RESUMO
Breast cancer (BC) is a complex disease comprising multiple distinct subtypes with different genetic features and pathological characteristics. Although a large number of antineoplastic compounds have been approved for clinical use, patient-to-patient variability in drug response is frequently observed, highlighting the need for efficient treatment prediction for individualized therapy. Several patient-derived models have been established lately for the prediction of drug response. However, each of these models has its limitations that impede their clinical application. Here, we report that the whole-tumor cell culture (WTC) ex vivo model could be stably established from all breast tumors with a high success rate (98 out of 116), and it could reassemble the parental tumors with the endogenous microenvironment. We observed strong clinical associations and predictive values from the investigation of a broad range of BC therapies with WTCs derived from a patient cohort. The accuracy was further supported by the correlation between WTC-based test results and patients' clinical responses in a separate validation study, where the neoadjuvant treatment regimens of 15 BC patients were mimicked. Collectively, the WTC model allows us to accomplish personalized drug testing within 10 d, even for small-sized tumors, highlighting its potential for individualized BC therapy. Furthermore, coupled with genomic and transcriptomic analyses, WTC-based testing can also help to stratify specific patient groups for assignment into appropriate clinical trials, as well as validate potential biomarkers during drug development.
Assuntos
Antineoplásicos , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Perfilação da Expressão Gênica , Biomarcadores , Técnicas de Cultura de Células , Microambiente TumoralRESUMO
Multiple myeloma (MM) is an incurable hematological cancer, in which immune checkpoint inhibition (ICI) with monoclonal antibodies (mAbs) has failed due to uncontrollable immune responses in combination therapies and lack of efficacy in monotherapies. Although NK cell-specific checkpoint targets such as NKG2A and KIRs are currently being evaluated in clinical trials, the clinical impact of NK cells on the PD1 cascade is less well understood compared to T cells. Furthermore, while NK cells have effector activity within the TME, under continuous ligand exposure, NK cell dysfunctionality may occur due to interaction of PD1 and its ligand PD-L1. Due to above-mentioned factors, we designed novel NK cell specific PD1-based chimeric switch receptors (PD1-CSR) by employing signaling domains of DAP10, DAP12 and CD3ζ to revert NK cell inhibition and retarget ICI. PD1-CSR modified NK cells showed increased degranulation, cytokine secretion and cytotoxicity upon recognition of PD-L1+ target cells. Additionally, PD1-CSR+ NK cells infiltrated and killed tumor spheroids. While primary NK cells (pNK), expressing native PD1, showed decreased degranulation and cytokine production against PD-L1+ target cells by twofold, PD1-CSR+ pNK cells demonstrated increased activity upon PD-L1+ target cell recognition and enhanced antibody-dependent cellular cytotoxicity. PD1-CSR+ pNK cells from patients with MM increased degranulation and cytokine expression against autologous CD138+PD-L1+ malignant plasma cells. Taken together, the present results demonstrate that PD1-CSR+ NK cells enhance and sustain potent anti-tumor activity in a PD-L1+ microenvironment and thus represent a promising strategy to advance adoptive NK cell-based immunotherapies toward PD-L1+ cancers.
Assuntos
Antígeno B7-H1 , Mieloma Múltiplo , Humanos , Antígeno B7-H1/metabolismo , Ligantes , Células Matadoras Naturais , Citocinas/metabolismo , Receptores de Células Matadoras Naturais/metabolismo , Imunoterapia/métodos , Microambiente TumoralRESUMO
BACKGROUND AIMS: Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38dim phenotype occurring during long-term cytokine stimulation of primary NK cells. METHODS: Primary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays. RESULTS: We verified the functionality of the αCD38-CAR-NK cells against CD38+ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo. CONCLUSIONS: Overall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.
Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Citocinas/metabolismo , Mieloma Múltiplo/terapia , Leucócitos Mononucleares/metabolismo , Células Matadoras Naturais , Fenótipo , Imunoterapia , Imunoterapia Adotiva/métodos , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Inadequate persistence of tumor-infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL-2 or IL-15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL-15, in contrast to IL-2, enriches for CD25+ /CD54+ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25+ /CD54+ NK cells for adoptive cell therapy should be considered.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Dinoprostona , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Citocinas , Humanos , Células Matadoras Naturais , Transdução de SinaisRESUMO
Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.
Assuntos
Imunoterapia/métodos , Linfócitos do Interstício Tumoral/metabolismo , Análise de Célula Única/métodos , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Imunoterapia Adotiva/métodos , Ativação Linfocitária/fisiologia , Modelos Animais , Metástase Neoplásica/patologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Peixe-ZebraRESUMO
Natural killer (NK) cells, which are innate lymphocytes endowed with potent cytotoxic activity, have recently attracted attention as potential anticancer therapeutics. While NK cells mediate encouraging responses in patients with leukemia, the therapeutic effects of NK cell infusion in patients with solid tumors are limited. Preclinical and clinical data suggest that the efficacy of NK cell infusion against solid malignancies is hampered by several factors including inadequate tumor infiltration and persistence/activation in the tumor microenvironment (TME). A number of metabolic features of the TME including hypoxia as well as elevated levels of adenosine, reactive oxygen species, and prostaglandins negatively affect NK cell activity. Moreover, cancer-associated fibroblasts, tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells actively suppress NK cell-dependent anticancer immunity. Here, we review the metabolic and cellular barriers that inhibit NK cells in solid neoplasms as we discuss potential strategies to circumvent such obstacles towards superior therapeutic activity.
Assuntos
Células Supressoras Mieloides , Neoplasias , Humanos , Células Matadoras Naturais , Microambiente Tumoral , Neoplasias/metabolismo , Células Supressoras Mieloides/metabolismoRESUMO
There is an urgent need for new treatment options in metastatic drug-resistant prostate cancer. Combining immunotherapy with other targeted therapies may be an effective strategy for advanced prostate cancer. In the present study, we sought to investigate to enhance the efficacy of anti-CTLA-4 therapy against prostate cancer by the combination with STAT3 inhibition.Male C57BL6 mice were subcutaneously inoculated with the murine prostate cancer cell line RM-1. Tumor progression was monitored following treatment with vehicle, the small molecule STAT3 inhibitor GPB730, anti-CTLA-4 or GPB730 + anti-CTLA-4. Treatment with anti-CTLA-4 or anti-CTLA-4 + GPB730 significantly inhibited tumor growth and enhanced survival compared to vehicle. Combining anti-CTLA-4 treatment with GPB730 resulted in a significantly prolonged survival compared to anti-CTLA-4 alone. GPB730 significantly increased infiltration of CD45 + cells in tumors of anti-CTLA-4-treated mice compared to anti-CTLA-4 alone. The levels of tumor-infiltrating Tregs were significantly decreased and the CD8:Treg ratio significantly increased by GPB730 treatment in combination with anti-CTLA-4 compared to anti-CTLA-4 alone. Immunohistochemical analysis showed a significant increase in CD45-positive cells in anti-CTLA-4 and anti-CTLA-4 + GPB730-treated tumors compared to vehicle or GPB730 monotherapy. Plasma levels of IL10 were significantly increased by anti-CTLA-4 compared to vehicle but no increase was observed when combining anti-CTLA-4 with GPB730.In conclusion, STAT3 inhibition by GPB730 enhances the antitumoral activity of anti-CTLA-4 and decreases the intratumoral Treg frequency in a prostate cancer mouse model. These results support the combination of STAT3 inhibition with anti-CTLA-4 therapy to increase clinical responses in patients with prostate cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Lactonas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Chemokines are soluble proteins that orchestrate cell migration in a regulated concentration gradient. During early stages of tumor development, chemokines shape the immune landscape of tumor microenvironment. CXCL9, also known as monokine induced by gamma-interferon (MIG), can be produced during inflammatory conditions by myeloid cells within the tumor microenvironment. It attracts cells expressing the CXCR3 receptor including activated T and NK cells and has been shown to play a role in responses to immune checkpoint therapy. Overexpression of CXCL9 has also shown to reduce tumor progression and metastasis via the inhibition of angiogenesis. Conversely, CXCL9 can act directly on tumor cells expressing the CXCR3 receptor to promote cell migration and epithelial mesenchymal transition. In this chapter we discuss the anti- and pro-tumoral features of CXCL9 within the tumor microenvironment.
Assuntos
Quimiocina CXCL9/metabolismo , Microambiente Tumoral , Animais , Humanos , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Receptores CXCR3/metabolismo , Linfócitos T/imunologiaRESUMO
Components of the tumor microenvironment (TME) are known to play an essential role during malignant progression, but often in a context-dependent manner. In bone and soft tissue sarcomas, disease-regulatory activities in the TME remain largely uncharacterized. This chapter introduces the cellular, structural, and chemical composition of the sarcoma TME from a pathobiological and therapeutic perspective.Sarcomas are malignant tumors with diverse features when it comes to primary tumor appearance, metastatic potential, and response to treatment. Many of the classic subtypes are mainly composed of malignant cells and are therefore assumed to be committed to autocrine signaling. Some of the tumors are infiltrated by immune cells and contain necrotic areas or excessive amounts of extracellular matrix (ECM) that regulates tissue stiffness and interstitial fluid pressure. Vascular invasion and blood vessel characteristics can in some instances be considered in the prognostic setting.Further insights into the disease-regulatory activities of the sarcoma TME will provide essential knowledge on how to develop successful combination treatments targeting not only malignant cells, but also their routes of nutrition and ability to shield themselves toward existing therapy.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Matriz Extracelular , Humanos , Sarcoma/terapia , Microambiente TumoralRESUMO
Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy.
Assuntos
Citotoxicidade Imunológica/imunologia , Imunoterapia Adotiva , Interleucina-15/farmacologia , Células Matadoras Naturais/imunologia , Neoplasias Experimentais/terapia , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Ativação Linfocitária , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Mitocondriais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Transdução de SinaisRESUMO
Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.
Assuntos
Células Dendríticas/imunologia , Interferon gama/farmacologia , Linfócitos T/imunologia , Receptores Toll-Like/agonistas , Diferenciação Celular , HumanosRESUMO
Dendritic cell (DC) vaccines induce T-cell responses in cancer patients. However, there is a paucity of data regarding the role of DC vaccines in shaping natural killer (NK) cell responses. Here, we observe that NK cells are less activated following DC vaccination. In vitro, DC-mediated inhibition of NK cells did not require cell-to-cell contact, but required increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation (pSTAT3) in DCs. When phosphorylation of STAT3 was inhibited in DCs, we found that DCs did not suppress NK cells, and observed an increase in the production of lymphotoxin-alpha (LTα) and interleukin-12 (IL-12) as well as reduced release of transforming growth factor beta (TGF-ß). The addition of recombinant LTα or IL-12 to the DC-NK-cell cocultures restored NK-cell activity, and neutralization of TGF-ß resulted in elevated production of LTα and IL-12 from DCs. Compared with LPS, DCs matured with a cocktail of R848, poly I:C, and IFN-γ showed reduced levels of pSTAT3 and higher levels of LTα and IL-12 and did not inhibit NK-cell activity. These results show that LTα, IL-12, and TGF-ß are involved in the cross-talk between NK cells and DCs. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfotoxina-alfa/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/imunologia , Vacinas Anticâncer , Comunicação Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Imunoterapia , Interferon gama/biossíntese , Interleucina-12/farmacologia , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfotoxina-alfa/farmacologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Fenótipo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca(2+) responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell-mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43-GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell-mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca(2+) influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.
Assuntos
Conexina 43/imunologia , Citotoxicidade Imunológica/imunologia , Células Dendríticas/imunologia , Junções Comunicantes/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Apoptose , Sinalização do Cálcio , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Conexina 43/antagonistas & inibidores , Células Dendríticas/ultraestrutura , Granzimas/fisiologia , Humanos , Vigilância Imunológica , Sinapses Imunológicas/imunologia , Testes de Liberação de Interferon-gama , Células Matadoras Naturais/ultraestruturaRESUMO
Adoptive infusion of natural killer (NK) cells is being increasingly explored as a therapy in patients with cancer, although clinical responses are thus far limited to patients with hematological malignancies. Inadequate homing of infused NK cells to the tumor site represents a key factor that may explain the poor anti-tumor effect of NK cell therapy against solid tumors. One of the major players in the regulation of lymphocyte chemotaxis is the chemokine receptor chemokine (C-X-C motif) receptor 3 (CXCR3) which is expressed on activated NK cells and induces NK cell migration toward gradients of the chemokine (C-X-C motif) ligand (CXCL9, 10 and 11). Here, we show that ex vivo expansion of human NK cells results in a tenfold increased expression of the CXCR3 receptor compared with resting NK cells (p = 0.04). Consequently, these NK cells displayed an improved migratory capacity toward solid tumors, which was dependent on tumor-derived CXCL10. In xenograft models, adoptively transferred NK cells showed increased migration toward CXCL10-transfected melanoma tumors compared with CXCL10-negative wild-type tumors, resulting in significantly reduced tumor burden and increased survival (median survival 41 vs. 32 days, p = 0.03). Furthermore, administration of interferon-gamma locally in the tumor stimulated the production of CXCL10 in subcutaneous melanoma tumors resulting in increased infiltration of adoptively transferred CXCR3-positive expanded NK cells. Our findings demonstrate the importance of CXCL10-induced chemoattraction in the anti-tumor response of adoptively transferred expanded NK cells against solid melanoma tumors.
Assuntos
Quimiocina CXCL10/imunologia , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Transferência Adotiva , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CXCL10/metabolismo , Quimiocinas CXC/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Humanos , Interferon gama/administração & dosagem , Interferon gama/metabolismo , Interferon gama/farmacologia , Células Matadoras Naturais/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Receptores CXCR3/metabolismo , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.
Assuntos
Citocinas/genética , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Compostos Heterocíclicos/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Benzilaminas , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Ciclamos , Citocinas/biossíntese , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Doença Enxerto-Hospedeiro/imunologia , Humanos , Imunofenotipagem , Teste de Cultura Mista de Linfócitos , Linfopoese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Quimera por Radiação , Receptores CXCR4/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologiaRESUMO
Natural killer (NK) cells are innate lymphocytes that are able to directly kill tumor cells through different mechanisms including ligation of TNF-related apoptosis-inducing ligand (TRAIL) receptors. Zoledronic acid (ZA) is a bisphosphonate known to upregulate the expression of TRAIL on human γδ T cells. Here, we investigated whether exposure to ZA would upregulate TRAIL expression on human NK cells and augment their cytotoxicity against tumor cells. When cocultured with monocytes, treatment with ZA and IL-2 resulted in a significant upregulation of TRAIL expression on human NK cells (p = 0.002). Consequently, ZA-primed NK cells were significantly more cytotoxic against TRAIL sensitive tumor cells (p < 0.0001). In the presence of ZA and IL-2, monocytes produced high levels of IFN-γ; when cultured in the presence of neutralizing antibodies to IFN-γ, TRAIL expression and TRAIL-mediated cytotoxicity of NK cells were significantly reduced. Furthermore, in tumor-bearing SCID/Beige mice, a significant delayed tumor progression and prolonged survival was observed after infusion of ZA-primed NK cells compared with that observed in mice infused with unprimed NK cells. These findings represent a novel approach to potentiate TRAIL-mediated apoptosis by adoptively infused NK cells that could improve the outcome in patients with cancer.
Assuntos
Citotoxicidade Imunológica , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Difosfonatos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Imidazóis/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/transplante , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos SCID , Neoplasias/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido ZoledrônicoRESUMO
Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1ß promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.
Assuntos
Neoplasias da Mama/imunologia , Testes Imunológicos de Citotoxicidade , Antígenos de Histocompatibilidade Classe I/biossíntese , Células Matadoras Naturais/imunologia , Receptor ErbB-2/fisiologia , Receptor ErbB-3/fisiologia , Transdução de Sinais/imunologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Sistema de Sinalização das MAP Quinases/imunologia , Receptor ErbB-2/biossíntese , Receptor ErbB-3/biossíntese , Transdução de Sinais/genética , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Evasão Tumoral/genéticaRESUMO
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand- receptor (TRAIL-R) family has emerged as a key mediator of cell fate and survival. Ligation of TRAIL ligand to TRAIL-R1 or TRAIL-R2 initiates the extrinsic apoptotic pathway characterized by the recruitment of death domains, assembly of the death-inducing signaling complex (DISC), caspase activation and ultimately apoptosis. Conversely the decoy receptors TRAIL-R3 and TRAIL-R4, which lack the pro-apoptotic death domain, function to dampen the apoptotic response by competing for TRAIL ligand. The tissue restricted expression of the decoy receptors on normal but not cancer cells provides a therapeutic rational for the development of selective TRAIL-mediated anti-tumor therapies. Recent clinical trials using agonistic antibodies against the apoptosis-inducing TRAIL receptors or recombinant TRAIL have been promising; however the number of patients in complete remission remains stubbornly low. The mechanisms of TRAIL resistance are relatively unexplored but may in part be due to TRAIL-R down-regulation or shedding of TRAIL-R by tumor cells. Therefore a better understanding of the mechanisms underlying TRAIL resistance is required. The ubiquitin-proteasome system (UPS) has been shown to regulate TRAIL-R members suggesting that pharmacological inhibition of the UPS may be a novel strategy to augment TRAIL-based therapies and increase efficacies. We recently identified b-AP15 as an inhibitor of proteasome deubiquitinase (DUB) activity. Interestingly, exposure of tumor cell lines to b-AP15 resulted in increased TRAIL-R2 expression and enhanced sensitivity to TRAIL-mediated apoptosis and cell death in vitro and in vivo. In conclusion, targeting the UPS may represent a novel strategy to increase the cell surface expression of pro-apoptotic TRAIL-R on cancer cells and should be considered in clinical trials targeting TRAIL-receptors in cancer patients.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Ubiquitina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Immune checkpoint inhibitors (ICIs) are linked to diverse immune-related adverse events (irAEs). Rare irAEs surface first in clinical practice. Here, we systematically studied the rare irAE, cytokine-release syndrome (CRS), in a cohort of 2672 patients treated with ICIs at Karolinska University Hospital in Stockholm, Sweden. We find that the risk of ICI-induced CRS - defined as fever, negative microbiological findings and absence of other probable causes within 30 days after ICI treatment - is approximately 1%, higher than previously reported. ICI-induced CRS was often mild and rechallenge with ICIs after mild CRS was generally safe. However, two out of 28 patients experienced high-grade CRS, and one was fatal. While C-reactive protein (CRP) and procalcitonin were not discriminative of fatal CRS, our data suggest that the quick Sequential Organ Failure Assessment (qSOFA) score might identify high-risk patients. These data provide a framework for CRS risk assessment and motivate multicenter studies to improve early CRS diagnosis.
Assuntos
Síndrome da Liberação de Citocina , Inibidores de Checkpoint Imunológico , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Masculino , Suécia/epidemiologia , Feminino , Pessoa de Meia-Idade , Idoso , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/sangue , Adulto , Estudos de Coortes , Hospitais Universitários , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: Tumor cells have the ability to invade and form small clusters that protrude into adjacent tissues, a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro, p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction. METHODS: Using epithelial cells transfected with inducible p60AmotL2, we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo, drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models, both in 2D and in 3D, and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells. RESULTS: We identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly, these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models, as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells. CONCLUSIONS: BET inhibitors specifically target p60AmotL2-expressing invasive cancer cells, likely by exploiting differences in chromatin accessibility, leading to cell death. Additionally, our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells.