The Impact of Focused Ultrasound in Two Tumor Models: Temporal Alterations in the Natural History on Tumor Microenvironment and Immune Cell Response

Abstract

Image-guided focused ultrasound (FUS) has been successfully employed as an ablative treatment for solid malignancies by exposing immune cells to tumor debris/antigens, consequently inducing an immune response within the tumor microenvironment (TME). To date, immunomodulation effects of non-ablative pulsed-FUS (pFUS) on the TME are poorly understood. In this study, the temporal differences of cytokines, chemokines, and trophic factors (CCTFs) and immune cell populations induced by pFUS were interrogated in murine B16 melanoma or 4T1 breast cancer cells subcutaneously inoculated into C57BL/6 or BALB/c mice. Natural history growth characteristics during the course of 11 days showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. With respect to tumor natural growth, pFUS applied to tumors on days 1, 5, or 9 demonstrated a decrease in the growth rate 24 h post-sonication. Flow cytometry analysis of tumors, LNs, and Sp, as well as CCTF profiles, relative DNA damage, and adaptive T-cell localization within tumors, demonstrated dynamic innate and adaptive immune-modulation following pFUS in early time points of B16 tumors and in advanced 4T1 tumors. These results provide insight into the temporal dynamics in the treatment-associated TME, which could be used to evaluate an immunomodulatory approach in different tumor types.

Cytosolic Ca2+ transients during pulsed focused ultrasound generate reactive oxygen species and cause DNA damage in tumor cells

“…pFUS, with or without microbubbles (MB), has been shown to activate inflammatory signaling pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), tumor necrosis factor-α (TNFα), and cyclo-oxygenase 2 (COX2) signaling pathways 6 - 9 that can result in release of cytokines, chemokines, and trophic factors (CCTF) from cells in tissues into the surrounding microenvironments of normal and diseased tissues 9 - 20 . Non-ablative pFUS alters CCTF and vascular cell adhesion molecule (CAM) expression for 24-48 h post-sonication in muscle 9 - 12 , kidney 7 , 13 - 15 , heart 16 , pancreas 17 , brain 8 , 18 , and various malignancies 19 , 20 . Microenvironmental changes from nondestructive pFUS prior to infusion of cellular products increases tropism to targeted sites to prevent or improve tissue damage 7 , 9 , 11 - 15 , 21 - 24 .…”

“…Microenvironmental changes from nondestructive pFUS prior to infusion of cellular products increases tropism to targeted sites to prevent or improve tissue damage 7 , 9 , 11 - 15 , 21 - 24 . We recently reported that pFUS at a peak negative pressure (PNP) of 6 MPa ( f 0 = 1.15 MHz; no MB contrast agents) to murine flank tumor models of B16 melanoma and 4T1 breast tumors resulted in numerous proteomic changes to the tumor microenvironment (TME) that were accompanied by infiltration of immune cells into tumors 19 , 20 . Moreover, non-ablative pFUS generated a shift toward an anti-tumor TME and reduced growth rates of B16 and 4T1 flank tumors over time 20 .…”

“…We recently reported that pFUS at a peak negative pressure (PNP) of 6 MPa ( f 0 = 1.15 MHz; no MB contrast agents) to murine flank tumor models of B16 melanoma and 4T1 breast tumors resulted in numerous proteomic changes to the tumor microenvironment (TME) that were accompanied by infiltration of immune cells into tumors 19 , 20 . Moreover, non-ablative pFUS generated a shift toward an anti-tumor TME and reduced growth rates of B16 and 4T1 flank tumors over time 20 . Histological evaluation of possible mechanisms behind the retarded growth rates of the flank tumors following pFUS revealed increased nuclear DNA damage using terminal deoxynucleotidyl transferase dUTP end-nick labeling (TUNEL) reactions 19 , 20 .…”

“…Moreover, non-ablative pFUS generated a shift toward an anti-tumor TME and reduced growth rates of B16 and 4T1 flank tumors over time 20 . Histological evaluation of possible mechanisms behind the retarded growth rates of the flank tumors following pFUS revealed increased nuclear DNA damage using terminal deoxynucleotidyl transferase dUTP end-nick labeling (TUNEL) reactions 19 , 20 . This observation was of particular interest as DNA damage and repair process can stimulate the innate immune system and alter the TME 25 .…”

“…Changes in cytosolic Ca 2+ alter mitochondrial biology and generate reactive oxygen species (ROS) that can ultimately induce DNA damage 35 . Our previous tumor studies 19 , 20 did not mechanistically investigate how pFUS caused DNA damage or how it might contribute to slowing tumor growth. This study investigated the biological mechanisms underpinning pFUS-induced DNA damage in vitro in multiple tumor cell lines: B16-F10 (B16), a murine melanoma line; 4T1, a murine breast tumor line; C6, a rat glioma line; and MDA-MB-231BRL a luciferase-transfected human breast tumor line with high brain metastatic potential.…”