RESUMEN
Tumor-treating fields (TTFields) are alternating electric fields in a specific frequency range (100-300 kHz) delivered to the human body through transducer arrays. In this study, we evaluated whether TTFields-mediated cell death can elicit antitumoral immunity and hence would be effectively combined with anti-PD-1 therapy. We demonstrate that in TTFields-treated cancer cells, damage-associated molecular patterns including high-mobility group B1 and adenosine triphosphate are released and calreticulin is exposed on the cell surface. Moreover, we show that TTFields treatment promotes the engulfment of cancer cells by dendritic cells (DCs) and DCs maturation in vitro, as well as recruitment of immune cells in vivo. Additionally, our study demonstrates that the combination of TTFields with anti-PD-1 therapy results in a significant decline of tumor volume and increase in the percentage of tumor-infiltrating leukocytes in two tumor models. In orthotopic lung tumors, these infiltrating leukocytes, specifically macrophages and DCs, showed elevated expression of PD-L1. Compatibly, cytotoxic T-cells isolated from these tumors demonstrated increased production of IFN-γ. In colon cancer tumors, T-cells infiltration was significantly increased following long treatment duration with TTFields plus anti-PD-1. Collectively, our results suggest that TTFields therapy can induce anticancer immune response. Furthermore, we demonstrate robust efficacy of concomitant application of TTFields and anti-PD-1 therapy. These data suggest that integrating TTFields with anti-PD-1 therapy may further enhance antitumor immunity, hence achieve better tumor control.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Carcinoma Hepatocelular/terapia , Carcinoma Pulmonar de Lewis/terapia , Terapia por Estimulación Eléctrica/métodos , Muerte Celular Inmunogénica , Linfocitos Infiltrantes de Tumor/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Apoptosis , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/patología , Proliferación Celular , Terapia Combinada , Femenino , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor Treating Fields (TTFields), an approved treatment modality for glioblastoma, are delivered via non-invasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application leads to abnormal mitosis, aneuploidy, and increased cell granularity, which are often associated with enhancement of autophagy. In this work, we evaluated whether TTFields effected the regulation of autophagy in glioma cells. We found that autophagy is upregulated in glioma cells treated with TTFields as demonstrated by immunoblot analysis of the lipidated microtubule-associated protein light chain 3 (LC3-II). Fluorescence and transmission electron microscopy demonstrated the presence of LC3 puncta and typical autophagosome-like structures in TTFields-treated cells. Utilizing time-lapse microscopy, we found that the significant increase in the formation of LC3 puncta was specific to cells that divided during TTFields application. Evaluation of selected cell stress parameters revealed an increase in the expression of the endoplasmic reticulum (ER) stress marker GRP78 and decreased intracellular ATP levels, both of which are indicative of increased proteotoxic stress. Pathway analysis demonstrated that TTFields-induced upregulation of autophagy is dependent on AMP-activated protein kinase (AMPK) activation. Depletion of AMPK or autophagy-related protein 7 (ATG7) inhibited the upregulation of autophagy in response to TTFields, as well as sensitized cells to the treatment, suggesting that cancer cells utilize autophagy as a resistance mechanism to TTFields. Combining TTFields with the autophagy inhibitor chloroquine (CQ) resulted in a significant dose-dependent reduction in cell growth compared with either TTFields or CQ alone. These results suggest that dividing cells upregulate autophagy in response to aneuploidy and ER stress induced by TTFields, and that AMPK serves as a key regulator of this process.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Neoplasias Encefálicas/patología , Estimulación Eléctrica/métodos , Glioblastoma/patología , Regulación hacia Arriba , Adenosina Trifosfato/metabolismo , Aneuploidia , Animales , Autofagosomas/metabolismo , Proteína 7 Relacionada con la Autofagia/antagonistas & inhibidores , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Supervivencia Celular , Terapia por Estimulación Eléctrica , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Glioblastoma/terapia , Proteínas de Choque Térmico/metabolismo , Humanos , Lisosomas/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Ratas , Factor A de Crecimiento Endotelial VascularRESUMEN
Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (Æ > 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.