Cytosolic Ca(2+) transients during pulsed focused ultrasound generate reactive oxygen species and cause DNA damage in tumor cells

Mechanical forces from non-ablative pulsed focused ultrasound (pFUS) generate pro-inflammatory tumor microenvironments (TME), marked by increased cytokines, chemokines, and trophic factors, as well as immune cell infiltration and reduced tumor growth. pFUS also causes DNA damage within tumors, which is a potent activator of immunity and could contribute to changes in the TME. This study investigated mechanisms behind the mechanotransductive effects of pFUS causing DNA damage in several tumor cell types. Methods: 4T1 (murine breast tumor), B16 (murine melanoma), C6 (rat glioma), or MDA-MB-231 (human breast tumor) cells were sonicated in vitro (1.1MHz; 6MPa PNP; 10ms pulses; 10% duty cycle; 300 pulses). DNA damage was detected by TUNEL, apoptosis was measured by immunocytochemistry for cleaved caspase-3. Calcium, superoxide, and H(2)O(2) were detected by fluorescent indicators and modulated by BAPTA-AM, mtTEMPOL, or Trolox, respectively. Results: pFUS increased TUNEL reactivity (range = 1.6-2.7-fold) in all cell types except C6 and did not induce apoptosis in any cell line. All lines displayed cytosolic Ca(2+) transients during sonication. pFUS increased superoxide (range = 1.6-2.0-fold) and H(2)O(2) (range = 2.3-2.8-fold) in all cell types except C6. BAPTA-AM blocked increased TUNEL reactivity, superoxide and H(2)O(2) formation, while Trolox also blocked increased TUNEL reactivity increased after pFUS. mtTEMPOL allowed H(2)O(2) formation and did not block increased TUNEL reactivity after pFUS. Unsonicated C6 cells had higher baseline concentrations of cytosolic Ca(2+), superoxide, and H(2)O(2), which were not associated with greater baseline TUNEL reactivity than the other cell lines. Conclusions: Mechanotransduction of pFUS directly induces DNA damage in tumor cells by cytosolic Ca(2+) transients causing formation of superoxide and subsequently, H(2)O(2). These results further suggest potential clinical utility for pFUS. However, the lack of pFUS-induced DNA damage in C6 cells demonstrates a range of potential tumor responses that may arise from physiological differences such as Ca(2+) or redox homeostasis.