Defining Molecular Signature of Pro-Immunogenic Radiotherapy Targets in Human Prostate Cancer Cells

Abstract

To understand the impact of clinically relevant radiation therapy (RT) on tumor immune gene expression and to utilize the changes that occur during treatment to improve cancer treatment outcome, we examined how immune response genes are modulated in prostate cancer cells of varying p53 status. LNCaP (p53 wild-type), PC3 (p53 null) and DU145 (p53 mutant) cells received a 10 Gy single dose or 1 Gy × 10 multifractionated radiation dose to simulate hypofractionated and conventionally fractionated prostate radiotherapy. Total RNA was isolated 24 h after multi-fractionated radiation treatment and single-dose treatments and subjected to microarray analysis and later validated by RT-PCR. RT-PCR was utilized to identify total-dose inflection points for significantly upregulated genes in response to multifractionated radiation therapy. Radiation-induced damage-associated molecular pattern molecules (DAMPs) and cytokine analyses were performed using bioluminescence and ELISA. Multifractionated doses activated immune response genes more robustly than single-dose treatment, with a relatively larger number of immune genes upregulated in PC3 compared to DU145 and LNCaP cells. The inflection point of multifractionated radiation-induced immune genes in PC3 cells was observed in the range of 8–10 Gy total radiation dose. Although both multifractionated and single-dose radiation-induced proinflammatory DAMPs and positively modulated the cytokine environment, the changes were of higher magnitude with multifractionated therapy. The findings of this study together with the gene expression data suggest that cells subjected to multifractionated radiation treatment would promote productive immune cell–tumor cell interactions.

Immunotherapy and stereotactic ablative radiotherapy (ISABR): a curative approach?

“…Conversely, use of higher doses of radiation, namely ≥15 Gy, increased the fraction of splenic regulatory T (T REG ) cells, which function to suppress tumour-specific immunity 26 . The importance of the radiation dose and fractionation schedule used was further corroborated in a study showing activation of immune-response-related genes, radiation-induced damage-associated molecular pattern molecules (DAMPs), and inflammatory cytokines in human prostate cancer cells when exposed to radiation in the range of 8–10 Gy 27 . Thus, these data suggest the existence of a threshold dose below which immune stimulation might be suboptimal and above which immunosuppression prevails.…”

Key mechanisms involved in ionizing radiation-induced systemic effects. A current review

“…Two genes overlapped between the two irradiation modalities, while the other 3 genes were involved in inflammatory responses by T cells and macrophages. 157 This study clearly demonstrated that the different irradiation modalities and doses not only induced a different activation level of the genes, but also the pattern of activated genes and thus the initiated immune and inflammatory mechanisms differed. Very similar results were obtained by Palayoor et al in human coronary artery endothelial cells.…”

“…157 Their data showed that multifractionated doses could activate immune response genes more robustly than single-dose treatment, with a relatively larger number of immune genes upregulated in p53-null cells compared to wild-type or p53 mutant cells. Although both single dose (1 × 10 Gy) and multifractionated dose (10 × 1 Gy) altered DAMPs and cytokine levels, the effect was more pronounced with multifractionated treatment.…”

Immunobiology of Radiotherapy: New Paradigms

“…Therefore, it is important to understand how immune genes respond to survival adaptation of irradiated tumor cells (during multifractionation as well as after single high-dose fraction) to best exploit radiation for combined immunotherapy approaches. Aryankalayil et al ( 2 ) demonstrated that both multifractionated as well single-dose radiation induced DAMPs and positively modulated the cytokine environment, with more observed in multifractionated radiotherapy. Kanagavelu et al ( 3 ) demonstrated the presence of immune modulation in partially irradiated contralateral syngeneic lung tumors.…”