Immunological Consequences Of Ultrasonic Stress In Tumor Cells After Exposure To Low Intensity Focused Ultrasound

Local tumor ablation can be achieved by physical energy-based therapies, such as, irradiation, ultrasound, microwave and radiofrequency. Although tumor antigens released from ablated tumors could induce an anti-tumoral immunity, antigen presentation from ablated tumors are not efficient in inducing a strong systemic immunity. Recently, we demonstrated that immunopriming by LOFU, followed by hypofractionated radiation, reversed immunological tolerance of T cells in draining lymph nodes, induced systemic anti-tumoral immunity, and produced local, regional and metastatic control of murine melanoma (J. Immunol. 196(4):1964-1976). LOFU uses low energy (∼500 W/cm2) and operating frequency of 1 MHz to induce membrane perturbation and mild hyperthermia at the focal zone, without ablating the tissue. Here, we investigate the effect of LOFU on gene expression and cell surface immunomodulation of murine tumor cells. LOFU parameters were set at 3-5W of total acoustic output power, 50% duty cycle, 1.5 seconds, and 1 mm spacing (TIPS, Philips Research). After LOFU treatment of a cell pellet, cells were cultured for various time points for RNA sequencing, qPCR, immunofluorescence and flow cytometry studies. Tumor cell lines included human DU145 prostate cancer cells and mouse RM1 and TPSA23 prostate cancer, Lewis lung carcinoma (3LL) and breast cancer (4T1). TPSA23 was derived from TRAMPC1 which was genetically engineered to express human PSA. Statistical evaluation distinguished gene expression changes by 1.5-fold, with false detection rate (FDR) <0.05. Wilcoxon rank sum was used to detect statistical significance in gene expression from RT-PCR results (p<0.05). Gene expression studies (RNAseq and qRT-PCR) demonstrate that LOFU (3W) induced key genes involved in the unfolded protein response (UPR) pathway. Compared to untreated DU145 cells, LOFU induced ERN1 three-folds, HSPA1B and HSPH1 by 8.79 folds and HSP105/110 by 6.18-fold within 6-24 hrs. In separate experiments, there was a significant increase in Hspa1 RNA expression (132-fold) and CHOP expression (2.5 fold) 24 h post-LOFU, in RM-1 and 4T1 cells, respectively. Flow cytometry and immunofluorescence in 3LL, 4T1 and RM1 cells demonstrated the translocation of stress proteins, BiP, calreticulin and HSP70 to the cell surface. There was induction of cell surface death receptors (CD40 and Fas), immune checkpoint inhibitors (PD-L1 and PD-L2) and class I MHC in 3LL, 4T1 and TPSA23 cell lines. These studies demonstrate that LOFU induces expression of genes involved in UPR and endoplasmic stress response. Translocation of stress chaperones to the cell surface along with increased expression of MHC I and death receptors contribute to the immunomodulatory properties of LOFU.