RESUMEN
Cancer immunotherapy induces a variety of autoinflammatory responses, including those against the thyroid gland, which can be exploited to predict clinical outcomes. Considering the paucity of information about thyroid autoimmunity in patients receiving cancer vaccines, we designed our study to assess the development of thyroglobulin antibodies (TgAbs) in patients treated with GVAX (vaccine made of a tumor cell type transfected with GM-CSF) and/or ipilimumab and correlated seroconversion with survival. Using both in house and commercial ELISA assays, we measured TgAbs in patients with pancreatic (No. = 53), prostate (No. = 35) or colon (No. = 8) cancer, before and after treatment with GVAX only (No. = 34), GVAX plus ipilimumab (No. = 42) or ipilimumab (No. = 20), and correlated their levels with patient's survival, disease status and T-cell surface markers. Antibodies to thyroperoxidase, myeloperoxidase, proteinase 3, insulin and actin were also measured. TgAbs specifically developed after GVAX, independent of the underlying cancer (81% in prostate, 75% colon cancer and 76% pancreatic cancer) and co-administration of ipilimumab (75% in GVAX only and 78% in GVAX plus ipilimumab). This TgAbs seroconversion could be detected mainly by the in house assay, suggesting that the thyroglobulin epitopes recognized by the antibodies induced by GVAX are different from the epitopes seen in the classic form of Hashimoto thyroiditis. Notably, TgAbs seroconversion was associated with significantly prolonged survival (p = 0.01 for pancreas and p = 0.005 for prostate cancer). In conclusion, GVAX immunotherapy induces the appearance of TgAbs that recognize a unique antigenic repertoire and associate with prolonged survival.
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Vacunas contra el Cáncer/administración & dosificación , Neoplasias del Colon/terapia , Neoplasias Pancreáticas/terapia , Neoplasias de la Próstata/terapia , Tiroglobulina/inmunología , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antineoplásicos/sangre , Antineoplásicos/administración & dosificación , Autoanticuerpos/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Estudios de Cohortes , Neoplasias del Colon/sangre , Neoplasias del Colon/inmunología , Neoplasias del Colon/mortalidad , Terapia Combinada , Humanos , Ipilimumab , Masculino , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/mortalidad , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/mortalidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Supervivencia , Tiroglobulina/genética , Tiroglobulina/metabolismo , Tirotropina/sangre , VacunaciónRESUMEN
BACKGROUND & AIMS: Premalignant lesions and early stage tumors contain immunosuppressive microenvironments that create barriers for cancer vaccines. Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which express an activated form of Kras in pancreatic tissues, develop pancreatic intraepithelial neoplasms (PanIN) that progress to pancreatic ductal adenocarcinoma (PDA). We used these mice to study immune suppression in PDA. METHODS: We immunized KPC and Kras(G12D/+);Pdx-1-Cre mice with attenuated intracellular Listeria monocytogenes (which induces CD4(+) and CD8(+) T-cell immunity) engineered to express Kras(G12D) (LM-Kras). The vaccine was given alone or in sequence with an anti-CD25 antibody (PC61) and cyclophosphamide to deplete T-regulatory (Treg) cells. Survival times were measured; pancreatic and spleen tissues were collected and analyzed by histologic, flow cytometry, and immunohistochemical analyses. RESULTS: Interferon γ-mediated, CD8(+) T-cell responses were observed in KPC and Kras(G12D/+);Pdx-1-Cre mice given LM-Kras, but not in unvaccinated mice. Administration of LM-Kras to KPC mice 4-6 weeks old (with early stage PanINs), depleted of Treg cells, significantly prolonged survival and reduced PanIN progression (median survival, 265 days), compared with unvaccinated mice (median survival, 150 days; P = .002), mice given only LM-Kras (median survival, 150 days; P = .050), and unvaccinated mice depleted of Treg cells (median survival, 170 days; P = .048). In 8- to 12-week-old mice (with late-stage PanINs), LM-Kras, alone or in combination with Treg cell depletion, did not increase survival time or slow PanIN progression. The combination of LM-Kras and Treg cell depletion reduced numbers of Foxp3(+)CD4(+) T cells in pancreatic lymph nodes, increased numbers of CD4(+) T cells that secrete interleukin 17 and interferon γ, and caused CD11b(+)Gr1(+) cells in the pancreas to acquire an immunostimulatory phenotype. CONCLUSIONS: Immunization of KPC mice with Listeria monocytogenes engineered to express Kras(G12D), along with depletion of Treg cells, reduces progression of early stage, but not late-stage, PanINs. This approach increases infiltration of the lesion with inflammatory cells. It might be possible to design immunotherapies against premalignant pancreatic lesions to slow or prevent progression to PDA.
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Vacunas contra el Cáncer/uso terapéutico , Carcinoma in Situ/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Listeria monocytogenes/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígeno CD11b/metabolismo , Vacunas contra el Cáncer/inmunología , Carcinoma in Situ/genética , Carcinoma in Situ/inmunología , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Ciclofosfamida/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Integrasas/genética , Integrasas/metabolismo , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores de Quimiocina/metabolismo , Linfocitos T Reguladores/metabolismo , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Preclinical murine models in which primary tumors spontaneously metastasize to distant organs are valuable tools to study metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line, NT2.5-lung metastasis (-LM), that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. Within one week of orthotopic implantation of NT2.5-LM in NeuN mice, distant metastases can be observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. Metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. We demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT) and enrichment in EMT-regulating pathways, suggestive of their enhanced metastatic potential. The new NT2.5-LM model provides more rapid and spontaneous development of widespread metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses targeting distant visceral metastases.
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Pancreatic cancer is more prevalent in older individuals and often carries a poorer prognosis for them. The relationship between the microenvironment and pancreatic cancer is multifactorial, and age-related changes in nonmalignant cells in the tumor microenvironment may play a key role in promoting cancer aggressiveness. Because fibroblasts have profound impacts on pancreatic cancer progression, we investigated whether age-related changes in pancreatic fibroblasts influence cancer growth and metastasis. Proteomics analysis revealed that aged fibroblasts secrete different factors than young fibroblasts, including increased growth/differentiation factor 15 (GDF-15). Treating young mice with GDF-15 enhanced tumor growth, whereas aged GDF-15 knockout mice showed reduced tumor growth. GDF-15 activated AKT, rendering tumors sensitive to AKT inhibition in an aged but not young microenvironment. These data provide evidence for how aging alters pancreatic fibroblasts and promotes tumor progression, providing potential therapeutic targets and avenues for studying pancreatic cancer while accounting for the effects of aging. SIGNIFICANCE: Aged pancreatic fibroblasts secrete GDF-15 and activate AKT signaling to promote pancreatic cancer growth, highlighting the critical role of aging-mediated changes in the pancreatic cancer microenvironment in driving tumor progression. See related commentary by Isaacson et al., p. 1185.
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Fibroblastos Asociados al Cáncer , Neoplasias Pancreáticas , Animales , Ratones , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/uso terapéutico , Proteínas Proto-Oncogénicas c-akt , Neoplasias Pancreáticas/patología , Páncreas/patología , Fibroblastos/patología , Microambiente Tumoral , Línea Celular Tumoral , Fibroblastos Asociados al Cáncer/patologíaRESUMEN
PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.
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Complejo CD3 , Endopeptidasas , Proteínas Ligadas a GPI , Inmunoterapia Adoptiva , Mesotelina , Neoplasias Pancreáticas , Receptores Quiméricos de Antígenos , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/inmunología , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Línea Celular Tumoral , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Adenocarcinoma/inmunología , Adenocarcinoma/terapia , Adenocarcinoma/patologíaRESUMEN
We previously reported that CD8(+) T cells are directed predominantly toward the immunodominant Her-2/neu (neu) epitope RNEU(420-429) in nontolerized FVB/N but not tolerized HER-2/neu (neu-N) mice. In this study, we screened overlapping peptides of the entire neu protein and identified six new epitopes recognized by vaccine-induced neu-N-derived T cells. Evaluation of individual nondominant responses by tetramer staining and IFN-γ secretion demonstrate that this repertoire is peripherally tolerized. To address the role that the complete CD8(+) T cell repertoire plays in vaccine-induced antitumor immunity, we created a whole-cell vaccine-expressing neu cDNA that has been mutated at the RNEU(420-429) anchor residue, thereby abrogating activation of immunodominant epitope responses. Studies comparing the mutated and nonmutated vaccines indicate that nondominant CD8(+) T cells can induce antitumor immunity when combined with regulatory T cell-depleting agents in both neu-N and FVB/N mice. Collectively, these studies demonstrate that the neu-directed T cell repertoire is not intrinsically incapable of eradicating tumors. Rather, they are suppressed by mechanisms of peripheral tolerance. Thus, these studies provide new insights into the function of the complete T cell repertoire directed toward a clinically relevant tumor Ag in tumor-bearing hosts.
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Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Epítopos de Linfocito T/metabolismo , Epítopos Inmunodominantes/administración & dosificación , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/terapia , Receptor ErbB-2/deficiencia , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta Inmunológica , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Tolerancia Inmunológica/genética , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Transgénicos , Células 3T3 NIH , Ratas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMEN
Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.
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Vacunas contra el Cáncer , Neoplasias Pancreáticas , Ratones , Animales , Linfocitos T CD4-Positivos , Antígenos de Neoplasias , Eficacia de las Vacunas , Neoplasias Pancreáticas/metabolismo , Microambiente TumoralRESUMEN
The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3-expressing CD8(+) T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti-tumor response. CD8(+) Foxp3(+) T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM-CSF secreting HER-2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen-specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3(+) and Foxp3(-) CD8(+) T cells have similar IFN-γ production and antigen-specific degranulation after stimulation with RNEU(420-429) , the immunodominant HER-2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU((420-429)) -specific effector T cells into neu-N mice (a model that results in immune tolerance to neu), confirm that CD8(+) Foxp3(+) T cells are present in tumors only if there is an existing pool of tumor-rejecting effector T cells. CD8(+) Foxp3(+) TILs mark the presence of tumor-rejecting antigen-specific T cells and their accumulation serves as a marker for an effective T cell response.
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Linfocitos T CD8-positivos/inmunología , Factores de Transcripción Forkhead/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Mamarias Experimentales/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Tolerancia Inmunológica , Activación de Linfocitos , Ratones , Ratones Transgénicos , Factor de Crecimiento Transformador beta/fisiología , Microambiente TumoralRESUMEN
A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8(+) T cell responses to a dominant peptide (RNEU(420-429)) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N-derived CD4(+)CD25(+) T cells. RNEU(420-429)-specific CD8(+) T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU(420-429)-specific CD8(+) T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4(+)CD25(+) T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8(+) T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.
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Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Neoplasias Mamarias Experimentales/inmunología , Animales , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/genética , Comunicación Celular/efectos de los fármacos , Comunicación Celular/inmunología , Ciclofosfamida/administración & dosificación , Citotoxicidad Inmunológica , Epítopos de Linfocito T/genética , Femenino , Genes erbB-2/inmunología , Inmunosupresores/administración & dosificación , Inmunoterapia Adoptiva , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Transgénicos , Receptores de Interleucina-2/inmunología , VacunaciónRESUMEN
The pathogenic nature of cancer is attributed, at least in part, to the ability of tumors cells to induce systemic and local mechanisms of immune tolerance. However, we previously reported that tumor-free survival in up to 100% of tolerized HER-2/neu transgenic mice can be achieved by administration of neu-specific mAb concurrently with a HER-2/neu-expressing, GM-CSF-secreting whole cell vaccine. In this report, we show that one mechanism of improved antitumor activity induced by the combination of these 2 neu-targeted interventions was enhanced Fc-mediated activation of APCs. Specifically, in vivo studies demonstrated localization of radiolabeled neu-specific mAb at the vaccine site. Subsequently, increased accumulation of neu-specific mAb at the vaccine-draining lymph node correlated with increased vaccine cell uptake by DCs in vivo. This led to enhancement of CD8(+) neu-specific T cell function in terms of proliferation, cytokine production, and central memory development. Thus, the administration of a neu-specific mAb with a neu-targeted GM-CSF-secreting tumor vaccine enhanced induction of neu-specific CD8(+) T cells through Fc-mediated activation of DCs. This multimodality attack on the same tumor antigen may have the potential to overcome tolerance to self antigens and weaken the immunosuppressive networks within the tumor microenvironment.
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Anticuerpos Monoclonales/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Receptor ErbB-2/inmunología , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígeno CD11c/inmunología , Linfocitos T CD8-positivos/citología , Línea Celular , Células Dendríticas/citología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Neoplasias/inmunología , Neoplasias/patología , Receptor ErbB-2/genética , Tasa de SupervivenciaRESUMEN
Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.
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BACKGROUND: The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC. RESULTS: By leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors. CONCLUSIONS: Determining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.
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Genómica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Transducción de Señal , Microambiente Tumoral/inmunología , Animales , Autopsia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Línea Celular Tumoral , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Terapia de Inmunosupresión , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/secundario , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias Pancreáticas/patología , Linfocitos T/inmunología , Microambiente Tumoral/genéticaRESUMEN
Tumor involvement of major vascular structures limits surgical options in pancreatic adenocarcinoma (PDAC), which in turn limits opportunities for cure. Despite advances in locoregional approaches, there is currently no role for incomplete resection. This study evaluated a gelatinized neoantigen-targeted vaccine applied to a grossly positive resection margin in preventing local recurrence. Incomplete surgical resection was performed in mice bearing syngeneic flank Panc02 tumors, leaving a 1 mm rim adherent to the muscle bed. A previously validated vaccine consisting of neoantigen peptides, a stimulator of interferon genes (STING) agonist and AddaVaxTM (termed PancVax) was embedded in a hyaluronic acid hydrogel and applied to the tumor bed. Tumor remnants, regional lymph nodes, and spleens were analyzed using histology, flow cytometry, gene expression profiling, and ELISPOT assays. The immune microenvironment at the tumor margin after surgery alone was characterized by a transient influx of myeloid-derived suppressor cells (MDSCs), prolonged neutrophil influx, and near complete loss of cytotoxic T cells. Application of PancVax gel was associated with enhanced T cell activation in the draining lymph node and expansion of neoantigen-specific T cells in the spleen. Mice implanted with PancVax gel demonstrated no evidence of residual tumor at two weeks postoperatively and healed incisions at two months postoperatively without local recurrence. In summary, application of PancVax gel at a grossly positive tumor margin led to systemic expansion of neoantigen-specific T cells and effectively prevented local recurrence. These findings support further work into locoregional adjuncts to immune modulation in PDAC.
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Adenocarcinoma , Vacunas contra el Cáncer , Neoplasias Pancreáticas , Adenocarcinoma/prevención & control , Adenocarcinoma/cirugía , Animales , Hidrogeles , Inmunoterapia , Ratones , Microambiente TumoralRESUMEN
Tumor-specific CD8(+) T cells can potentially be activated by two distinct mechanisms of major histocompatibility complex class I-restricted antigen presentation as follows: direct presentation by tumor cells themselves or indirect presentation by professional antigen-presenting cells (APCs). However, controversy still exists as to whether indirect presentation (the cross-priming mechanism) can contribute to effective in vivo priming of tumor-specific CD8(+) T cells that are capable of eradicating cancer in patients. A clinical trial of vaccination with granulocyte macrophage-colony stimulating factor-transduced pancreatic cancer lines was designed to test whether cross-presentation by locally recruited APCs can activate pancreatic tumor-specific CD8(+) T cells. Previously, we reported postvaccination delayed-type hypersensitivity (DTH) responses to autologous tumor in 3 out of 14 treated patients. Mesothelin is an antigen demonstrated previously by gene expression profiling to be up-regulated in most pancreatic cancers. We report here the consistent induction of CD8(+) T cell responses to multiple HLA-A2, A3, and A24-restricted mesothelin epitopes exclusively in the three patients with vaccine-induced DTH responses. Importantly, neither of the vaccinating pancreatic cancer cell lines expressed HLA-A2, A3, or A24. These results provide the first direct evidence that CD8 T cell responses can be generated via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination site.
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Células Presentadoras de Antígenos/fisiología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Glicoproteínas de Membrana/inmunología , Neoplasias Pancreáticas/inmunología , Línea Celular , Proteínas Ligadas a GPI , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Antígeno HLA-A2/análisis , Antígeno HLA-A3/análisis , Antígeno HLA-A3/fisiología , Humanos , Hipersensibilidad Tardía/etiología , Mesotelina , Neoplasias Pancreáticas/terapia , VacunaciónRESUMEN
Subclinical doses of Paclitaxel (PTX) given 1day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu(+) tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8(+) T cells with enhanced lytic activity against neu(+) tumors. PTX treatment also enhances maturation marker expression on CD11c(+) DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8(+) T cells with greater lytic capability than DC's from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-gamma-secreting CD8(+) T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer , Células Dendríticas/efectos de los fármacos , Paclitaxel/farmacología , Animales , Antígenos CD/biosíntesis , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Quimioterapia Adyuvante , Ciclofosfamida/administración & dosificación , Ciclofosfamida/farmacología , Citocinas/genética , Citocinas/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Quimioterapia Combinada , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias/inmunología , Neoplasias/terapia , Receptor ErbB-2/inmunología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Carga Tumoral/efectos de los fármacosRESUMEN
In prior studies, we delineated the landscape of neoantigens arising from nonsynonymous point mutations in a murine pancreatic cancer model, Panc02. We developed a peptide vaccine by targeting neoantigens predicted using a pipeline that incorporates the MHC binding algorithm NetMHC. The vaccine, when combined with immune checkpoint modulators, elicited a robust neoepitope-specific antitumor immune response and led to tumor clearance. However, only a small fraction of the predicted neoepitopes induced T cell immunity, similarly to that reported for neoantigen vaccines tested in clinical studies. While these studies have used binding affinities to MHC I as surrogates for T cell immunity, this approach does not include spatial information on the mutated residue that is crucial for TCR activation. Here, we investigate conformational alterations in and around the MHC binding groove induced by selected minimal neoepitopes, and we examine the influence of a given mutated residue as a function of its spatial position. We found that structural parameters, including the solvent-accessible surface area (SASA) of the neoepitope and the position and spatial configuration of the mutated residue within the sequence, can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines.
Asunto(s)
Antígenos de Neoplasias/química , Vacunas contra el Cáncer/química , Epítopos/química , Simulación del Acoplamiento Molecular , Animales , Afinidad de Anticuerpos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Células Cultivadas , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunogenicidad Vacunal , Masculino , Ratones , Ratones Endogámicos C57BL , MutaciónRESUMEN
Immunotherapy has shown limited success in prostate cancer; this may be partially explained by its immunosuppressive tumor microenvironment (TME). Although androgen-deprivation therapy (ADT), the most common treatment for prostate cancer, initially promotes a robust T cell infiltrate, T cell responses are later attenuated. Based on the castration-sensitive Myc-CaP model, we developed an antigen-specific system to study CD8 T cell tolerance to prostate tumors. This model is unique in that CD8 T cells recognize a bona-fide tumor antigen (Her-2/neu), rather than an overexpressed xenogenic antigen like chicken ovalbumin or influenza hemagglutinin. Using this novel model, we demonstrate robust tolerance that is not alleviated by TLR agonists or ADT. This model may serve as a novel and useful tool to further interrogate methods by which to augment anti-tumor cancer immune responses to prostate cancer. Significance: Prostate cancer is a leading cause of cancer-related death in men worldwide, with an estimated 33,000 deaths projected in the U.S. in 2020. Although primary (localized) tumors can be cured by surgery or radiation, approximately 40% of patients eventually develop recurrent disease. While initially responsive to androgen-deprivation, many patients with recurrent prostate cancer eventually progress to a more advanced disease state known as metastatic castration-resistant prostate cancer (mCRPC); this is the lethal phenotype. These studies describe a novel androgen-responsive murine cell line that expresses a bona-fide tumor antigen (Her-2/neu). Pre-clinical work with this model shows robust and antigen-specific CD8 T cell tolerance, providing a novel preclinical model to study CD8 T cell tolerance to prostate tumors.
Asunto(s)
Antagonistas de Andrógenos , Neoplasias de la Próstata , Animales , Linfocitos T CD8-positivos , Humanos , Masculino , Ratones , Recurrencia Local de Neoplasia , Oncogenes , Neoplasias de la Próstata/terapia , Microambiente TumoralRESUMEN
Immunotherapies that modulate T cell function have been firmly established as a pillar of cancer therapy, whereas the potential for B cells in the antitumor immune response is less established. B cell-activating factor (BAFF) is a B cell-activating cytokine belonging to the TNF ligand family that has been associated with autoimmunity, but little is known about its effects on cancer immunity. We find that BAFF upregulates multiple B cell costimulatory molecules; augments IL-12a expression, consistent with Be-1 lineage commitment; and enhances B cell antigen-presentation to CD4+ Th cells in vitro. In a syngeneic mouse model of melanoma, BAFF upregulates B cell CD40 and PD-L1 expression; it also modulates T cell function through increased T cell activation and TH1 polarization, enhanced expression of the proinflammatory leukocyte trafficking chemokine CCR6, and promotion of a memory phenotype, leading to enhanced antitumor immunity. Similarly, adjuvant BAFF promotes a memory phenotype of T cells in vaccine-draining lymph nodes and augments the antitumor efficacy of whole cell vaccines. BAFF also has distinct immunoregulatory functions, promoting the expansion of CD4+Foxp3+ Tregs in the spleen and tumor microenvironment (TME). Human melanoma data from The Cancer Genome Atlas (TCGA) demonstrate that BAFF expression is positively associated with overall survival and a TH1/IFN-γ gene signature. These data support a potential role for BAFF signaling as a cancer immunotherapy.
Asunto(s)
Factor Activador de Células B/inmunología , Inmunidad Celular , Subunidad p35 de la Interleucina-12/inmunología , Melanoma Experimental/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Animales , Factor Activador de Células B/genética , Interferón gamma/inmunología , Subunidad p35 de la Interleucina-12/genética , Melanoma Experimental/genética , Melanoma Experimental/patología , RatonesRESUMEN
Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Mutación , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Apoptosis , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferación Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pronóstico , Células Tumorales CultivadasRESUMEN
The factors that influence nanoparticle fate in vivo following systemic delivery remain an area of intense interest. Of particular interest is whether labeling with a cancer-specific antibody ligand ("active targeting") is superior to its unlabeled counterpart ("passive targeting"). Using models of breast cancer in three immune variants of mice, we demonstrate that intratumor retention of antibody-labeled nanoparticles was determined by tumor-associated dendritic cells, neutrophils, monocytes, and macrophages and not by antibody-antigen interactions. Systemic exposure to either nanoparticle type induced an immune response leading to CD8+ T cell infiltration and tumor growth delay that was independent of antibody therapeutic activity. These results suggest that antitumor immune responses can be induced by systemic exposure to nanoparticles without requiring a therapeutic payload. We conclude that immune status of the host and microenvironment of solid tumors are critical variables for studies in cancer nanomedicine and that nanoparticle technology may harbor potential for cancer immunotherapy.