RESUMO
The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Camundongos , Animais , Vetores Genéticos/genética , Camundongos Transgênicos , Terapia Genética , Transgenes , Dependovirus/genética , Transdução GenéticaRESUMO
Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.
Assuntos
Orthoreovirus Mamífero 3/genética , Terapia Viral Oncolítica/métodos , Replicação Viral/genética , Aminoácidos/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Variação Genética/genética , Humanos , Orthoreovirus Mamífero 3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Orthoreovirus de Mamíferos/genética , Orthoreovirus de Mamíferos/metabolismo , Filogenia , Reoviridae/genética , Proteínas Virais/metabolismoRESUMO
The efficacy of oncolytic viruses (OVs), such as reovirus, is dictated by host immune responses, including those mediated by the pro- versus anti-inflammatory macrophages. As such, a detailed understanding of the interaction between reovirus and different macrophage types is critical for therapeutic efficacy. To explore reovirus-macrophage interactions, we performed tandem mass tag (TMT)-based quantitative temporal proteomics on mouse bone marrow-derived macrophages (BMMs) generated with two cytokines, macrophage colony stimulating factor (M-CSF) and granulocytic-macrophage colony stimulating factor (GM-CSF), representing anti- and proinflammatory macrophages, respectively. We quantified 6863 proteins across five time points in duplicate, comparing M-CSF (M-BMM) and GM-CSF (GM-BMM) in response to OV. We find that GM-BMMs have lower expression of key intrinsic proteins that facilitate an antiviral immune response, express higher levels of reovirus receptor protein JAM-A, and are more susceptible to oncolytic reovirus infection compared to M-BMMs. Interestingly, although M-BMMs are less susceptible to reovirus infection and subsequent cell death, they initiate an antireovirus adaptive T cell immune response comparable to that of GM-BMMs. Taken together, these data describe distinct proteome differences between these two macrophage populations in terms of their ability to mount antiviral immune responses.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Fator Estimulador de Colônias de Macrófagos , Animais , Medula Óssea , Células da Medula Óssea , Células Cultivadas , Camundongos , ProteomaRESUMO
Oncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.
Assuntos
Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Proteômica/métodos , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/patologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia , Interferon gama/genética , Interferon gama/imunologia , Ligantes , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MHC-I peptides are intracellular-cleaved peptides, usually 8-11 amino acids in length, which are presented on the cell surface and facilitate CD8+ T cell responses. Despite the appreciation of CD8+ T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8+ T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8+ T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8+ T-cell-based immunotherapies.
Assuntos
Antibióticos Antineoplásicos/análise , Neoplasias do Colo/terapia , Doxorrubicina/análise , Antígenos de Histocompatibilidade Classe I/análise , Linfoma/terapia , Animais , Antibióticos Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Doxorrubicina/farmacologia , Descoberta de Drogas , Células HCT116 , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia , Ligantes , Linfoma/imunologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais CultivadasRESUMO
BACKGROUND/AIMS: The oxidative stress sensor transient receptor potential melastatin-2 (TRPM2) ion channel has recently gained attention in many types of cancer. The lung tissue is highly susceptible to oxidative stress-mediated injury and diseases; therefore, we aimed to determine whether TRPM2 plays an essential role in protecting lung cancer cells from oxidative damage while promoting cancer cell survival and metastasis. METHODS: We used two non-small cell lung (NSCLC) cell lines A549 and H1299 as a lung cancer model. We investigated the functional expression of TRPM2 using electrophysiology, qRT-PCR and Western blots. CFSE and flow cytometry were used to study TRPM2 role in proliferation, cell cycle and apoptosis. Gap closure chambers and Three-Tiered Chemotaxis Chamber were used to study the role of TRPM2 in metastasis. SCID mice were used to study the role of TRPM2 in lung tumor growth and metastasis. RESULTS: we demonstrate that TRPM2 is functionally expressed in NSCLC cells and that its downregulation significantly inhibits cell proliferation and promotes apoptosis. These results were concomitant with an induction in DNA damage and G2/M cell cycle arrest. TRPM2 silencing inhibits also lung cancer cells invasion ability and alters EMT processes. Mechanistically, TRPM2 downregulation causes an increase in the intracellular levels of reactive oxygen (ROS) and nitrogen (RNS) species, which in turn causes DNA damage and JNK activation leading to G2/M arrest, and an ultimate cell death. Finally, TRPM2 downregulation suppresses the growth of human lung tumour xenograft in SCID mice and TRPM2 depleted tumours exhibited a significant reduction in the mRNA expression level of EMT markers compared to the control tumors. CONCLUSION: Our data provide new insights on the functional expression of TRPM2 in lung cancer, its essential role in tumour growth and metastasis through the control of JNK signaling pathway, and that TRPM2 could be exploited for targeted lung cancer therapies.
Assuntos
Apoptose , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Antracenos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Pontos de Checagem da Fase M do Ciclo Celular , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos SCID , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genéticaRESUMO
Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor+ population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor+ tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor+ population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44+ CD24- do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor+ CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor+ breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654.
Assuntos
Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Neoplasias da Mama/imunologia , Transformação Celular Neoplásica/imunologia , Epigênese Genética , Evasão da Resposta Imune/imunologia , Células-Tronco Neoplásicas/citologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Inativação Gênica , Humanos , Camundongos , Células-Tronco Neoplásicas/imunologiaRESUMO
Reticulon-4 (RTN4), commonly known as a neurite outgrowth inhibitor (Nogo), is emerging as an important player in human cancers. Clinically, we found lower RTN4 expression in patient-derived tumors was associated with significantly better survival in lung, breast, cervical, and renal cancer patients. To identify the role of RTN4 in cancer biology, we performed mass spectrometry-based quantitative proteomic analysis on cancer cells following RTN4 knockdown and found its link with pro-survival as well as cytoskeleton-related processes. Subsequent mechanistic investigations revealed that RTN4 regulates lipid homeostasis, AKT signaling, and cytoskeleton modulation. In particular, downregulation of RTN4 reduced sphingomyelin synthesis and impaired plasma membrane localization of AKT, wherein AKT phosphorylation, involved in many cancers, was significantly reduced without any comparable effect on AKT-related upstream kinases, in a sphingolipid-dependent manner. Furthermore, knockdown of RTN4 retarded proliferation of cancer cells in vitro as well as tumor xenografts in mice. Finally, RTN4 knockdown affected tubulin stability and promoted higher cytotoxic effects with chemotherapeutic paclitaxel in cancer cells both in vitro and in vivo. In summary, RTN4 is involved in carcinogenesis and represents a molecular candidate that may be targeted to achieve desired antitumor effects in clinics.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Citoesqueleto/metabolismo , Técnicas de Silenciamento de Genes/métodos , Proteínas Nogo/genética , Paclitaxel/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Paclitaxel/farmacologia , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional "no enzyme" searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Espectrometria de Massas/métodos , Peptídeos/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia , Ligantes , Camundongos , Peptídeos/genéticaRESUMO
Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host-virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G-, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G-, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral-host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection.
Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Células Mieloides/metabolismo , Proteômica/métodos , Infecções por Reoviridae/genética , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Biomarcadores/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Diferenciação Celular , Proliferação de Células , Deleção de Genes , Regulação da Expressão Gênica , Ontologia Genética , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Células Mieloides/imunologia , Células Mieloides/virologia , Orthoreovirus de Mamíferos/crescimento & desenvolvimento , Orthoreovirus de Mamíferos/patogenicidade , Receptores CCR2/genética , Receptores CCR2/imunologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/virologia , Transdução de Sinais , Fatores de TempoRESUMO
Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies.
Assuntos
Vetores Genéticos , Imunomodulação , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/imunologia , Vírus Oncolíticos , Fenótipo , Animais , Antígenos Ly/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Antígeno CD11b/metabolismo , Diferenciação Celular , Quimiotaxia/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/imunologia , Camundongos , Células Mieloides/citologia , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Receptores de Quimiocinas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Immunosuppression associated with ovarian cancer (OC) and resultant peritoneal carcinomatosis (PC) hampers the efficacy of many promising treatment options, including immunotherapies. It is hypothesized that oncolytic virus-based therapies can simultaneously kill OC and mitigate immunosuppression. Currently, reovirus-based anticancer therapy is undergoing phase I/II clinical trials for the treatment of OC. Hence, this study was focused on characterizing the effects of reovirus therapy on OC and associated immune microenvironment. Our data shows that reovirus efficiently killed OC cells and induced higher expression of the molecules involved in antigen presentation including major histocompatibility complex (MHC) class I, ß2-microglobulin (ß2M), TAP-1, and TAP-2. In addition, in the presence of reovirus, dendritic cells (DCs) overcame the OC-mediated phenotypic suppression and successfully stimulated tumor-specific CD8+ T cells. In animal studies, reovirus targeted local and distal OC, alleviated the severity of PC and significantly prolonged survival. These therapeutic effects were accompanied by decreased frequency of suppressive cells, e.g., Gr1.1+, CD11b+ myeloid derived suppressor cells (MDSCs), and CD4+, CD25+, FOXP3+ Tregs, tumor-infiltration of CD3+ cells and higher expression of Th1 cytokines. Finally, reovirus therapy during early stages of OC also resulted in the postponement of PC development. This report elucidates timely information on a therapeutic approach that can target OC through clinically desired multifaceted mechanisms to better the outcomes.
Assuntos
Carcinoma/terapia , Imunomodulação , Terapia Viral Oncolítica/métodos , Neoplasias Ovarianas/terapia , Neoplasias Peritoneais/terapia , Reoviridae/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Microambiente Celular , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Feminino , Vetores Genéticos , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Reoviridae/imunologiaRESUMO
The recruitment of monocytes and their differentiation into immunosuppressive cells is associated with the low efficacy of preclinical nonconformal radiotherapy (RT) for tumors. However, nonconformal RT (non-CRT) does not mimic clinical practice, and little is known about the role of monocytes after RT modes used in patients, such as conformal RT (CRT). Here, we investigated the acute immune response induced by after CRT. Contrary to non-CRT approaches, we found that CRT induces a rapid and robust recruitment of monocytes to the tumor that minimally differentiate into tumor-associated macrophages or dendritic cells but instead up-regulate major histocompatibility complex II and costimulatory molecules. We found that these large numbers of infiltrating monocytes are responsible for activating effector polyfunctional CD8+ tumor-infiltrating lymphocytes that reduce tumor burden. Mechanistically, we show that monocyte-derived type I interferon is pivotal in promoting monocyte accumulation and immunostimulatory function in a positive feedback loop. We also demonstrate that monocyte accumulation in the tumor microenvironment is hindered when RT inadvertently affects healthy tissues, as occurs in non-CRT. Our results unravel the immunostimulatory function of monocytes during clinically relevant modes of RT and demonstrate that limiting the exposure of healthy tissues to radiation has a positive therapeutic effect on the overall antitumor immune response.
Assuntos
Interferon Tipo I , Neoplasias , Humanos , Monócitos , Neoplasias/radioterapia , Diferenciação Celular , Interferon Tipo I/farmacologia , Linfócitos do Interstício Tumoral , Microambiente TumoralRESUMO
CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.
Assuntos
NAD , Viroses , Humanos , Linfócitos T CD8-Positivos , Antígenos , AntiviraisRESUMO
In this issue of JEM, Gschwend et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210745) reveal the indispensable role of alveolar epithelial cells type 2 in controlling the density of alveolar macrophages. This study highlights the intricate crosstalk that lung stroma and macrophages undergo to maintain homeostasis.
Assuntos
Macrófagos Alveolares , Macrófagos , Células EpiteliaisRESUMO
BACKGROUND: Immunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described. METHODS: We used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells. RESULTS: Our study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ and CD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon γ (IFNγ)/tumor necrosis factor α (TNFα). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth. CONCLUSIONS: Our results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy.
Assuntos
Anticorpos/administração & dosagem , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Inibidores de Checkpoint Imunológico/administração & dosagem , Melanoma Experimental/tratamento farmacológico , Poli I-C/administração & dosagem , Receptor de Morte Celular Programada 1/metabolismo , Pele/citologia , Animais , Anticorpos/farmacologia , Antígenos CD40/antagonistas & inibidores , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptor Celular 2 do Vírus da Hepatite A/genética , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Estadiamento de Neoplasias , Poli I-C/farmacologia , Receptor de Morte Celular Programada 1/genética , Análise de Sequência de RNA , Pele/efeitos dos fármacos , Pele/imunologiaRESUMO
Transient Receptor Potential Melastatin-2 (TRPM2) ion channel is emerging as a great therapeutic target in many types of cancer, including gastric cancer - a major health threat of cancer related-death worldwide. Our previous study demonstrated the critical role of TRPM2 in gastric cancer cells bioenergetics and survival; however, its role in gastric cancer metastasis, the major cause of patient death, remains unknown. Here, using molecular and functional assays, we demonstrate that TRPM2 downregulation significantly inhibits the migration and invasion abilities of gastric cancer cells, with a significant reversion in the expression level of metastatic markers. These effects were concomitant with decreased Akt and increased PTEN activities. Finally, TRPM2 silencing resulted in deregulation of metastatic markers and abolished the tumor growth ability of AGS gastric cancer cells in NOD/SCID mice. Taken together, our results provide compelling evidence on the important function of TRPM2 in the modulation of gastric cancer cell invasion likely through controlling the PTEN/Akt pathway.
Assuntos
Movimento Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Canais de Cátion TRPM/metabolismo , Animais , Cálcio/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Citosol/metabolismo , Regulação para Baixo , Ativação Enzimática , Transição Epitelial-Mesenquimal , Inativação Gênica , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade NeoplásicaRESUMO
Oncolytic viruses (OV) such as reovirus preferentially infect and kill cancer cells. Thus, the mechanisms that dictate the susceptibility of cancer cells to OV-induced cytotoxicity hold the key to their success in clinics. Here, we investigated whether cancer cell metabolism defines its susceptibility to OV and if OV-induced metabolic perturbations can be therapeutically targeted. Using mass spectrometry-based metabolomics and extracellular flux analysis on a panel of cancer cell lines with varying degrees of susceptibility to reovirus, we found that OV-induced changes in central energy metabolism, pyruvate metabolism, and oxidative stress correlate with their susceptibility to reovirus. In particular, reovirus infection accentuated Warburg-like metabolic perturbations in cell lines relatively resistant to oncolysis. These metabolic changes were facilitated by oxidative stress-induced inhibitory phosphorylation of pyruvate dehydrogenase (PDH) that impaired the routing of pyruvate into the tricarboxylic acid cycle and established a metabolic state unsupportive of OV replication. From the therapeutic perspective, reactivation of PDH in cancer cells that were weakly sensitive for reovirus, either through PDH kinase (PDK) inhibitors dichloroacetate and AZD7545 or short hairpin RNA-specific depletion of PDK1, enhanced the efficacy of reovirus-induced oncolysis in vitro and in vivo. These findings identify targeted metabolic reprogramming as a possible combination strategy to enhance the antitumor effects of OV in clinics. SIGNIFICANCE: This study proposes targeted metabolic reprogramming as a valid combinatorial strategy to enhance the translational efficacy of oncolytic virus-based cancer therapies.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/15/3824/F1.large.jpg.
Assuntos
Metabolômica/métodos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Reoviridae/patogenicidade , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Proteoglycans are promising therapeutic targets in Multiple Sclerosis (MS), because they regulate many aspects of the immune response. This was studied using surfen, an agent that binds both heparan sulphate proteoglycans (HSPGs) and chondroitin sulphate proteoglycans (CSPGs). Initial cell culture work on bone marrow derived macrophages (BMDMs) found that surfen reduced concentrations of the chemokines CCL2, CCL4 and CCL5, with reduced messenger (m)RNA expression for Tumor Necrosis Factor, IL-6, IL-1ß and inducible nitric oxide synthase. These data were further explored using Experimental Autoimmune Encephalomyelitis (EAE) in mice. Surfen reduced clinical signs during EAE when administered from disease onset, and reduced infiltration by CD4 positive T cells and macrophages into the central nervous system. These mice also showed reduced mRNA expression for the chemokines CCL3 and CCL5, with reduced concentrations of CCL2, CCL3 and CCL5. During EAE, surfen treatment induced a persistent increase in Interleukin (IL)-4 concentrations which may enhance T helper 2 responses. During EAE, surfen treatment reduced mRNA expression for HSPGs (NDST1, agrin, syndecan-4, perlecan, serglycin, syndecan-1) and the CSPG versican. By contrast, surfen increased mRNA expression for the CSPG aggrecan, with no effect on neurocan. During EAE, significant positive correlations were found between mRNA expression and clinical score for syndecan-4, serglycin and syndecan-1 and a significant negative correlation for aggrecan. These correlations were absent in surfen treated mice. Repair in the later stages of MS involves remyelination, which was modeled by injecting lysolecithin (lysophosphatidylcholine, LPC) into mouse corpus callosum to create regions of demyelination. When surfen was injected 2 days after LPC, it delayed remyelination of the lesions, but had no effect when injected 7 days after LPC. The delayed remyelination was associated with local increases in CSPG expression. Therefore surfen suppresses inflammation but inhibits remyelination in these models. A mechanism in common may be increased CSPG expression.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Fatores Imunológicos/farmacologia , Inflamação/tratamento farmacológico , Remielinização/efeitos dos fármacos , Ureia/análogos & derivados , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Medula Óssea/fisiologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/fisiologia , Células Cultivadas , Quimiocinas/metabolismo , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/patologia , Corpo Caloso/fisiopatologia , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Fatores Imunológicos/administração & dosagem , Inflamação/patologia , Inflamação/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Macrófagos/fisiologia , Camundongos Endogâmicos C57BL , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Remielinização/fisiologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Ureia/efeitos adversos , Ureia/farmacologiaRESUMO
Oncolytic viruses selectively target and kill cancer cells in an immunogenic fashion, thus supporting the establishment of therapeutically relevant tumor-specific immune responses. In 2015, the US Food and Drug Administration (FDA) approved the oncolytic herpes simplex virus T-VEC for use in advanced melanoma patients. Since then, a plethora of trials has been initiated to assess the safety and efficacy of multiple oncolytic viruses in patients affected with various malignancies. Here, we summarize recent preclinical and clinical progress in the field of oncolytic virotherapy.