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Background: MHC class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. Methods: We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis. Results: Spatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (DFS) (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1×10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFNγ+/- NK cells and T cells. We discovered that both IFNγ+ NK and CD8 T cells were more frequently associated with other IFNγ+ lymphocytes in comparison to IFNγ- NK cells and CD8 T cells (p<1×10-30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells. Conclusions: Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Co-association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent co-localization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.
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Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.
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Linfonodos , Consumo de Oxigênio , Animais , Consumo de Oxigênio/fisiologia , Linfonodos/metabolismo , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , Oxigênio/análise , Linfócitos T/metabolismo , Linfócitos T/citologiaRESUMO
Background: Boiling histotripsy (BH), a mechanical focused ultrasound ablation strategy, can elicit intriguing signatures of anti-tumor immunity. However, the influence of BH on dendritic cell function is unknown, compromising our ability to optimally combine BH with immunotherapies to control metastatic disease. Methods: BH was applied using a sparse scan (1 mm spacing between sonications) protocol to B16F10-ZsGreen melanoma in bilateral and unilateral settings. Ipsilateral and contralateral tumor growth was measured. Flow cytometry was used to track ZsGreen antigen and assess how BH drives dendritic cell behavior. Results: BH monotherapy elicited ipsilateral and abscopal tumor control in this highly aggressive model. Tumor antigen presence in immune cells in the tumor-draining lymph nodes (TDLNs) was ~3-fold greater at 24h after BH, but this abated by 96h. B cells, macrophages, monocytes, granulocytes, and both conventional dendritic cell subsets (i.e. cDC1s and cDC2s) acquired markedly more antigen with BH. BH drove activation of both cDC subsets, with activation being dependent upon tumor antigen acquisition. Our data also suggest that BH-liberated tumor antigen is complexed with damage-associated molecular patterns (DAMPs) and that cDCs do not traffic to the TDLN with antigen. Rather, they acquire antigen as it flows through afferent lymph vessels into the TDLN. Conclusion: When applied with a sparse scan protocol, BH monotherapy elicits abscopal melanoma control and shapes dendritic cell function through several previously unappreciated mechanisms. These results offer new insight into how to best combine BH with immunotherapies for the treatment of metastatic melanoma.
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Ablação por Ultrassom Focalizado de Alta Intensidade , Melanoma , Humanos , Melanoma/terapia , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Antígenos de Neoplasias , Células DendríticasRESUMO
Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.
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Background: Boiling histotripsy (BH), a mechanical focused ultrasound ablation strategy, can elicit intriguing signatures of anti-tumor immunity. However, the influence of BH on dendritic cell function is unknown, compromising our ability to optimally combine BH with immunotherapies to control metastatic disease. Methods: BH was applied using a sparse scan (1 mm spacing between sonications) protocol to B16F10-ZsGreen melanoma in bilateral and unilateral settings. Ipsilateral and contralateral tumor growth was measured. Flow cytometry was used to track ZsGreen antigen and assess how BH drives dendritic cell behavior. Results: BH monotherapy elicited ipsilateral and abscopal tumor control in this highly aggressive model. Tumor antigen presence in immune cells in the tumor-draining lymph nodes (TDLNs) was ~3-fold greater at 24h after BH, but this abated by 96h. B cells, macrophages, monocytes, granulocytes, and both conventional dendritic cell subsets (i.e. cDC1s and cDC2s) acquired markedly more antigen with BH. BH drove activation of both cDC subsets, with activation being dependent upon tumor antigen acquisition. Our data also suggest that BH-liberated tumor antigen is complexed with damage-associated molecular patterns (DAMPs) and that cDCs do not traffic to the TDLN with antigen. Rather, they acquire antigen as it flows through afferent lymph vessels into the TDLN. Conclusion: When applied with a sparse scan protocol, BH monotherapy elicits abscopal melanoma control and shapes dendritic cell function through several previously unappreciated mechanisms. These results offer new insight into how to best combine BH with immunotherapies for the treatment of metastatic melanoma.
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Pharmacological methods for promoting mitochondrial elongation suggest that effector T cells can be altered to support a memory T cell-like metabolic state. Such mitochondrial elongation approaches may enhance the development of immunological memory. Therefore, we hypothesized that deletion of the mitochondrial fission protein dynamin-related protein 1 (DRP1) would lead to mitochondrial elongation and generate a large memory T cell population, an approach that could be exploited to enhance vaccination protocols. We find that, as expected, while deletion of DRP1 from T cells in dLckCre × Drp1flfl does compromise the magnitude and functionality of primary effector CD8+ T cells, a disproportionately large pool of memory CD8+ T cells does form. In contrast to primary effector CD8+ T cells, DRP1-deficient memory dLckCre × Drp1flfl CD8+ T cells mount a secondary response comparable to control memory T cells with respect to kinetics, magnitude, and effector capabilities. Interestingly, the relative propensity to form memory cells in the absence of DRP1 was associated with neither differentiation toward more memory precursor CD8+ T cells nor decreased cellular death of effector T cells. Instead, the tendency to form memory CD8+ T cells in the absence of DRP1 is associated with decreased T cell receptor expression. Remarkably, in a competitive environment with DRP1-replete CD8+ T cells, the absence of DRP1 from CD8+ T cells compromised the generation of primary, memory, and secondary responses, indicating that approaches targeting DRP1 need to be carefully tailored.
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Células T de Memória , Dinâmica Mitocondrial , Dinaminas/metabolismo , Linfócitos T CD8-Positivos , Mitocôndrias/metabolismoRESUMO
Focused ultrasound (FUS) is a powerful emerging tool for non-invasive, non-ionizing targeted destruction of tumors. The last two decades have seen a growing body of preclinical and clinical literature supporting the capacity of FUS to increase nascent immune responses to tumors and to potentiate cancer immunotherapies (e.g. checkpoint inhibitors) through a variety of means, including immune modulation and drug delivery. With the rapid acceleration of this field and a multitude of FUS immunotherapy clinical trials having now been deployed worldwide, there is a need to streamline and standardize the methodology for immunological analyses field-wide. Recently, the Focused Ultrasound Foundation and Cancer Research Institute partnered to convene a group of over 85 leaders to discuss the nexus of FUS and immuno-oncology. The guidelines documented herein were assembled in response to recommendations that emerged from this discussion, emphasizing the urgent need for heightened accessibility of immune analysis methods and standardized protocols unique to the field. These guidelines are designated for existing stakeholders in the FUS immuno-oncology domain or those newly entering the field, to provide guidance on collection, storage, and immunological profiling of tissue or blood specimens in the context of FUS immunotherapy studies, and additionally offer templates for standardized deployment of these methods based on collective experience gained within the field to date. These guidelines are tumor-agnostic and provide evidence-based, consensus-based recommendations for both preclinical and clinical immune analysis of tissue and blood specimens.
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Imunoterapia , Neoplasias , Humanos , Imunoterapia/métodos , Sistemas de Liberação de Medicamentos/métodos , Neoplasias/terapiaRESUMO
High-grade gliomas are malignant brain tumors, and patient outcomes remain dismal despite the emergence of immunotherapies aimed at promoting tumor elimination by the immune system. A robust antitumor immune response requires the presentation of tumor antigens by dendritic cells (DC) to prime cytolytic T cells. However, there is a paucity of research on dendritic cell activity in the context of high-grade gliomas. As such, this review covers what is known about the role of DC in the CNS, DC infiltration of high-grade gliomas, tumor antigen drainage, the immunogenicity of DC activity, and DC subsets involved in the antitumor immune response. Finally, we consider the implications of suboptimal DC function in the context of immunotherapies and identify opportunities to optimize immunotherapies to treat high-grade gliomas.
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Immune checkpoint blockade immunotherapy has radically changed patient outcomes in multiple cancer types. Pancreatic cancer is one of the notable exceptions, being protected from immunotherapy by a variety of mechanisms, including the presence of a dense stroma and immunosuppressive myeloid cells. Previous studies have demonstrated that CD40 stimulation can remodel the tumor microenvironment in a manner that promotes effector immune cell responses and can cooperate with immune checkpoint inhibition for durable tumor control mediated by T cells. Here we confirm the capability of this combination therapy to dramatically, and durably, control pancreatic cancer growth in an orthotopic model and that the immune memory to this cancer is primarily a function of CD4+ T cells. We extend this understanding by demonstrating that recruitment of recently primed T cells from the draining lymph nodes is not necessary for the observed control, suggesting that the pre-existing intra-tumoral cells respond to the combination therapy. Further, we find that the efficacy of CD40 stimulation is not dependent upon CD70, which is commonly induced on dendritic cells in response to CD40 agonism. Finally, we find that directly targeting the receptor for CD70, CD27, in combination with the TLR3 agonist polyIC, provides some protection despite failing to increase the frequency of interferon gamma-secreting T cells.
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PURPOSE: Glioblastoma (GB) poses formidable challenges to systemic immunotherapy approaches owing to the paucity of immune infiltration and presence of the blood brain/tumor barriers (BBB/BTB). We hypothesize that BBB/BTB disruption (BBB/BTB-D) with focused ultrasound (FUS) and microbubbles (MB) increases immune infiltration in GB. As a prelude to rational combination of FUS with ITx, we herein investigate the impact of localized BBB/BTB-D on innate and adaptive immune responses in an orthotopic murine GB model. METHODS: Mice with GL261 gliomas received i.v. MB and underwent FUS BBB/BTB-D (1.1 MHz, 0.5 Hz pulse repetition frequency, 10 ms bursts, 0.4-0.6 MPa). Brains, meninges, and peripheral lymphoid organs were excised and examined by flow cytometry 1-2 weeks following FUS. RESULTS: The number of dendritic cells (DC) was significantly elevated in GL261 tumors and draining cervical LN in response to sonication. CD86 + DC frequency was also upregulated with 0.6 MPa FUS, suggesting increased maturity. While FUS did not significantly alter CD8 + T cell frequency across evaluated organs, these cells upregulated checkpoint molecules at 1 week post-FUS, suggesting increased activation. By 2 weeks post-FUS, we noted emergence of adaptive resistance mechanisms, including upregulation of TIGIT on CD4 + T cells and CD155 on non-immune tumor and stromal cells. CONCLUSIONS: FUS BBB/BTB-D exerts mild, transient inflammatory effects in gliomas-suggesting that its combination with adjunct therapeutic strategies targeting adaptive resistance may improve outcomes. The potential for FUS-mediated BBB/BTB-D to modify immunological signatures is a timely and important consideration for ongoing clinical trials investigating this regimen in GB.
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Neoplasias Encefálicas , Glioblastoma , Terapia por Ultrassom , Animais , Barreira Hematoencefálica/patologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Glioblastoma/imunologia , Glioblastoma/patologia , Glioblastoma/terapia , Imageamento por Ressonância Magnética/métodos , CamundongosRESUMO
The substantial advances attained by checkpoint blockade immunotherapies have driven an expansion in the approaches used to promote T cell access to the tumor microenvironment to provide targets for checkpoint immunotherapy. Inherent in any T cell response to a tumor antigen is the capacity of dendritic cells to initiate and support such responses. Here, the rationale and early immunobiology of CD40 as a master regulator of dendritic cell activation is reviewed, with further contextualization and appreciation for the role of CD40 stimulation not only in cancer vaccines but also in other contemporary immune-oncology approaches.
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Vacinas Anticâncer , Neoplasias , Adjuvantes Imunológicos , Antígenos de Neoplasias , Antígenos CD40 , Imunoterapia , Neoplasias/terapiaRESUMO
Dendritic cells are potently activated by the synergistic action of CD40 stimulation in conjunction with signaling through toll like receptors, subsequently priming T cells. Cancer vaccines targeting the activation of dendritic cells in this manner show promise in murine models and are being developed for human patients. While the efficacy of vaccines based on CD40 and toll like receptor stimulation has been established, further investigation is needed to understand the mechanism of tumor control and how vaccination alters tumor infiltrating immune cells. In this study we vaccinated mice bearing established murine melanoma tumors with agonistic anti-CD40, polyI:C, and tumor antigen. Vaccination led to increased intratumoral T cell numbers and delayed tumor growth, yet did not require trafficking of T cells from the periphery. Pre-existing intratumoral T cells exhibited an acute burst in proliferation but became less functional in response to vaccination. However, the increased intratumoral T cell numbers yielded increased numbers of effector T cells per tumor. Together, our data indicate that the existing T cell response and intratumoral dendritic cells are critical for vaccination efficacy. It also suggests that circulating T cells responding to vaccination may not be an appropriate biomarker for vaccine efficacy.
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Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Vacinação/métodosRESUMO
The capacity of the immune system to influence tumor progression has been a long-standing notion that first generated clinical traction over a 100 years ago when Dr. William Coley injected disaggregated bacterial components into sarcomas and noted that the ensuing inflammation commonly associated with tumor regression.1 Since then, our understanding of the individual components and the overall interaction of the immune system has expanded exponentially. This has led to the development of a robust understanding of how components of innate and adaptive immunity recognize and respond to tumors and leveraging this information for the development of tumor immunotherapies. However, clinical failures have also deepened our knowledge of how tumors might adapt/be selected to avoid or inhibit immune responses, which, in turn, has led to the further iteration of immunotherapies. In this tutorial, the established elements of tumor immunity are explained, and areas where our knowledge base is too thin is highlighted. The principles of tumor immunity that guide the development of cancer vaccines are further illustrated, and potential considerations of how to integrate cancer vaccines with conventional therapies and other immunotherapies are proposed.
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Vacinas Anticâncer/administração & dosagem , Imunoterapia/métodos , Neoplasias/terapia , Linfócitos T/imunologia , Evasão Tumoral , Imunidade Adaptativa , Animais , Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Imunogenicidade da Vacina , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Neoplasias/imunologia , Microambiente Tumoral/imunologiaRESUMO
Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2, such as TEPP-46 and DASA-58, limit tumorigenesis and inflammation. To understand how these compounds alter T cell function, we assessed their therapeutic activity in a mouse model of T cell-mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology, in part, through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17-producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition, we found that activation of PKM2 interfered with TGF-ß1 signaling, which is necessary for the development of TH17 and regulatory T cells. Collectively, our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function.
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Diferenciação Celular/imunologia , Esclerose Múltipla/imunologia , Piruvato Quinase/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Piridazinas/farmacologia , Pirróis/farmacologia , Piruvato Quinase/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologiaRESUMO
BACKGROUND: Immune checkpoint inhibitors are being considered for locally advanced cervical cancer (LACC) together with standard-of-care pelvic chemoradiation (CRT). However, the safety of the combination and its optimal schedule are unknown. Defining the safety of the combination is a primary objective of a study examining concurrent and sequential schedules. This article presents a safety analysis that was fully accrued and met reporting requirements. METHODS: Pembrolizumab was given after CRT (arm 1) or during CRT (arm 2) according to a randomized phase 2 design. Patients who were 18 years old or older and had LACC (stages IB-IVA according to the 2009 International Federation of Gynecology and Obstetrics system) were randomized 1:1 to the treatment regimens. The CRT was identical in the 2 arms. Pembrolizumab was administered every 3 weeks for 3 doses; no maintenance was allowed. All patients receiving any treatment were evaluated for safety. Safety assessments included the incidence and severity of adverse events (AEs) and the occurrence of protocol-defined dose-limiting toxicity (DLT) through 30 days after the last pembrolizumab infusion. RESULTS: As of August 2019, 52 of the 88 planned patients had completed treatment and were evaluable for toxicity. Treatment-related grade 2 or higher toxicity was experienced by 88%; 11 had at least 1 grade 4 AE, and another 23 had at least 1 grade 3 AE. Grade 1 or higher diarrhea was reported in 34 patients (65%; 50% of these were grade 1), and there was no difference between arms (63% in arm 1 vs 68% in arm 2). Two patients experienced 3 DLTs. Most patients completed cisplatin (100% in arm 1 vs 82% in arm 2); 83% in both arms completed all pembrolizumab. CONCLUSIONS: Preliminary results support the safety and feasibility of adding pembrolizumab to pelvic CRT concurrently or sequentially. LAY SUMMARY: Pembrolizumab is a humanized antibody against programmed cell death protein 1 that is used in cancer immunotherapy. Preliminary data suggest that pembrolizumab can be safely combined with chemotherapy and pelvic radiation in the treatment of locally advanced cervical cancer. Future studies of the addition of immunotherapy to traditional chemoradiation are planned to determine the best way to deliver the treatment and whether any improvement is seen with the addition of immunotherapy to traditional therapy.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Pelve/patologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/radioterapia , Anticorpos Monoclonais Humanizados/farmacologia , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) remains recalcitrant to most targeted therapy approaches. However, recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy. We therefore tested a strategy for immune sensitization of murine TNBC (4T1 tumors) through combination of focused ultrasound (FUS) thermal ablation and a chemotherapy, gemcitabine (GEM), known to attenuate myeloid-derived suppressor cells (MDSCs). METHODS: We applied a sparse-scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM. We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity. We also tested this combination in Rag1-/- mice or T cell-depleted wild-type mice to determine the essentiality of adaptive immunity. Further, we layered Programmed cell death protein 1 (PD-1) blockade onto this combination to evaluate its impact on tumor outgrowth and survival. RESULTS: The immune-modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1, consistent with the dominant MDSC-driven immunosuppression evident in this model. The combination of FUS+GEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice. Tumor control correlated with increased circulating antigen-experienced T cells and was entirely dependent on T cell-mediated immunity. The ability of FUS+GEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti-PD-1. CONCLUSION: Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth, improves survival and enhances immunogenicity in a murine metastatic TNBC model. This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluation. TRIAL REGISTRATION NUMBERS: NCT03237572 and NCT04116320.
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Imunidade Adaptativa/imunologia , Desoxicitidina/análogos & derivados , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Imunossupressores/uso terapêutico , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Feminino , Humanos , Imunossupressores/farmacologia , GencitabinaRESUMO
Background: Focused ultrasound (FUS) activation of microbubbles (MBs) for blood-brain (BBB) and blood-tumor barrier (BTB) opening permits targeted therapeutic delivery. While the effects of FUS+MBs mediated BBB opening have been investigated for normal brain tissue, no such studies exist for intracranial tumors. As this technology advances into clinical immunotherapy trials, it will be crucial to understand how FUS+MBs modulates the tumor immune microenvironment. Methods and Results: Bulk RNA sequencing revealed that FUS+MBs BTB/BBB opening (1 MHz, 0.5 MPa peak-negative pressure) of intracranial B16F1cOVA tumors increases the expression of genes related to proinflammatory cytokine and chemokine signaling, pattern recognition receptor signaling, and antigen processing and presentation. Flow cytometry revealed increased maturation (i.e. CD86) of dendritic cells (DCs) in the meninges and altered antigen loading of DCs in both the tumor and meninges. For DCs in tumor draining lymph nodes, FUS+MBs had no effect on maturation and elicited only a trend towards increased presentation of tumor-derived peptide by MHC. Neither tumor endothelial cell adhesion molecule expression nor homing of activated T cells was affected by FUS+MBs. Conclusion: FUS+MBs-mediated BTB/BBB opening elicits signatures of inflammation; however, the response is mild, transient, and unlikely to elicit a systemic response independent of administration of immune adjuvants.
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Neoplasias Encefálicas/terapia , Células Dendríticas/metabolismo , Melanoma Experimental/terapia , Ovalbumina/imunologia , Terapia por Ultrassom/métodos , Animais , Barreira Hematoencefálica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Regulação Neoplásica da Expressão Gênica , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Microbolhas , Análise de Sequência de RNA , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Diacylglycerol lipase-ß (DAGLß) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLß regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive T cell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLß in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor α secretion. Cell-based vaccination studies revealed that DC maturation ex vivo and immunogenicity in vivo was surprisingly unaffected by DAGLß inactivation. Collectively, we identify DAGLß pathways as a means for attenuating DC inflammatory signaling while sparing critical adaptive immune functions and further expand the utility of targeting lipid pathways for immunomodulation.