COX-2 expression positively correlates with PD-L1 expression in human melanoma cells

Abstract

Background: The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma.

Methods: Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAF^V600E A375 and NRAS^Q61R SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro.

Results: BRAF^V600E/V600K and NRAS^Q61R/Q61L were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAF^V600E A375 and NRAS^Q61R SK-MEL-2 melanoma cell lines.

Conclusions: COX-2 expression correlates with and modulates PD-L1 expression in melanoma cells. These findings have clinical relevance since they provide a rationale to implement novel clinical trials to test COX-2 inhibition as a potential treatment to prevent melanoma progression and immune evasion as well as to enhance the anti-tumor activity of PD-1/PD-L1 based immunotherapy for the treatment of melanoma patients with or without BRAF/NRAS mutations.

Keywords: COX-2; Celecoxib; Immune checkpoint molecules; Immunotherapy; Melanoma; PD-L1.

Fig. 1

Representative IHC staining patterns with PD-L1-specific mAb (clone SP-142) of FFPE primary (a, b, c) and metastatic lymph node (d, e, f) from a total of 44 primary and 45 not matched metastatic melanoma tumors. PD-L1 expression was reviewed and enumerated independently and blindly by two experienced pathologists (GB and AA). PD-L1 expression was scored as negative (0+) (a, d), light positive (1+) (b, e) and positive (2+) (c, f) when the PD-L1 score in an entire lesion was 0, 1–5, and >5% respectively. Magnification is indicated. Arrows indicate examples of PD-L1 positive melanoma cells

Fig. 2

Representative IHC staining patterns with COX-2-specific mAb (clone CX-294) of FFPE primary (a, b, c) and metastatic lymph node (d, e, f) from a total of 44 primary and 45 not matched metastatic melanoma tumors. COX-2 expression was reviewed and enumerated independently and blindly by two experienced pathologists (GB and AA). COX-2 expression was scored as variations in the percentage of stained cells at 10% intervals. Staining was graded as a semi-quantitative variable. Results ranged from 0 to 80% of positive melanoma cells. Percentage of COX-2 positive cells, tumor margin (dotted line) and magnification are indicated. Arrows indicate examples of COX-2 positive melanoma cells

Fig. 3

Correlation between PD-L1 and COX-2 expression in primary (a) and metastatic lymph node (b) of melanoma tumors. COX-2 expression, graded as a semi-quantitative variable, was correlated with PD-L1 expression scored as negative (0+), light positive (1+) and positive (2+) when the PD-L1 score in an entire lesion was 0, 1–5, and >5% respectively. Difference in the expression of COX-2 in according to PD-L1 groups was analyzed using the Kruskal–Wallis rank test. On each box, the central mark is the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points not considered outliers, and outliers are plotted individually. P value is indicated

Fig. 4

Representative IHC (a, b, f, g) and matched immunofluorescent staining (c, d, e, h, i, l) patterns of FFPE primary (a, b, c, d, e) and not matched metastatic lymph node (f, g, h, i, l) of melanoma tumors with COX-2-specific mAb (clone CX-294) (a, c, f, h), PD-L1-specific mAb (clone SP-142) (b, d, g, i) and both COX-2- (clone CX-294) and PD-L1-specific mAbs (clone SP-142) (e, l). Immunofluorescent staining matches to squared field of IHC staining. COX-2 was detected by goat anti-mouse IgG dylight 488 (green). Immunofluorescent staining of PD-L1 was detected by goat anti-rabbit IgG dylight 594 (red). Nuclei were stained by DAPI (blue). Negative controls are provided in Additional file 1: Figure S1. Arrows indicate examples of positive cells. Tumor margin (dotted line) and magnification are indicated

Fig. 5

Effect of celecoxib on the in vitro proliferation of BRAF^V600E and NRAS^Q61R melanoma cell lines. a BRAF^V600E A375 and NRAS^Q61R SK-MEL-2 melanoma cells were seeded at the density of 3 × 10³ per well in a 96-well plate and incubated with the indicated concentrations of celecoxib. Untreated cells were used as a control. DMSO (vehicle of celecoxib) concentration was maintained at 0.02% in all wells. Following a 24 h incubation at 37 °C in a 5% CO₂ atmosphere, growth inhibition was determined by MTT assay. Data are expressed as mean percent of proliferation ± SD of treated cells as compared to untreated control cells. Mean percent of proliferation and SD were calculated from three independent experiments performed in triplicate. Difference between doses of celecoxib was calculated using unpaired t-test. *** indicate P < 0.001. b BRAF^V600E A375 and NRAS^Q61R SK-MEL-2 melanoma cells were seeded at the density of 4 × 10⁵ per well in a 75 cm² tissue culture flask and incubated with celecoxib (60 μM). Untreated cells were used as a control. DMSO (vehicle of celecoxib) concentration was maintained at 0.02% in all wells. Following a 24 h incubation at 37 °C in a 5% CO₂ atmosphere, viability of cells was determined by trypan blue assay. Data are expressed as mean percentage of viable (negative) and death cells (positive) of treated cells as compared to untreated control cells. *** indicate P < 0.001

Fig. 6

Down-regulation of PD-L1 by celecoxib in BRAF^V600E and NRAS^Q61R melanoma cell lines. BRAF^V600E A375 and NRAS^Q61R SK-MEL-2 melanoma cells were seeded at the density of 4 × 10⁵ per well in a 75 cm² tissue culture flask and incubated with celecoxib (60 μM). Untreated cells were used as a control. DMSO (vehicle of celecoxib) concentration was maintained at 0.02% in all wells. a Following a 24 h incubation at 37 °C in a 5% CO₂ atmosphere, cells were harvested and lysed. Cell lysates were analyzed by western blot with the indicated mAbs. PD-L1 was detected using the PD-L1-specific mAb (clone SP-142). GAPDH was used as a loading control. Representative results are shown (upper panel). The levels of PD-L1 normalized to GAPDH are plotted and expressed as mean ± SD of the results obtained in three independent experiments (bottom panel). *** indicate P < 0.001. b Following a 24 h incubation at 37 °C in a 5% CO₂ atmosphere, cells were harvested and cell surface stained with the PE-conjugated PD-L1-specific mouse mAb [clone MIH1 (RUO)]. PE-conjugated mouse IgG1 was used as a specificity control. Representative results are shown