mediated response against the breast cancer cells. Overall, our results demonstrate the efficacy of tissue engineering approaches to study species-specific cancer-bone interactions. Further studies using genetically modified hematopoietic stem cells and bioengineered microenvironments will enable us to address the specific roles of signaling molecules regulating hematopoietic niches and cancer metastasis in vivo.
Assuntos
Bioengenharia , Neoplasias Ósseas/imunologia , Neoplasias da Mama/imunologia , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/citologia , Sistema Imunitário/imunologia , Transplante de Células-Tronco Mesenquimais , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Ósseas/terapia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Proliferação de Células , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A productive immune response requires transient upregulation of the microRNA miR-155 in hematopoietic cells mediating innate and adaptive immunity. In order to investigate miR-155 in the context of tumor-associated immune responses, we stably knocked down (KD) miR-155 in the myeloid compartment of MMTV-PyMT mice, a mouse model of spontaneous breast carcinogenesis that closely mimics tumor-host interactions seen in humans. Notably, miR-155/KD significantly accelerated tumor growth by impairing classic activation of tumor-associated macrophages (TAMs). This created an imbalance toward a protumoral microenvironment as evidenced by a lower proportion of CD11c(+) TAMs, reduced expression of activation markers, and the skewing of immune cells within the tumor toward an macrophage type 2/T helper 2 response. This study highlights the importance of tumor-infiltrating hematopoietic cells in constraining carcinogenesis and establishes an antitumoral function of a prototypical oncomiR.
Assuntos
Imunidade Inata/genética , Macrófagos/imunologia , Macrófagos/metabolismo , MicroRNAs/genética , Neoplasias/genética , Neoplasias/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ordem dos Genes , Vetores Genéticos , Inflamação/genética , Inflamação/imunologia , Macrófagos/patologia , Camundongos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Carga Tumoral/genética , Carga Tumoral/imunologiaRESUMO
Neoantigen-based cancer vaccines have emerged as a promising immunotherapeutic approach to treat cancer. Nevertheless, the high degree of heterogeneity in tumors poses a significant hurdle for developing a vaccine that targets the therapeutically relevant neoantigens capable of effectively stimulating an immune response as each tumor contains numerous unique putative neoantigens. Understanding the complexities of tumor heterogeneity is crucial for the development of personalized neoantigen-based vaccines, which hold the potential to revolutionize cancer treatment and improve patient outcomes. In this review, we discuss recent advancements in the design of neoantigen-based cancer vaccines emphasizing the identification, validation, formulation, and targeting of neoantigens while addressing the challenges posed by tumor heterogeneity. The review highlights the application of cutting-edge approaches, such as single-cell sequencing and artificial intelligence to identify immunogenic neoantigens, while outlining current limitations and proposing future research directions to develop effective neoantigen-based vaccines.
Assuntos
Vacinas Anticâncer , Neoplasias , Humanos , Vacinas Anticâncer/uso terapêutico , Antígenos de Neoplasias/genética , Inteligência Artificial , Neoplasias/tratamento farmacológico , ImunoterapiaRESUMO
Cellular plasticity in cancer enables adaptation to selective pressures and stress imposed by the tumor microenvironment. This plasticity facilitates the remodeling of cancer cell phenotype and function (such as tumor stemness, metastasis, chemo/radio resistance), and the reprogramming of the surrounding tumor microenvironment to enable immune evasion. Epithelial plasticity is one form of cellular plasticity, which is intrinsically linked with epithelial-mesenchymal transition (EMT). Traditionally, EMT has been regarded as a binary state. Yet, increasing evidence suggests that EMT involves a spectrum of quasi-epithelial and quasi-mesenchymal phenotypes governed by complex interactions between cellular metabolism, transcriptome regulation, and epigenetic mechanisms. Herein, we review the complex cross-talk between the different layers of epithelial plasticity in cancer, encompassing the core layer of transcription factors, their interacting epigenetic modifiers and non-coding RNAs, and the manipulation of cancer immunogenicity in transitioning between epithelial and mesenchymal states. In examining these factors, we provide insights into promising therapeutic avenues and potential anti-cancer targets.
RESUMO
Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4+ and CD8+ T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models.
Assuntos
Vacinas Anticâncer , Neoplasias , Animais , Camundongos , Citometria de Fluxo , Células Matadoras Naturais , Neoplasias/terapia , Fenótipo , Antígenos de NeoplasiasRESUMO
Vaccines have been hailed as one of the most remarkable medical advancements in human history, and their potential for treating cancer by generating or expanding anti-tumor T cells has garnered significant interest in recent years. However, the limited efficacy of therapeutic cancer vaccines in clinical trials can be partially attributed to the inadequacy of current preclinical mouse models in recapitulating the complexities of the human immune system. In this study, we developed two innovative humanized mouse models to assess the immunogenicity and therapeutic effectiveness of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141+ dendritic cells (DCs). Both models were based on the transference of human peripheral blood mononuclear cells (PBMCs) into immunocompromised HLA-A*02-NSG mice (NSG-A2), where the use of fresh PBMCs boosted the engraftment of human cells up to 80%. The dynamics of immune cells in the PBMC-hu-NSG-A2 mice demonstrated that T cells constituted the vast majority of engrafted cells, which progressively expanded over time and retained their responsiveness to ex vivo stimulation. Using the PBMC-hu-NSG-A2 system, we generated a hyperplastic skin graft model expressing the HPV16-E7 oncogene. Remarkably, human cells populated the skin grafts, and upon vaccination with a DNA vaccine encoding an HPV16-E6/E7 protein, rapid rejection targeted to the E7-expressing skin was detected, underscoring the capacity of the model to mount a vaccine-specific response. To overcome the decline in DC numbers observed over time in PBMC-hu-NSG-A2 animals, we augmented the abundance of CD141+ DCs, the specific targets of our tailored nanoemulsions (TNEs), by transferring additional autologous PBMCs pre-treated in vitro with the growth factor Flt3-L. The Flt3-L treatment bolstered CD141+ DC numbers, leading to potent antigen-specific CD4+ and CD8+ T cell responses in vivo, which caused the regression of pre-established triple-negative breast cancer and melanoma tumors following CD141+ DC-targeting TNE vaccination. Notably, using HLA-A*02-matching PBMCs for humanizing NSG-A2 mice resulted in a delayed onset of graft-versus-host disease and enhanced the efficacy of the TNE vaccination compared with the parental NSG strain. In conclusion, we successfully established two humanized mouse models that exhibited strong antigen-specific responses and demonstrated tumor regression following vaccination. These models serve as valuable platforms for assessing the efficacy of therapeutic cancer vaccines targeting HPV16-dysplastic skin and diverse tumor antigens specifically delivered to CD141+ DCs.
Assuntos
Vacinas Anticâncer , Melanoma , Humanos , Animais , Camundongos , Transplante de Pele , Leucócitos Mononucleares , Hiperplasia , Anticorpos , Modelos Animais de Doenças , Antígenos de Neoplasias , Células Dendríticas , Antígenos HLA-ARESUMO
Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.
RESUMO
Transforming growth factor-beta (TGF-beta) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-beta is often released as part of an inactive tripartite complex consisting of TGF-beta, the TGF-beta propeptide, and a molecule of latent TGF-beta binding protein (LTBP). The interaction of TGF-beta and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-beta from this state is crucial for signaling. To examine the contribution of LTBP to TGF-beta function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1(C33S/C33S) mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-beta1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1(-/-) animals. Tgfb1(C33S/C33S) mice exhibited decreased levels of active TGF-beta1, decreased TGF-beta signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-beta complex is important for proper TGF-beta1 function and that Tgfb1(C33S/C33S) mice are hypomorphs for active TGF-beta1. Moreover, although mechanisms exist to activate latent TGF-beta1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.
Assuntos
Inflamação/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Neoplasias/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Inflamação/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células de Langerhans/citologia , Células de Langerhans/metabolismo , Proteínas de Ligação a TGF-beta Latente/genética , Camundongos , Camundongos Knockout , Neoplasias/patologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/genéticaRESUMO
The blood brain barrier (BBB) and blood tumour barrier (BTB) remain a major roadblock for delivering therapies to treat brain cancer. Amongst brain cancers, glioblastoma (GBM) is notoriously difficult to treat due to the challenge of delivering chemotherapeutic drugs across the BBB and into the tumour microenvironment. Consequently, GBM has high rates of tumour recurrence. Currently, limited numbers of chemotherapies are available that can cross the BBB to treat GBM. Nanomedicine is an attractive solution for treating GBM as it can augment drug penetration across the BBB and into the heterogeneous tumour site. However, very few nanomedicines exist that can easily overcome both the BBB and BTB owing to difficulty in synthesizing nanoparticles that meet the small size and surface functionality restrictions. In this study, we have developed for the first-time, a room temperature protocol to synthesise ultra-small size with large pore silica nanoparticles (USLP, size â¼30 nm, pore size >7 nm) with the ability to load high concentrations of chemotherapeutic drugs and conjugate a targeting moiety to their surface. The nanoparticles were conjugated with lactoferrin (>80 kDa), whose receptors are overexpressed by both the BBB and GBM, to achieve additional active targeting. Lactoferrin conjugated USLP (USLP-Lf) were loaded with doxorubicin - a chemotherapy agent that is known to be highly effective against GBM in vitro but cannot permeate the BBB. USLP-Lf were able to selectively permeate the BBB in vitro, and were effectively taken up by glioblastoma U87 cells. When compared to the uncoated USLP-NPs, the coating with lactoferrin significantly improved penetration of USLP into U87 tumour spheroids (after 12 hours at 100 µm distance, RFU value 19.58 vs. 49.16 respectively). Moreover, this USLP-Lf based delivery platform improved the efficacy of doxorubicin-mediated apoptosis of GBM cells in both 2D and 3D models. Collectively, our new nano-platform has the potential to overcome both the BBB and BTB to treat GBM more effectively.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Barreira Hematoencefálica , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Humanos , Lactoferrina , Dióxido de Silício/uso terapêutico , Microambiente TumoralRESUMO
Glioblastoma (GBM) is one of the most aggressive cancers of the brain. Despite extensive research over the last several decades, the survival rates for GBM have not improved and prognosis remains poor. To date, only a few therapies are approved for the treatment of GBM with the main reasons being: 1) significant tumour heterogeneity which promotes the selection of resistant subpopulations 2) GBM induced immunosuppression and 3) fortified location of the tumour in the brain which hinders the delivery of therapeutics. Existing therapies for GBM such as radiotherapy, surgery and chemotherapy have been unable to reach the clinical efficacy necessary to prolong patient survival more than a few months. This comprehensive review evaluates the current and emerging therapies including those in clinical trials that may potentially improve both targeted delivery of therapeutics directly to the tumour site and the development of agents that may specifically target GBM. Particular focus has also been given to emerging delivery technologies such as focused ultrasound, cellular delivery systems nanomedicines and immunotherapy. Finally, we discuss the importance of developing novel materials for improved delivery efficacy of nanoparticles and therapeutics to reduce the suffering of GBM patients.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Animais , HumanosRESUMO
The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p < 0.01) as well as in their microenvironment (p < 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA (p < 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 (p < 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.
RESUMO
PURPOSE: Preclinical radiotherapy applications require dedicated irradiation systems which are expensive and not widely available. In this work, a clinical dual source 137 Cs cell irradiator was adapted to deliver 1-cm diameter preclinical treatment beams using a lead and stainless steel custom-made collimator to treat one or two mice at a time. METHODS: The dosimetric characteristics of all the different components of the system (including collimator, phantoms, and radiation sources) have been simulated with EGSnrc Monte Carlo methods. The collimator was constructed based on these simulations and the calculated results were verified against dosimetric measurements with MOSKin detectors, GAFchromic films, and dosimetric gels. RESULTS: The comparisons showed an agreement, in terms of full width half maximum values, between the simulated and the measured dose cross profiles at the midline within 4% for both gel dosimetry and GAFchromic films. Out of beam dose, measured in air at the collimator midplane with MOSFET detectors was between 6% and 10% of the beam axis dose. The dimensions of the beam are constant along the vertical axis of the collimator and also the simulated and measured Percentage Depth Dose (PDD) curves show an agreement within 1%. CONCLUSIONS: The collimator design developed in this work allows the creation of a beam with the necessary characteristics for ablative radiotherapy treatments on small animals using a standard clinical cell irradiator. This collimator design will make advanced preclinical studies with ablative beams possible for all those institutions which do not have collimated preclinical irradiators available.
Assuntos
Radiometria , Planejamento da Radioterapia Assistida por Computador , Animais , Camundongos , Método de Monte Carlo , Imagens de Fantasmas , Dosagem RadioterapêuticaRESUMO
The interaction of Prep1 and Pbx homeodomain transcription factors regulates their activity, nuclear localization, and likely, function in development. To understand the in vivo role of Prep1, we have analyzed an embryonic lethal hypomorphic mutant mouse (Prep1(i/i)). Prep1(i/i) embryos die at embryonic day 17.5 (E17.5) to birth with an overall organ hypoplasia, severe anemia, impaired angiogenesis, and eye anomalies, particularly in the lens and retina. The anemia correlates with delayed differentiation of erythroid progenitors and may be, at least in part, responsible for intrauterine death. At E14.5, Prep1 is present in fetal liver (FL) cMyb-positive cells, whose deficiency causes a marked hematopoietic phenotype. Prep1 is also localized to FL endothelial progenitors, consistent with the observed angiogenic phenotype. Likewise, at the same gestational day, Prep1 is present in the eye cells that bear Pax6, implicated in eye development. The levels of cMyb and Pax6 in FL and in the retina, respectively, are significantly decreased in Prep1(i/i) embryos, consistent with the hematopoietic and eye phenotypes. Concomitantly, Prep1 deficiency results in the overall decrease of protein levels of its related family member Meis1 and its partners Pbx1 and Pbx2. As both Prep1 and Meis interact with Pbx, the overall Prep1/Meis-Pbx DNA-binding activity is strongly reduced in whole Prep1(i/i) embryos and their organs. Our data indicate that Prep1 is an essential gene that acts upstream of and within a Pbx-Meis network that regulates multiple aspects of embryonic development.
Assuntos
Embrião de Mamíferos , Proteínas de Homeodomínio/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Fenótipo , Fatores de Transcrição/metabolismo , Alantoide/citologia , Anemia/genética , Animais , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Olho/anatomia & histologia , Olho/embriologia , Olho/patologia , Feminino , Marcação de Genes , Idade Gestacional , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Meis1 , Proteínas de Neoplasias/genética , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Gravidez , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Urokinase-type plasminogen activator (uPA) binding to uPAR induces migration, adhesion, and proliferation through multiple interactions with G proteins-coupled receptor FPRL1, integrins, or the epidermal growth factor (EGF) receptor (EGFR). At least two forms of uPAR are present on the cell surface: full-length and cleaved uPAR, each specifically interacting with one or more transmembrane proteins. The connection between these interactions and the effects on the signaling pathways activation is not clear. We have exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. This mutant is not cleaved by proteases, is glycosylphosphatidylinositol anchored, and binds uPA with a normal K(d). Both wild-type (wt) and hcr-uPAR are able to mediate uPA-induced migration, are constitutively associated with the EGFR, and associate with alpha3beta1 integrin upon uPA binding. However, they engage different pathways in response to uPA. wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to alpha3beta1 results in uPAR cleavage and extracellular signal-regulated kinase (ERK) activation. On the contrary, hcr-uPAR does not activate ERK and does not engage FPRL1 or any other G protein-coupled receptor, but it activates an alternative pathway initiated by the formation of a triple complex (uPAR-alpha3beta1-EGFR) and resulting in the autotyrosine phosphorylation of EGFR.
Assuntos
Movimento Celular/efeitos dos fármacos , Mutação/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Integrinas/metabolismo , Camundongos , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Receptores de Superfície Celular/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The efficacy of several therapeutic strategies against cancer, including cytotoxic drugs, radiotherapy, targeted immunotherapies and oncolytic viruses, depend on intact type I interferon (IFN) signaling for the promotion of both direct (tumor cell inhibition) and indirect (anti-tumor immune responses) effects. Malfunctions of this pathway in tumor cells or in immune cells may be responsible for the lack of response or resistance. Although type I IFN signaling is required to trigger anti-tumor immunity, emerging evidence indicates that chronic activation of type I IFN pathway may be involved in mediating resistance to different cancer treatments. The plastic and dynamic features of type I IFN responses should be carefully considered to fully exploit the therapeutic potential of strategies targeting IFN signaling. Here, we review available evidence supporting the involvement of type I IFN signaling in mediating resistance to various cancer therapies and highlight the most promising modalities that are being tested to overcome resistance.
RESUMO
Non-antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalized neoepitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen-anti-Clec9A mAb conjugates target cross-presenting DCs, adjuvant must be codelivered for cytotoxic T lymphocyte (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating OVA antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation and CTL and antibody responses. OVA-Clec9A-TNE-induced DC activation required CD4 and CD8 epitopes, CD40, and IFN-α. Clec9A-TNE encapsulating HPV E6/E7 significantly suppressed HPV-associated tumor growth, while E6/E7-CpG did not. Clec9A-TNE loaded with pooled B16-F10 melanoma neoepitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neoepitopes. Clec9A-TNE encapsulating 6 neoepitopes significantly suppressed B16-F10 melanoma growth in a CD4+ T cell-dependent manner. Thus, cross-presenting DCs targeted with antigen-Clec9A-TNE stimulate therapeutically effective tumor-specific immunity, dependent on T cell help.