Photodynamic therapy reduces the inhibitory effect of osteosarcoma cells on dendritic cells by upregulating HSP70

Abstract

Osteosarcoma is the most common primary bone tumor and predominantly affects children and adolescents. The prognosis for patients with osteosarcoma is poor. Therefore, the development of novel treatments for osteosarcoma is required. Photodynamic therapy (PDT) is a disease site-specific treatment that utilizes a photosensitizing agent along with light to kill cancer cells. This agent only works following activation by certain wavelengths of light. After the agent is absorbed by the cancer cells, light is then applied to the area to be treated. The light causes the drug to react with oxygen, which produces radical and reactive oxygen species that kill the cells. However, the immune reaction that occurs following PDT remains unknown. The present study demonstrated that the necrosis of osteosarcoma cells inhibited the function of dendritic cells. However, treatment of osteosarcoma cells with PDT restored the function of dendritic cells by upregulating heat shock protein 70. Taken together, the results of the present study provided insight into the subsequent molecular reaction following PDT treatment of osteosarcoma at the molecular level.

Keywords: HSP70; dendritic cells; osteosarcoma; photodynamic therapy.

Figure 1.

The remnants of LM8 decreased the co-stimulatory molecules, and inhibited IL-12 and IL-6 levels, increased IL-10 levels, and inhibited the ability of DCs to stimulate T-cell proliferation. (A) The DCs were isolated following a 48-h co-culture with the remnants of LM8 cells. The cells were then labeled with antibodies against CD11c, MHC-2, CD40, CD86, CD80 and CCR7 for phenotypic analysis by flow cytometry. The numbers in the histograms indicate the geometric mean fluorescence intensity. (B) Following isolation from the co-culture system, the DCs were cultured for 12 h. Subsequently, the expression levels of IL-12, IL-6 and IL-10 in the supernatant were analyzed by ELISA. (C) CD4⁺ T cells from DO11.10 OVA_323-339-specific (TCR-transgenic × C57BL/6) F1 hybrid mice were co-cultured with DCs or mDCs (control) in the presence of OVA peptides. Five days later, the total number of viable CD4⁺ T (CD4⁺ 7-AAD⁻) cells in each well was measured by flow cytometry. Data represent one of at least three experiments with similar results. *P<0.05 LM8 DC/T vs. Control DC/T. IL, interleukin; DCs, dendritic cells; CD, cluster of differentiation; MHC-2, major histocompatibility complex 2; CCR7, C-C chemokine receptor 7; DC/T, DCs and T cell co-culture; mDCs, mature DCs.

Figure 2.

PDT treatment partly reversed the effect of LM8 remnants on the phenotype of DCs and their ability to stimulate T cells proliferation. (A) The LM8 cells were pre-treated with PDT, and then labeled with antibodies against MHC-2, CD11c, CD40, CD86, CD80 and CCR7, for phenotypic analysis by flow cytometry. (B) The numbers in the histograms indicate the geometric mean fluorescence intensity. *P<0.05 PDT-LM8 vs LM8. (C) CD4⁺ T cells from DO11.10 OVA_323-339-specific (TCR-transgenic × C57BL/6) F1 hybrid mice were co-cultured with DCs or mDCs (control) in the presence of OVA peptides. Five days later, the total number of viable CD4⁺ T (CD4⁺ 7-AAD⁻) cells in each well was measured by flow cytometry. Data represent one of at least three experiments with similar results. *P<0.05 PDT-LM8 DC/T vs LM8 DC/T. PDT, photodynamic therapy; DCs, dendritic cells; MHC-2, major histocompatibility complex 2; CD, cluster of differentiation; CCR7, C-C chemokine receptor 7; DC/T, DCs and T cell co-culture.

Figure 3.

PDT treatment upregulated the HSP70 expression in LM8 cells and promoted upregulation of HSP70-activated DCs. (A) The LM8 cells with and without PDT pre-treatment were collected for RNA sequencing analysis. (B) The expression of HSP70, ATF3, Bcl-2, P53, P21, P16 and P27 was analyzed by reverse transcription-quantitative PCR. (C) LM8 cells were transfected with HSP70 small interfering RNA, and the DCs were then co-cultured with LM8 for 48 h, prior to being labeled with an antibody against CD86 for phenotypic analysis by flow cytometry. The numbers in the histograms indicate the geometric mean fluorescence intensity. Data represent one of at least three experiments with similar results. *P<0.05 HSP70-si-PDT-LM8 vs PDT-LM8. PDT, photodynamic therapy; HSP70, heat shock protein 70; DCs, dendritic cells; ATF3, activating transcription factor 3; Bcl-2, B-cell lymphoma 2; PCR, polymerase chain reaction; CD, cluster of differentiation.