Strategy of differentiation therapy: effect of dual-frequency ultrasound on the induction of liver cancer stem-like cells on a HA-based multilayer film system.

Cancer stem cells (CSCs) and normal stem cells share the ability to self-renew and drive tumor formation, recurrence, and distant metastasis and are resistant to chemotherapeutic drugs. One potential therapeutic approach for targeting CSCs is to induce CSCs to differentiate into normal cancer cells to eliminate self-renewal and enhance drug sensitivity. We developed a hyaluronic acid (HA)-based multilayer film system for selecting CSC-like hepatocellular carcinoma (HCC) cell colonies. Herein, we assess the differentiation therapy of HCC CSCs using dual-frequency low-intensity ultrasound (LIUS). HA-based multilayer films of poly (allylamine hydrochloride), (PAH/HA)6, were used to isolate CSC colonies. Colony formation, maintenance, and CSC marker expression were identified. The colony-formation rate was investigated, and putative CSC markers for CD44/CD133 expression after 7 days of culture were upregulated on (PAH/HA)6 multilayer films. Dual-frequency LIUS was used to induce CSC colony differentiation, and the phenotype variation, CSC marker expression, gene expression, drug-resistance ability, and invasion ability of CSC colonies with/without LIUS stimulation were compared. The numbers of colonies and CD44/CD133 double-positive cells and the expression levels of stem cell-related genes and proteins associated with stemness all decreased due to differentiation after LIUS exposure. Furthermore, a significant reduction in CSC drug resistance and invasion ability was observed. These results indicate that dual-frequency LIUS induces CSC differentiation and reduces drug resistance and invasion ability. Differentiation of CSCs provides an alternative therapeutic strategy to reverse CSC stemness and force their loss of self-renewal ability. CSC-targeted therapy holds great promise as an effective therapeutic approach for the treatment of human tumors.