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 multifractionated 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.

Figures

FIG. 1

Inflection point kinetics of immune genes in multifractionated treated PC3 and DU145 cells as assessed by real-time RT-PCR. PC3 and DU145 cells were exposed to 1–10 Gy of radiation delivered as 1 Gy multifractionated treatment. Fold change in IFI27, MX1, CXCL11, BST2, HSH2D and IL1A expression 24 h after cells received 1–10 Gy of radiation was determined by RT-PCR. Data shown are fold change (AV ± SEM) of 3 biologically distinct experiments. *P < 0.05, **P < 0.01.

FIG. 2

Radiation induces proinflammatory DAMPs and positively modulates the cytokine environment. Prostate carcinoma cells (DU145, PC3, LNCaP) were exposed to 10 Gy fractionated treatment (1 Gy × 10, multifractionated, gray bars) or 10 Gy single-dose treatment (10 Gy, single dose, black bars) or were mock irradiated (0 Gy, open bars). Culture supernatant was analyzed in triplicates after 48 and 72 h. Panel A: secreted DAMPs ATP and HMGB1, and panel B: modulation of tumor derived cytokines IL-8, GM-CSF and IL-6. *Denotes statistical significance vs. control (0 Gy). °Denotes marked modulation (≥twofold) relative controls (0 Gy).