RESUMEN
Radiotherapy offers an effective treatment for advanced cancer but local and distant failures remain a significant challenge. Here, we treated melanoma and pancreatic carcinoma in syngeneic mice with ionizing radiation (IR) combined with the poly(ADP-ribose) polymerase inhibitor (PARPi) veliparib to inhibit DNA repair and promote accelerated senescence. Based on prior work implicating cytotoxic T lymphocytes (CTLs) as key mediators of radiation effects, we discovered that senescent tumor cells induced by radiation and veliparib express immunostimulatory cytokines to activate CTLs that mediate an effective antitumor response. When these senescent tumor cells were injected into tumor-bearing mice, an antitumor CTL response was induced which potentiated the effects of radiation, resulting in elimination of established tumors. Applied to human cancers, radiation-inducible immunotherapy may enhance radiotherapy responses to prevent local recurrence and distant metastasis.
Asunto(s)
Bencimidazoles/farmacología , Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia/métodos , Melanoma Experimental/terapia , Neoplasias Pancreáticas/terapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Vacunas contra el Cáncer/inmunología , Senescencia Celular/efectos de los fármacos , Senescencia Celular/efectos de la radiación , Terapia Combinada , Citocinas/biosíntesis , Citocinas/inmunología , Citotoxicidad Inmunológica , Femenino , Humanos , Activación de Linfocitos , Melanoma Experimental/inmunología , Melanoma Experimental/mortalidad , Ratones , Trasplante de Neoplasias , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/mortalidad , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Tasa de Supervivencia , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
Patients with locally advanced cancer or distant metastasis frequently receive prolonged treatment with chemotherapy and/or fractionated radiotherapy (RT). Despite the initial clinical response, treatment resistance frequently develops and cure in these patients is uncommon. Developments in RT technology allow for the use of high-dose (or ablative) RT to target local tumors, with limited damage to the surrounding normal tissue. We report that reduction of tumor burden after ablative RT depends largely on T-cell responses. Ablative RT dramatically increases T-cell priming in draining lymphoid tissues, leading to reduction/eradication of the primary tumor or distant metastasis in a CD8(+) T cell-dependent fashion. We further demonstrate that ablative RT-initiated immune responses and tumor reduction are abrogated by conventional fractionated RT or adjuvant chemotherapy but greatly amplified by local immunotherapy. Our study challenges the rationale for current RT/chemotherapy strategies and highlights the importance of immune activation in preventing tumor relapse. Our findings emphasize the need for new strategies that not only reduce tumor burden but also enhance the role of antitumor immunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/radioterapia , Animales , Presentación de Antígeno , Quimioterapia Adyuvante , Terapia Combinada , Ratones , Radioterapia , Carga Tumoral/inmunologíaRESUMEN
Ad.Egr-TNF is a radioinducible adenovector currently in phase 3 trials for inoperable pancreatic cancer. The combination of Ad.Egr-TNF and ionizing radiation (IR) contributes to local tumor control through the production of tumor necrosis factor-alpha (TNFalpha) in the tumor microenvironment. Moreover, clinical and preclinical studies with Ad.Egr-TNF/IR have suggested that this local approach suppresses the growth of distant metastatic disease; however, the mechanisms responsible for this effect remain unclear. These studies have been performed in wild-type (WT) and TNFR1,2(-/-) mice to assess the role of TNFalpha-induced signaling in the suppression of draining lymph node (DLN) metastases. The results demonstrate that production of TNFalpha in the tumor microenvironment induces expression of interferon (IFNbeta). In turn, IFNbeta stimulates the production of chemokines that recruit CD8(+) T cells to the tumor. The results further demonstrate that activation of tumor antigen-specific CD8(+) CTLs contributes to local antitumor activity and suppression of DLN metastases. These findings support a model in which treatment of tumors with Ad.Egr-TNF and IR is mediated by local and distant immune-mediated antitumor effects that suppress the development of metastases.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Metástasis de la Neoplasia/prevención & control , Metástasis de la Neoplasia/terapia , Radiación Ionizante , Factor de Necrosis Tumoral alfa/metabolismo , Adenoviridae/genética , Animales , Proliferación Celular , Vectores Genéticos/genética , Humanos , Interferón beta/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Metástasis de la Neoplasia/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
This article aims at investigating the feasibility and potential of natural killer (NK) cells to treat corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). Through retrospective analysis of existing anti-virus researches and clinical application results of NK cells, and based on the characteristics and properties of this cell, we designed a plan for NK cells to treat COVID-19. As an important part of the immune system, NK cells have clear anti-viral ability. In addition, it also plays the role in immune defense, immune surveillance and immune regulation. Many clinical applications have proved the safety and effectiveness of NK cell application. Currently COVID-19 is highly contagious and harmful to people, and easy to cause irreversible lung damage. Although quarantine can effectively prevent and control viral infections, there is no effective drug treatment available. To avoid further greater losses, it is tremendously necessary to investigate an effective treatment plan.
Asunto(s)
Infecciones por Coronavirus , Células Asesinas Naturales , Pandemias , Neumonía Viral , Betacoronavirus , COVID-19 , Infecciones por Coronavirus/terapia , Humanos , Neumonía Viral/terapia , Estudios Retrospectivos , SARS-CoV-2RESUMEN
Previous studies found that lysophosphatidic acid (LPA) upregulated Fas ligand (FasL) presentation on the ovarian cancer cell surface and lead to apoptosis of activated lymphocytes. In this report, we investigated the role of selective induction of cyclooxygenase-2 (Cox-2) in FasL cell surface presentation stimulated by LPA. Ovarian cancer cells pretreated with general aspirin derivative acetylsalicylic acid and specific Cox-2 inhibitor (NS-398) before stimulation with LPA, FasL cell surface presentation was significantly blocked, so was the apoptosis of activated lymphocytes mediated by increasing FasL on the ovarian cancer cell surface. Using the specific inhibitors PD98059, AG1478 or dominant-negative epidermal-growth-factor receptor (EGFR-DN) plasmid, we found that the activation of ERK1/2 played a role in Cox-2 induction, and the transactivation of EGFR worked as an upstream signaling pathway in ERK1/2 phosphorylation. This study first revealed the selective induction of Cox-2 by LPA led to FasL presentation on ovarian cancer cell surface and provide cancer cell immune privilege, and might provide important information of Cox-2 in cancer progression and Cox-2 inhibitors' application in cancer chemoprevention.
Asunto(s)
Presentación de Antígeno , Antígenos de Superficie/metabolismo , Membrana Celular/metabolismo , Ciclooxigenasa 2/metabolismo , Lisofosfolípidos/metabolismo , Glicoproteínas de Membrana/metabolismo , Factores de Necrosis Tumoral/metabolismo , Animales , Apoptosis/fisiología , Aspirina/metabolismo , Línea Celular Tumoral , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa/metabolismo , Activación Enzimática , Inducción Enzimática , Inhibidores Enzimáticos/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Ligando Fas , Femenino , Flavonoides/metabolismo , Humanos , Activación de Linfocitos , Linfocitos/metabolismo , Nitrobencenos/metabolismo , Neoplasias Ováricas/inmunología , Quinazolinas , Transducción de Señal/fisiología , Sulfonamidas/metabolismo , Tirfostinos/metabolismoRESUMEN
Conflicting reports exist on the effect of actin depolymerization in anti-Fas-induced apoptosis. Lysophosphatidic acid (LPA) has been found to inhibit apoptosis in variable cell types. In this study, we evaluated LPA's protective effects on anti-Fas-induced apoptosis enhanced by actin depolymerization and possible mechanisms in epithelial ovarian cancer. OVCAR3 cells were pretreated with vehicle or LPA, then treated with Cytochalasin D (Cyto D), followed with anti-Fas mAb to induce apoptosis. Cells were stained with apoptotic markers and analyzed by flow cytometry. We report that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization. Immunoprecipition of Fas death-inducing signaling complex (DISC) and Western blot suggested that the actin depolymerization accelerated caspase-8 activation, while LPA inhibited the association and activation of caspase-8 at the DISC. LPA inhibited caspase-3 and 7 activation induced by anti-Fas and/or Cyto D in cytosols. Phosphorylation of ERK and Bad112 by LPA may play a role in preventing caspase-3 activation through mitochondrial pathway induced by Cyto D. Our investigation found that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization, and LPA may protect epithelial ovarian cancer from immune cell attack and cytoskeleton disrupting reagents induced apoptosis through multiple pathways.
Asunto(s)
Actinas/metabolismo , Apoptosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Lisofosfolípidos/farmacología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Receptor fas/metabolismo , Anticuerpos/inmunología , Proteínas Portadoras/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular Tumoral , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Fosforilación/efectos de los fármacos , Proteína Letal Asociada a bcl , Receptor fas/inmunologíaRESUMEN
BACKGROUND: There is genuine need to develop interventional treatment options for management of lung tumors. Radiofrequency ablation (RFA) is one such alternative being promoted to treat lung tumors recently. Larger studies should help define RFA's further development. Furthermore fluorodeoxyglucose positron emission tomography (PET) has been reported to be an accurate indicator of treatment response in variety of tumors. This study focuses on the evaluating the feasibility of RFA and usefulness of PET scan in lung tumors after RFA procedure. PATIENTS AND METHODS: Between November 1999 and May 2002, 50 patients with primary or metastasis pulmonary tumors underwent RFA procedure. The electrode was guided to the target areas using computerized tomography (CT). Tumors smaller than 3.5 cm were given single RFA, while tumors larger than 3.5 cm received RFA to multiple sites. Maximum 4 lesions or 6 target areas were treated during one operating procedure. Whole body and/or lung PET images were acquired; identical site CT images and chest X-ray were taken 1 week before and after RFA. RESULTS: Of the 50 patients, 17 had single lesions while rest had multiple lesions. Tumors smaller than 3.5 cm were completely dissipated after RFA. In tumors larger than 3.5 cm, the part within 3.5 cm diameter dissipated. While CT showed that tumor image became larger 1 to 2 weeks after RFA procedure. PET demonstrated tumor destruction in 70% cases, compared to 38% in CT. CONCLUSION: The present study shows RFA to be safe and effective treatment option for lung tumors. PET is superior to CT in evaluation the effectiveness of RFA treatment shortly after the procedure.
RESUMEN
Tumor escape from immune-mediated destruction has been associated with immunosuppressive mechanisms that inhibit T cell activation. Although evidence for an active immune response, including infiltration with CD8(+) T cells, can be found in a subset of patients, those tumors are nonetheless not immunologically rejected. In the current report, we show that it is the subset of T cell-inflamed tumors that showed high expression of three defined immunosuppressive mechanisms: indoleamine-2,3-dioxygenase (IDO), PD-L1/B7-H1, and FoxP3(+) regulatory T cells (T(regs)), suggesting that these inhibitory pathways might serve as negative feedback mechanisms that followed, rather than preceded, CD8(+) T cell infiltration. Mechanistic studies in mice revealed that up-regulated expression of IDO and PD-L1, as well as recruitment of T(regs), in the tumor microenvironment depended on the presence of CD8(+) T cells. The former was driven by interferon-γ and the latter by a production of CCR4-binding chemokines along with a component of induced proliferation. Our results argue that these major immunosuppressive pathways are intrinsically driven by the immune system rather than being orchestrated by cancer cells, and imply that cancer immunotherapy approaches targeting negative regulatory immune checkpoints might be preferentially beneficial for patients with a preexisting T cell-inflamed tumor microenvironment.
Asunto(s)
Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Melanoma/inmunología , Melanoma/patología , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Regulación hacia Arriba , Animales , Proliferación Celular , Quimiocinas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Melanoma/enzimología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Metástasis de la Neoplasia , Regulación hacia Arriba/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Most cancer patients receive radiotherapy during the course of their disease. Improvements in the therapeutic index have been based mainly on physical improvements in delivery, as radiosensitizer development to target tumor cells has yet to yield effective agents. Recent investigations have focused on the tumor stroma as a target for radiosensitization. Here, we report that depletion of tumor-associated macrophages (TAMvarphi) by systemic or local injection of the macrophage-depleting liposomal clodronate before radiotherapy can increase the antitumor effects of ionizing radiation (IR), either as a large single dose (20 Gy) or as a fractionated dose (2 Gy x 10). Coimplantation of tumor cells with bone marrow-derived macrophages (BMDMvarphi) increased tumor radioresistance. Studies using mice with germline deletions in tumor necrosis factor receptors 1 and 2 (TNFR1,2(-/-)) or TNFalpha (TNF(-/-)), or treatment of wild-type mice with a soluble TNF receptor fusion protein (Enbrel), revealed that radioresistance mediated by BMDMvarphi required intact TNFalpha signaling. Radiation exposure upregulated vascular endothelial growth factor (VEGF) in macrophages and VEGF-neutralizing antibodies enhanced the antitumor response to IR. Thus, the radioprotective effect of TNFalpha was mediated by TAM-produced VEGF. Our findings offer a mechanistic basis to target macrophage populations generally or TNFalpha-induced macrophage VEGF specifically as tractable strategies to improve the efficacy of radiotherapy.
Asunto(s)
Inmunoglobulina G/farmacología , Macrófagos/efectos de los fármacos , Neoplasias/inmunología , Neoplasias/radioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Proliferación Celular/efectos de los fármacos , Ácido Clodrónico/administración & dosificación , Ácido Clodrónico/farmacología , Evaluación Preclínica de Medicamentos , Etanercept , Liposomas/administración & dosificación , Liposomas/farmacología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/patología , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Receptores del Factor de Necrosis Tumoral , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Despite the frequent detection of circulating tumor antigen-specific T cells, either spontaneously or following active immunization or adoptive transfer, immune-mediated cancer regression occurs only in the minority of patients. One theoretical rate-limiting step is whether effector T cells successfully migrate into metastatic tumor sites. Affymetrix gene expression profiling done on a series of metastatic melanoma biopsies revealed a major segregation of samples based on the presence or absence of T-cell-associated transcripts. The presence of lymphocytes correlated with the expression of defined chemokine genes. A subset of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) was confirmed by protein array and/or quantitative reverse transcription-PCR to be preferentially expressed in tumors that contained T cells. Corresponding chemokine receptors were found to be up-regulated on human CD8(+) effector T cells, and transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8(+) effector cells in vitro. Screening by chemokine protein array identified a subset of melanoma cell lines that produced a similar broad array of chemokines. These melanoma cells more effectively recruited human CD8(+) effector T cells when implanted as xenografts in nonobese diabetic/severe combined immunodeficient mice in vivo. Chemokine blockade with specific antibodies inhibited migration of CD8(+) T cells. Our results suggest that lack of critical chemokines in a subset of melanoma metastases may limit the migration of activated T cells, which in turn could limit the effectiveness of antitumor immunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Quimiocinas/biosíntesis , Melanoma/inmunología , Melanoma/metabolismo , Línea Celular Tumoral , Movimiento Celular/inmunología , Quimiocinas/genética , Quimiocinas/inmunología , Expresión Génica , Humanos , Melanoma/genética , Familia de Multigenes , Análisis por Matrices de ProteínasRESUMEN
The identification of tumor-expressed antigens that can be recognized by specific T lymphocytes has made it possible both to study the properties of T cells participating in anti-tumor immune responses in patients and also to develop antigen-specific immunotherapies as a treatment modality. Interestingly, moves toward intervention have proceeded at a faster pace than have investigations toward understanding. In melanoma in particular, many clinical trials of active immunization have been performed, and many of these have shown increases in tumor antigen-specific T cells circulating in the blood. However, clinical responses have been infrequent, arguing that mechanisms of resistance downstream from initial T cell priming may be dominant in many cases. In fact, may patients show spontaneous generation of immune effector cells and/or antibodies, implying that the priming phase has occurred already in such individuals even without vaccination. Recent attention has turned toward mechanisms of immune evasion at the effector phase of the anti-tumor immune response, predominantly within the tumor microenvironment. Evidence is accumulating that T cell-intrinsic hyporesponsiveness or anergy, extrinsic suppression by regulatory cell populations, inhibitory ligands such as PD-L1, soluble factors such as TGF-beta, and the activity of nutrient-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO), may contribute to immune escape in different settings. Murine preclinical models have shown that interfering with each of these processes can translate into T cell-mediated tumor control. Clinical studies to estimate the frequency of specific immune evasion mechanisms in individual patients, to correlate specific events with clinical outcome, and to develop strategies to counter resistance mechanisms should receive a high priority.
Asunto(s)
Neoplasias/inmunología , Animales , Antígenos de Neoplasias/química , Vacunas contra el Cáncer , Humanos , Inmunización , Terapia de Inmunosupresión , Ligandos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Although melanoma tumors usually express antigens that can be recognized by T cells, immune-mediated tumor rejection is rare. In many cases this is despite the presence of high frequencies of circulating tumor antigen-specific T cells, suggesting that tumor resistance downstream from T cell priming represents a critical barrier. Analyzing T cells directly from the melanoma tumor microenvironment, as well as the nature of the microenvironment itself, is central for understanding the key downstream mechanisms of tumor escape. In the current report we have studied tumor-associated lymphocytes from a patient with metastatic melanoma and large volume malignant ascites. The ascites fluid showed abundant tumor cells that expressed common melanoma antigens and retained expression of class I MHC and antigen processing machinery. The ascites fluid contained the chemokines CCL10, CCL15, and CCL18 which was associated with a large influx of activated T cells, including CD8(+) T cells recognizing HLA-A2 tetramer complexes with peptides from Melan-A and NA17-A. However, several functional defects of these tumor antigen-specific T cells were seen, including poor production of IFN-gamma in response to peptide-pulsed APC or autologous tumor cells, and lack of expression of perforin. Although these defects were T cell intrinsic, we also observed abundant CD4(+)CD25(+)FoxP3(+) T cells, as well as transcripts for FoxP3, IL-10, PD-L1/B7-H1, and indoleamine-2,3-dioxygenase (IDO). Our observations suggest that, despite recruitment of large numbers of activated CD8(+) T cells into the tumor microenvironment, T cell hyporesponsiveness and additional negative regulatory mechanisms can limit the effector phase of the anti-tumor immune response.
Asunto(s)
Ascitis/inmunología , Líquido Ascítico/inmunología , Linfocitos T CD8-positivos/inmunología , Melanoma/inmunología , Adulto , Presentación de Antígeno/inmunología , Antígenos CD , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/uso terapéutico , Antígenos de Neoplasias/inmunología , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Ascitis/etiología , Líquido Ascítico/química , Líquido Ascítico/citología , Antígeno CTLA-4 , Vacunas contra el Cáncer , Quimiocinas/inmunología , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunosupresores/inmunología , Inmunosupresores/uso terapéutico , Inmunoterapia , Interleucina-2/inmunología , Interleucina-2/uso terapéutico , Activación de Linfocitos/inmunología , Masculino , Melanoma/complicaciones , Melanoma/terapia , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/inmunología , Escape del Tumor , Antígeno gp100 del MelanomaRESUMEN
Memory CD8(+) T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8(+) T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8(+) T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA(-)CD8(+) T cells, or of CD8(+) T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA(+) and CD45RA(-) cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7(+) subpopulations, with poorest proliferation being observed in the CD45RA(-)CCR7(-) effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8(+) T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Ciclo Celular/inmunología , Gránulos Citoplasmáticos/inmunología , Pruebas Inmunológicas de Citotoxicidad , Memoria Inmunológica , Serina Endopeptidasas/biosíntesis , Subgrupos de Linfocitos T/inmunología , Anticuerpos Monoclonales/fisiología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Linfocitos T CD8-positivos/enzimología , Células Cultivadas , Gránulos Citoplasmáticos/enzimología , Pruebas Inmunológicas de Citotoxicidad/métodos , Granzimas , Humanos , Inmunofenotipificación , Serina Endopeptidasas/sangre , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/enzimología , Regulación hacia Arriba/inmunologíaRESUMEN
It is now little disputed that most if not all cancer cells express antigens that can be recognized by specific CD8(+) T lymphocytes. However, a central question in the field of anti-tumor immunity is why such antigen-expressing tumors are not spontaneously eliminated by the immune system. While in some cases, this lack of rejection may be due to immunologic ignorance, induction of anti-tumor T-cell responses in many patients has been detected in the peripheral blood, either spontaneously or in response to vaccination, without accompanying tumor rejection. These observations argue for the importance of barriers downstream from initial T-cell priming that need to be addressed to translate immune responses into clinical tumor regression. Recent data suggest that the proper trafficking of effector T cells into the tumor microenvironment may not always occur. T cells that do effectively home to tumor metastases are often found to be dysfunctional, pointing toward immunosuppressive mechanisms in the tumor microenvironment. T-cell anergy due to insufficient B7 costimulation, extrinsic suppression by regulatory cell populations, inhibition by ligands such as programmed death ligand-1, metabolic dysregulation by enzymes such as indoleamine-2,3-dioxygenase, and the action of soluble inhibitory factors such as transforming growth factor-beta have all been clearly implicated in generating this suppressive microenvironment. Identification of these downstream processes points to new therapeutic targets that should be manipulated to facilitate the effector phase of anti-tumor immune responses in concert with vaccination or T-cell adoptive transfer.
Asunto(s)
Tolerancia Inmunológica/fisiología , Neoplasias/inmunología , Linfocitos T/inmunología , Animales , Humanos , Metástasis de la Neoplasia/inmunologíaRESUMEN
Previous reports support that lysophosphatidic acid (LPA) upregulates Fas ligand (FasL) cell surface presentation on the ovarian cancer cells. In this study, we aim to investigate soluble FasL (sFasL) secretion associated with the small membrane microvesicles upon LPA stimulation, and to analyze the roles of cytoskeletal reorganization in FasL transport induced by LPA. Ovarian cancer cells were stimulated with LPA and spent media were harvested, concentrated, and ultracentrifugated to collect the supernatant and pellet. Western blot suggested that sFasL released from ovarian cancer cells were the mature form, and these sFasL are released with the small membrane microvesicles. Flow cytometry showed that the majority of microvesicles secreted contained FasL on their membrane, and these small membrane microvesicles are bioactive against activated human T lymphocytes. The microtubule-disrupting reagent nocodazole, not the actin-filament-disrupting reagent cytochalasin D pretreatment blocked FasL-expressing small membrane microvesicle release stimulated by LPA, suggesting that microtubules play an essential role in FasL microvesicle transport and exocytosis. LPA may promote ovarian cancer metastasis by counterattacking peritoneal cavity anti-tumor immunity.
Asunto(s)
Lisofosfolípidos/farmacología , Glicoproteínas de Membrana/biosíntesis , Metástasis de la Neoplasia/fisiopatología , Neoplasias Ováricas/patología , Western Blotting , Citoesqueleto/fisiología , Ensayo de Inmunoadsorción Enzimática , Proteína Ligando Fas , Femenino , Citometría de Flujo , Humanos , Microtúbulos/fisiología , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
OBJECTIVES: Alterations in the expression of Fas have been demonstrated in various cancers as a mechanism for tumor cells to escape from immune surveillance. In this study, we observed the effect of lysophosphatidic acid (LPA) on Fas expression and function in ovarian cancer cells. METHODS: Ovarian cancer cell lines were incubated with or without LPA and Fas cell surface presentations were detected by flow cytometry. Anti-Fas IgM was added for induction and analysis of apoptosis by flow cytometry. Cell lysis and subcellular fractions were probed for protein expression by Western blot. Cells were also stained with human anti-Fas Ab, followed with Rhodamine red-X-conjugated goat anti-mouse IgG, and immunofluorescence images were acquired on a Nikon digital camera. RESULTS: Following treatment with LPA, ovarian cancer cells showed significant rapid reduction of Fas presentation on the cell surface. LPA protected ovarian cancer cells from anti-Fas-induced apoptosis. Cell lysis and subcellular fractionations proved that LPA treatment induced a translocation of Fas receptors, along with phosphorylated ezrin, from the membrane anchored to the actin cytoskeleton, to the cytosol. Translocation of the Fas receptor reduced Fas concentration in the membrane and may inhibit its clustering and internalization during early apoptosis induced by anti-Fas. DISC staining proved that LPA inhibited Fas receptor aggregation and caspase-8 activation at the membrane, which further inhibited caspase-3 and 7 activation in the cytosol. CONCLUSIONS: Our studies suggest that LPA induces translocation of Fas from the cell membrane to the cytosol, which may provide a mechanism by which ovarian cancer cells evade FasL-bearing immune cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Lisofosfolípidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Receptor fas/metabolismo , Apoptosis/fisiología , Caspasas/metabolismo , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Citosol/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática , Proteína Ligando Fas , Femenino , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/farmacología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Invasividad Neoplásica , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Receptor fas/inmunología , Receptor fas/fisiologíaRESUMEN
OBJECTIVE: Constitutive expression and upregulation of FasL by malignant epithelial cells counterattack infiltrating natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) and induce apoptosis of normal cells within the tumor, which may induce metastasis. As little is known about the mechanisms that regulate expression of Fas ligand and the subsequent release of FasL in epithelial ovarian cancer cells (EOC), we investigated the effects of lysophosphatidic acid (LPA) on FasL expression and associated signaling pathways. METHODS: We used established EOC cell lines that were incubated with or without LPA and FasL expression was detected by flow cytometry. Cells were additionally lysed and detected for total protein expression. Activated CD4+ T cells, after coculture with or without EOC, were collected for apoptosis staining and analysis by flow cytometry. RESULTS: Flow cytometry showed that LPA strongly upregulated FasL expression on the OVCAR3 cell surface (P < 0.01), yet in Dov13 cells, LPA significantly upregulated FasL expression only in the presence of the general matrix metalloproteinase (MMP) inhibitors GM6001 and MMP inhibitor II (P < 0.01). The MEK/ERK1/2 kinase cascade is required for FasL upregulation, since the MEK inhibitor PD98059 significantly inhibited FasL upregulation induced by LPA (P < 0.01). Type II secretory phospholipase A2 (sPLA2-II), which promotes protein exocytosis from secretory vesicles and gelatinase granules, affects FasL translocation from intracellular to the cell surface. Pretreatment of Dov13 cells with LPA increased activated T cell apoptosis in cocultures. CONCLUSIONS: These data suggest that upregulation of FasL by LPA provides EOC immune-privilege and leads to apoptosis of activated T lymphocytes.