Hypotonia-induced cell swelling enhances ultrasound-induced mechanical damage to cancer cells

It has been shown that killing of suspended cells by low-intensity ultrasound (0.08-0.11 W/cm(2)) can be enhanced by a mild non-lethal hypotonic (146 mOsm) medium. In this study we wished to determine whether hypotonia-induced cell swelling of suspension cells was directly related to enhancement of ultrasound-mediated cell killing, and to verify whether similar effects could be observed on circulating and attached cells. U937 cells under mild hypotonia were exposed to ultrasound for different times with real-time monitoring of cell size using a particle-size-distribution analyzer. To study the effect on attached cells, HeLa cells were exposed to ultrasound while under hypotonia in an in vivo-simulated set-up. The result showed that the enhanced cell killing (up to more than twice) was directly proportional to hypotonia-induced cell swelling. Similar membrane damage based on PI staining could be observed on HeLa cells treated with hypotonia. An in vivo-simulated circulating system also showed similar findings for hypotonia-enhanced ultrasound cell killing. These findings showed that mild hypotonia can be used to augment the effect of ultrasound in the treatment of cancers, particularly leukemia. The results showing that such enhancement is related to cell swelling could guide us toward proper timing of sonication while under hypotonic treatment.