RESUMO
Bromodomain-containing protein 4 (BRD4) is an intracellular protein that regulates expression of various cellular functions. This study investigated whether BRD4 inhibition can alter the immunomodulatory and antitumor effects of radiation therapy (RT). A murine breast cancer cell line was implanted into BALB/c mice. The dual-tumor model was used to evaluate the abscopal effects of RT. A total of 24 Gy was delivered and BRD4 inhibitor was injected intravenously. Tumor size was measured, and in vivo imaging was performed to evaluate tumor growth. Flow cytometry and immunohistochemistry were performed to examine immunologic changes upon treatment. The combination of BRD4 inhibitor and RT significantly suppressed tumor growth compared to RT alone. BRD4 inhibitor reduced the size of the unirradiated tumor, indicating that it may induce systemic immune responses. The expression of HIF-1α and PD-L1 in the tumor was significantly downregulated by the BRD4 inhibitor. The proportion of M1 tumor-associated macrophages (TAMs) increased, and the proportion of M2 TAMs decreased upon BRD4 inhibition. BRD4 inhibitor expanded CD4+ and CD8+ T cell populations in the tumor microenvironment. Additionally, splenic monocytic myeloid derived suppressor cells, which were increased by RT, were reduced upon the addition of BRD4 inhibitor. Therefore, the addition of BRD4 inhibitor significantly enhanced the systemic antitumor responses of local RT.
Assuntos
Neoplasias da Mama , Neoplasias , Proteínas Nucleares , Animais , Camundongos , Linfócitos T CD8-Positivos , Citometria de Fluxo , Imunomodulação , Camundongos Endogâmicos BALB C , Modelos Animais de Doenças , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapiaRESUMO
PURPOSE: In this study, we investigated whether local radiotherapy (RT) and an anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) agonist could increase the efficacy of PD-L1 blockade. METHODS AND MATERIALS: We analyzed a breast cancer dataset from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) to determine the role of GITR in breast cancer. We used the 4T1 murine TNBC model (primary and secondary tumors) to investigate the efficacy of PD-L1 blockade, local RT, anti-GITR agonist, and their combinations. We assessed tumor growth by tumor volume measurements, in vivo bioluminescence imaging, and metastatic lung nodule counts to evaluate the effects of these treatments. Flow cytometry and immunohistochemistry determined the proportions and phenotypes of CD8+ T-cells and regulatory T-cells (Tregs) in the tumors and spleen. Plasma cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: In the METABRIC cohort, patients with high expression of TNFRSF18, which encodes GITR, had significantly better survival than those with low expression. Adding local RT or anti-GITR agonist to PD-L1 blockade did not significantly augment efficacy compared to PD-L1 blockade alone; however, adding both to PD-L1 blockade significantly reduced tumor growth and lung metastasis. The benefits of the triple combination were accompanied by increased CD8+ T-cells and decreased Tregs in the tumor microenvironment and spleen. CONCLUSIONS: The combination of local RT and an anti-GITR agonist significantly enhanced the anti-tumor immune responses induced by PD-L1 blockade. These results provide the preclinical rationale for the combination of therapy.
Assuntos
Antígeno B7-H1 , Neoplasias da Mama , Humanos , Animais , Camundongos , Feminino , Linfócitos T CD8-Positivos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Glucocorticoides/farmacologia , Receptores do Fator de Necrose Tumoral , Microambiente Tumoral , Linhagem Celular TumoralRESUMO
BACKGROUND AND PURPOSE: Local radiotherapy (RT) exerts immunostimulatory effects by inducing immunogenic cell death. However, it remains unknown whether in vitro-irradiated tumor cells can elicit anti-tumor responses and enhance the efficacy of local RT and immune checkpoint inhibitors when injected in vivo. METHODS AND MATERIALS: We tested the "in vitro-irradiated cancer vaccine (ICV)", wherein tumor cells killed by varying doses of irradiation and their supernatants are intravenously injected. We examined the efficacy of combining local RT (24 Gy in three fractions), PD-L1 blockade, and the ICV in a murine breast cancer model. The immune cell profiles were analyzed via flow cytometry and immunohistochemistry. The cytokine levels were measured by multiplex immunoassays. RESULTS: The ICV significantly increased the effector memory phenotype and interferon-γ production capacity in splenic CD8+ T cells. The in vitro-irradiated products contained immune response-related molecules. When combined with local RT and PD-L1 blockade, the ICV significantly delayed the growth of irradiated and non-irradiated tumors. The triple combination therapy increased the proportions of CD8+ T cells and effector memory CD8+ T cells while decreasing the proportion of CTLA-4+ exhausted CD8+ T cells within tumor microenvironment. Additionally, plasma level of interferon-γ and proliferation of effector T cells in the spleen and tumor-draining lymph nodes were significantly increased by the triple combination therapy. CONCLUSIONS: The ICV enhanced the therapeutic efficacy of local RT and PD-L1 blockade by augmenting anti-tumor immune responses. Our findings suggest a therapeutic potential of in vitro-irradiation products of tumor cells.
RESUMO
Immune checkpoint inhibitors have been successful in a wide range of tumor types but still have limited efficacy in immunologically cold tumors, such as breast cancers. We hypothesized that the combination of agonistic anti-OX40 (α-OX40) co-stimulation, PD-1 blockade, and radiotherapy would improve the therapeutic efficacy of the immune checkpoint blockade in a syngeneic murine triple-negative breast cancer model. Murine triple-negative breast cancer cells (4T1) were grown in immune-competent BALB/c mice, and tumors were irradiated with 24 Gy in three fractions. PD-1 blockade and α-OX40 were administered five times every other day. Flow cytometric analyses and immunohistochemistry were used to monitor subsequent changes in the immune cell repertoire. The combination of α-OX40, radiotherapy, and PD-1 blockade significantly improved primary tumor control, abscopal effects, and long-term survival beyond 2 months (60%). In the tumor microenvironment, the ratio of CD8+ T cells to CD4 + FOXP3+ regulatory T cells was significantly elevated and exhausted CD8+ T cells (PD-1+, CTLA-4+, TIM-3+, or LAG-3+ cells) were significantly reduced in the triple combination group. Systemically, α-OX40 co-stimulation and radiation significantly increased the CD103+ dendritic cell response in the spleen and plasma IFN-γ, respectively. Together, our results suggest that the combination of α-OX40 co-stimulation and radiation is a viable approach to overcome therapeutic resistance to PD-1 blockade in immunologically cold tumors, such as triple-negative breast cancer.
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INTRODUCTION: We hypothesised that the combined use of radiation therapy and a phosphoinositide 3-kinaseγδ inhibitor to reduce immune suppression would enhance the efficacy of an immune checkpoint inhibitor. METHODS: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumours were irradiated by 3 fractions of 24 Gy. A PD-1 blockade and a phosphoinositide 3-kinase (PI3K)γδ inhibitor were then administered every other day for 2 weeks. The same experiments were performed in humanised patient-derived breast cancer xenograft model and its tumour was sequenced to identify immune-related pathways and profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas pan-cancer cohort, and the deconvolution algorithm was used to profile immune cell repertoire. RESULTS: Using a PI3Kγδ inhibitor, radiation therapy (RT) and PD-1 blockade significantly delayed primary tumour growth, boosted the abscopal effect and improved animal survival. RT significantly increased CD8+cytotoxic T-cell fractions, immune-suppressive regulatory T cells (Tregs), myeloid-derived suppressor cells and M2 tumour-associated macrophages (TAMs). However, the PI3Kγδ inhibitor significantly lowered the proportions of Tregs, myeloid-derived suppressor cells and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumour CD8+ T-cell populations after triple combination therapy. In a humanised patient-derived breast cancer xenograft model, triple combination therapy significantly delayed tumour growth and decreased immune-suppressive pathways. In The Cancer Genome Atlas cohort, high Treg/CD8+ T cell and M2/M1 TAM ratios were associated with poor overall patient survival. CONCLUSION: These findings indicate PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME, and combining PI3Kγδ inhibitor, RT and PD-1 blockade may overcome the therapeutic resistance of immunologically cold tumours. SYNOPSIS: Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may be a viable clinical approach, helping to overcome the therapeutic resistance of immunologically cold tumours such as breast cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/terapia , Quimiorradioterapia/métodos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Sinergismo Farmacológico , Feminino , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos da radiação , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/efeitos da radiação , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The poor response of breast cancer to immune checkpoint blockade might result from low immunogenicity and the immune-suppressive tumor microenvironment. We hypothesized that in situ tumor vaccination via radiation therapy (RT) and suppression of immune tolerance via phosphoinositide 3-kinase δ (PI3Kδ) inhibition would enhance the efficacy of immune checkpoint blockade. METHODS AND MATERIALS: 4T1 murine breast cancer cells were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated with 24 Gy in 3 fractions. A PD-1 blockade and a PI3Kαδ inhibitor were then administered every other day for 2 weeks. Fluorescence-activated cell sorting and immunohistochemistry served to monitor subsequent changes in immune cell repertoire. RESULTS: The triple combination of RT, PD-1 blockade, and PI3Kαδ inhibitor significantly delayed tumor growth. The immune-deficient syngeneic 4T1 murine tumor model failed to show this tumor growth delay. Use of RT and PI3Kαδ inhibitor increased the proportions of CD8+ T cells; PI3Kαδ inhibitor led to a decrease in regulatory T cells and polymorphonuclear myeloid-derived suppressor cells. The triple combination resulted in a remarkable increase in cytotoxic CD8+ T cells, suggesting a prominent immune-modulatory effect. The abscopal effect was most prominent in the triple-combination therapy group, and it correlated with splenic CD8+ T cell accumulation. CONCLUSIONS: These findings collectively indicate that combining RT, PI3Kαδ inhibitor, and PD-1 blockade could be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors, such as breast cancer, with an immunosuppressive tumor microenvironment.