Long-term expression changes of immune-related genes in prostate cancer after radiotherapy

Abstract

The expression of immune-related genes in cancer cells can alter the anti-tumor immune response and thereby impact patient outcomes. Radiotherapy has been shown to modulate immune-related genes dependent on the fractionation regimen. To identify long-term changes in gene expression after irradiation, PC3 (p53 deleted) and LNCaP (p53 wildtype) prostate cancer cells were irradiated with either a single dose (SD, 10 Gy) or a fractionated regimen (MF) of 10 fractions (1 Gy per fraction). Whole human genome arrays were used to determine gene expression at 24 h and 2 months after irradiation. Immune pathway activation was analyzed with Ingenuity Pathway Analysis software. Additionally, 3D colony formation assays and T-cell cytotoxicity assays were performed. LNCaP had a higher basal expression of immunogenic genes and was more efficiently killed by cytotoxic T-cells compared to PC3. In both cell lines, MF irradiation resulted in an increase in multiple immune-related genes immediately after irradiation, while at 2 months, SD irradiation had a more pronounced effect on radiation-induced gene expression. Both immunogenic and immunosuppressive genes were upregulated in the long term in PC3 cells by a 10 Gy SD irradiation but not in LNCaP. T-cell-mediated cytotoxicity was significantly increased in 10 Gy SD PC3 cells compared to the unirradiated control and could be further enhanced by treatment with immune checkpoint inhibitors. Irradiation impacts the expression of immune-related genes in cancer cells in a fractionation-dependent manner. Understanding and targeting these changes may be a promising strategy for primary prostate cancer and recurrent tumors.

Keywords: Checkpoint inhibitor; Immune modulation; Immune therapy; Long-term effect; Prostate cancer; Radiation therapy.

Fig. 1

Basal expression of immune-related genes is cell line-dependent. a Basal expression of immunogenic and b immunosuppressive genes in unirradiated PC3 and LNCaP prostate cancer cells. c. Human T-lymphocytes were isolated from human blood, activated with anti-CD3 and anti-CD28 antibodies for 24 h, and cultured for 10 days in IL2-containing media. T-lymphocytes and prostate cancer cells were co-cultured at different ratios (0:1, 1:1, 2:1, 4:1). LDH concentration in the supernatant was measured at 4 h or 20 h as a marker of tumor cell killing. Results show mean ± STDEV (n = 3). d Schematic of single-dose (SD) and multifractionated irradiation (MF) and 3D clonogenic survival of PC3 and LNCaP cells after SD irradiation (2, 4, 6, 8 Gy) or MF irradiation with 2 fractions of 1 Gy per day (2 × 1 Gy, 4 × 1 Gy, 6 × 1 Gy, 8 × 1 Gy). Results show mean ± STDEV (n = 3)

Fig. 2

Long-term upregulation of immune-related genes after single-dose irradiation. a Time line for single-dose (SD) and multifractionated (MF) irradiation. Gene expression was analyzed at 24 h (short term, ST) or 2 months (long term, LT) after the final irradiation dose. b Heat map of radiation-induced immune-related genes in PC3 and LNCaP prostate cancer cells. The data show fold change to the unirradiated control. c Pathway activation (z-score) of PC3 and LNCaP cells at 2 months after a single dose of 10 Gy determined with the Ingenuity Pathway Analysis (IPA) software (Qiagen). For a list of differentially expressed genes, see also Suppl. Figure S4

Fig. 3

SD irradiation activates the interferon pathway in a cell line-dependent manner. Gene expression analysis of PC3 and LNCaP cells at 2 months (long term, LT) after a single-dose (SD) irradiation of 10 Gy was analyzed with Ingenuity Pathway Analysis (IPA) software from Qiagen. Red color indicates upregulation, green color indicates downregulation, and white color indicates no change. Arrows and lines between molecules indicate associations and interactions

Fig. 4

The fractionation regimen impacts the expression of pro-immunogenic and immunosuppressive genes. a Heat map of radiation-induced pro-immunogenic genes in PC3 and LNCaP prostate cancer cells at 24 h (short term, ST) or 2 months (long term, LT) after a single-dose (SD) irradiation of 10 Gy or a multifractionated (MF) irradiation of 10 times 1 Gy (2 fractions per day, see also Fig. 2a). The data show fold change to the unirradiated control. b Normalized gene expression of PC3 and LNCaP at 2 months after a single dose of 10 Gy. Results show mean ± STDEV (n = 3). c Heat map and d relative gene expression of immunosuppressive genes. Results show mean ± STDEV (n = 3, * P < 0.05, ** P < 0.01, Student’s t test)

Fig. 5

Long-term SD tumor cells are more sensitive to T-lymphocyte-mediated cell death. a PC3 cells were irradiated either with a single dose (SD) of 10 Gy or with multifractionated (MF) irradiation of 10 times 1 Gy and cultured for 2 months (long term, LT). Unirradiated cells were used as control. Human T-lymphocytes were isolated from human blood, activated with anti-CD3 and anti-CD28 antibodies for 24 h, and cultured for 10 days in IL2-containing media. T-lymphocytes and tumor cells were co-cultured with different ratios (0:1, 1:1, 2:1). LDH concentration was measured at 4 h or 20 h as a marker of tumor cell killing. Results show mean ± STDEV (n = 3, * P < 0.05, ** P < 0.01, Student’s t test). b T-lymphocytes and tumor cells were co-cultured with a ration of 2:1 and concurrently treated with the checkpoint inhibitors Pembrolizumab, Nivolumab, Durvalumab, and Ipilumumab (all 10 μg/ml) for 20 h. Results show mean ± STDEV (n = 3, * P < 0.05, ** P < 0.01, Student’s t test)