In vitro and in vivo studies suggest treatment could eliminate resistant cancer cell population.

Scientists claim nanoparticle-mediated photothermal therapy could provide an effective therapeutic option for killing off cancer stem cells (CSCs) that are resistant to traditional forms of radiation and chemotherapy. Wake Forest School of Medicine researchers have found that while cancer stem cells from otherwise hard to treat triple negative breast cancer are largely resistant to traditional forms of hyperthermia, the local high levels of heat generated by carbon nanotubes subjected to laser light causes cell necrosis and rapid cell death of both CSCs and the bulk cancer cells.

Reporting their results in Biomaterials, Suzy V. Torti, Ph.D., M.D., and colleagues found that the treatment led to complete tumor regression and increased survival in tumor-bearing mice. Dr. Torti et al.’s published paper is titled “The resistance of breast cancer stem cells to conventional hyperthermia and their sensitivity to nanoparticle-mediated photothermal therapy.”

The Wake Forest team carried out a series of studies on triple-negative breast cancer cells breast (BCSCs) and bulk (nonstem) cancer cells. Initial tests using water baths showed that while hyperthermia significantly reduced the viability of bulk cancer cells, the BCSCs were 2.7–5.6-fold more resistant to heating and persisted after treatment. One clue as to the basis of the resistance came with the finding that the BCSCs expressed much higher levels of the heat shock protein HSP90 than the bulk cancer cells. In fact, treating the BSCs with an HSP90 inhibitor partially sensitized the cells to hyperthermia.

The researchers then evalauted whether BCSCs might show increased susceptibility to the more intense, localized subcellular heat generated by nanoparticles, even at the same temperatures tested using the water bath technique. Populations of BCSCs and bulk cancer cells were mixed with multiwalled carbon nanotubes (MWCNTs), immediately exposed to near-infrared light for the time needed to generate temperatures of 43°, 45°, 47°, or 49° Celsius,  and then washed to remove MWCNTs and replated.

Viability tests confirmed that the combination of MWCNTs and laser heating resulted in significant, temperature-dependent decreases in the viability of both bulk breast cancer cells and BCSCs. Both cell types were found to be equally sensitive to the nanotube-mediated thermal therapy (NMTT). Interestingly, this increased susceptibility of BCSCs to NMTT wasn’t due to the more rapid rate of temperature increase (ROTI) generated by laser heating.  Notably, mammosphere formation assays in addition confirmed that BCSCs that did survive following NMTT didn’t retain their long-term proliferative capacity.

One possible reason why BCSCs are more sensitive to NMTT than traditional hypothermia is that the intense localized heat generated by NIR-stimulation of MWCNTs causes critical membrane damage to cells adjacent to the nanotubes, resulting in necrotic cell death. This possibility contrasts with the apoptotic cell death that traditional hyperthermia therapy induces. The investigators evaluated this possibility by treating stem and bulk breast cancer cells using either rapid ROTI water bath hyperthermia, or NMTT, and then at different time points evaluating annexin V labeling as a marker of apoptosis and 7AAD permeability, which is an indicator of plasma membrane integrity.

The results confirmed that while the ROTI water bath therapy didn’t greatly impact the membrane permeability of either cell type or cause high levels of cell death, up to 80% of NMTT-treated bulk cancer cells and BCSCs demonstrated membrane damage over the same time period. Neither cell type exhibited siginficantly increased levels of apoptosis markers, indicating that the major form of cell death observed following NMTT was necrosis.

“Induction of necrotic death may be therapeutically advantageous, since mechanisms of resistance to apoptotic cell death are bypassed,” the authors state. “Although further studies will be required to elucidate the details of the interaction between NIR-stimulated nanotubes and the cancer cell surface that leads to cell death, it is likely that the high surface temperature of NIR-stimulated MWCNTs irreversibly permeabilizes cell membranes.”

Encouragingly, NMTT showed promising effects in vivo. Athymic mice were implanted subcutaneously with BCSCs and given intratumoral injections fo MWCNTs, followed by laser exposure. Control animals were either given no therapy or treated using a vehicle plus laser, or MWCNTs without laser developed. Animals in all these cohorts developed significant tumor burdens, and only 11–22% were still alive 45 days post treatment. In contrast, mice treated using MWCNTs plus laser therapy led to complete tumor regression and survival was boosted by 100% relative to the control groups.

“Our results demonstrate that breast cancer stem cells are highly resistant to conventional thermal treatments,” the authors write. “Nanotube-mediated hyperthermia may serve as a simple therapy that simultaneously eliminates both the stem cells and bulk cancer cells that constitute a breast tumor.”

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