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. 2021 Jan 1;11(2):602-613.
doi: 10.7150/thno.48353. eCollection 2021.

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

Robert B Rosenblatt  1 , Joseph A Frank  1   2 , Scott R Burks  1
Affiliations

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

Robert B Rosenblatt et al. Theranostics. .

Abstract

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 H2O2 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 Ca2+ transients during sonication. pFUS increased superoxide (range = 1.6-2.0-fold) and H2O2 (range = 2.3-2.8-fold) in all cell types except C6. BAPTA-AM blocked increased TUNEL reactivity, superoxide and H2O2 formation, while Trolox also blocked increased TUNEL reactivity increased after pFUS. mtTEMPOL allowed H2O2 formation and did not block increased TUNEL reactivity after pFUS. Unsonicated C6 cells had higher baseline concentrations of cytosolic Ca2+, superoxide, and H2O2, 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 Ca2+ transients causing formation of superoxide and subsequently, H2O2. 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 Ca2+ or redox homeostasis.

Keywords: DNA damage; calcium; focused ultrasound; reactive oxygen species; tumor.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
pFUS increases TUNEL reactivity without apoptosis in tumor cells. A) Representative imaging (TUNEL-positive nuclei in red) and B) quantification of TUNEL reactivity in tumor cells with or without pFUS (n = 9 per group per cell line) at 6 h post-pFUS. C) Representative imaging of ICC for cleaved (activated) caspase-3 (green) in tumor cells with or without pFUS at 6 h post-pFUS. D) Quantification of activated caspase-3 in each group (n = 9 per group per cell line). H2O2 group in each panel represents a positive controls for each measurement where cells were incubated with H2O2 (1 mM) for 6 h. Asterisks represent p < 0.05 by ANOVA comparisons performed on all groups for each cell line. Scale bars = 50 μm.
Figure 2
Figure 2
pFUS activates cytosolic Ca2+ transients in tumor cells and intracellular Ca2+ chelation suppresses pFUS-induced TUNEL reactivity. A) Representative imaging of Fluo-4 fluorescence before and during sonication (psuedocolor; scale bars = 50 μm) and B) fluorometric traces of Fluo-4 intensity before and during sonication (n = 69-130 per cell type; dashed line represents sonication time). Groups with like symbols are statistically similar to each other and significantly different from groups denoted by other symbols following ANOVA comparing peak magnitudes of each group. C) Quantification of TUNEL reactivity in cells with or without pFUS in the presence of intracellular BAPTA (n = 9 per group per cell type). Statistical significance was tested between control and treated groups using t-tests for each cell line.
Figure 3
Figure 3
pFUS increases Ca2+-dependent superoxide formation in cells, but neutralization of superoxide by SOD mimetic does not reduce pFUS-induced increases in TUNEL reactivity. A) Representative imaging of Mitosox fluorescence intensity (red; scale bars = 100 μm) and B) quantification with or without pFUS and in the presence or absence of intracellular BAPTA at 2 h post-sonication (n = 15 per group per cell line). Mitosox was loaded immediately prior to pFUS. Asterisks represent p < 0.05 from ANOVA comparisons performed on all groups from each cell line. C) Quantification of TUNEL reactivity with or without pFUS following incubation with the SOD mimetic mtTEMPOL (n = 9 per group per cell line). Asterisks represent p < 0.05 by t-tests between treated and control groups for each cell line.
Figure 4
Figure 4
pFUS increases Ca2+-dependent formation of H2O2 and neutralization of H2O2 with Trolox suppresses pFUS-induced increases in TUNEL reactivity. A) Representative images of intracellular H2O2 indicator (MAK-164, MilliporeSigma) fluorescence (green; scale bars = 100 μm) and B) quantification with or without pFUS and in the presence or absence of intracellular BAPTA or mtTEMPOL at 2 h post-sonication. The H2O2 indicator was loaded immediately after pFUS. Asterisks represent p < 0.05 by ANOVA comparisons performed on all groups from each cell line. C) Quantification of TUNEL reactivity in cells with or with pFUS in the presence of intracellular Trolox (n = 9 per group per cell type). Statistical significance was tested between control and treated groups using t-tests for each cell line.
Figure 5
Figure 5
Unsonicated C6 cells have elevated concentrations of cytosolic Ca2+, superoxide, and H2O2 compared to other unsonicated cells types, not higher levels of TUNEL reactivity. A) Ca2+ quantification in cytosolic volumes by Fluo-4 following cell permeabilization with digitonin (20 μM) (n = 8-9 per cell type). B) Ca2+ quantification in intracellular volumes by Fluo-4 following cell permeabilization with Triton X-100 (0.1% v/v) (n = 5 per cell type). C) Quantification of Mitosox fluorescence following 2 h intracellular incubation (n = 15 per cell type). D) Quantification of intracellular H2O2 indicator (MAK-164, MilliporeSigma) fluorescence following 2 h intracellular incubation (n = 15 per cell type). E) Quantification of TUNEL reactivity in all cell types without sonication (n = 9 per cell type). Asterisks in all graphs represent p < 0.05 by ANOVA for each measurement. Groups with asterisks in (E) were statistically similar (p < 0.05).
Figure 6
Figure 6
Schematic representation of cellular Ca2+ and ROS dynamics from pFUS to tumor cells. 1) pFUS causes increases in cytosolic Ca2+. Both plasma-membrane and store-release mechanisms (i.e., CICR and IP3-mediated release) are hypothesized to be involved. 2) Elevated cytosolic Ca2+ levels increase mitochondrial Ca2+ influx through the MCU leading to increased superoxide formation. 3) Reduction of superoxide forms H2O2 which 4) diffuses from the mitochondria to induce nuclear DNA damage.
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