PCSK9 regulates the efficacy of immune checkpoint therapy in lung cancer

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by tumors was reported as a deleterious factor that led to the reduction of lymphocyte infiltration and the poorer efficacy of ICIs in vivo. This study aimed to explore whether PCSK9 expression in tumor tissue could predict the response of advanced non-small cell lung cancer (NSCLC) to anti-PD-1 immunotherapy and the synergistic antitumor effect of the combination of the PCSK9 inhibitor with the anti-CD137 agonist. One hundred fifteen advanced NSCLC patients who received anti-PD-1 immunotherapy were retrospectively studied with PCSK9 expression in baseline NSCLC tissues detected by immunohistochemistry (IHC). The mPFS of the PCSK9^lo group was significantly longer than that of the PCSK9^hi group [8.1 vs. 3.6 months, hazard ratio (HR): 3.450; 95% confidence interval (CI), 2.166-5.496]. A higher objective response rate (ORR) and a higher disease control rate (DCR) were observed in the PCSK9^lo group than in the PCSK9^hi group (54.4% vs. 34.5%, 94.7% vs. 65.5%). Reduction and marginal distribution of CD8⁺ T cells were observed in PCSK9^hi NSCLC tissues. Tumor growth was retarded by the PCSK9 inhibitor and the anti-CD137 agonist alone in the Lewis lung carcinoma (LLC) mice model and further retarded by the PCSK9 inhibitor in combination with the CD137 agonist with long-term survival of the host mice with noticeable increases of CD8⁺ and GzmB⁺ CD8⁺ T cells and reduction of Tregs. Together, these results suggested that high PCSK9 expression in baseline tumor tissue was a deleterious factor for the efficacy of anti-PD-1 immunotherapy in advanced NSCLC patients. The PCSK9 inhibitor in combination with the anti-CD137 agonist could not only enhance the recruitment of CD8⁺ and GzmB⁺ CD8⁺ T cells but also deplete Tregs, which may be a novel therapeutic strategy for future research and clinical practice.

Keywords: PCSK9; advanced non-small cell lung cancer; anti-CD137 agonist; immune infiltration; immunohistochemical markers; immunotherapy; proprotein convertase subtilisin/kexin type 9.

Figure 1

Immunohistochemistry of proprotein convertase subtilisin/kexin type 9 (PCSK9) in advanced non-small cell lung cancer (NSCLC) tissues. (A) PCSK9 expression in NSCLC tissues was associated with the N status of advanced NSCLC patients. (B) Kaplan–Meier estimate for PFS of the PCSK9^hi and PCSK9^lo groups by a proper cutoff of mean IOD of PCSK9 expression. The data cutoff was 30 April 2022. (C) Immunohistochemistry staining for PCSK9 of NSCLC tissues from advanced NSCLC patients before receiving anti-PD-1 immunotherapy. *ns, No significant difference, *P < 0.05, **P < 0.01.

Figure 2

High expression of PCSK9 in NSCLC tissues was associated with poorer efficacy of anti-PD-1 immunotherapy in advanced NSCLC. (A) Best response of the PCSK9^hi and PCSK9^lo groups. (B) ORR of the PCSK9^hi and PCSK9^lo groups. (C) DCR of the PCSK9^hi and PCSK9^lo groups. (D) Univariate and multivariate analyses of factors associated with PFS. PD-L1 negative, TPS < 1%; PD-L1 low, TPS < 50%; PD-L1 high, TPS ≥ 50%.

Figure 3

CD8⁺ T cells increased and moved into the tumor-cell-rich areas in PCSK9^lo tissue, while they stayed in the margin of tumor areas in PCSK9^hi tissue. (A, B) Multiplexed immunofluorescent staining of CK, CD8, and DAPI in the tumor microenvironment (bottom left panel and right panel, ×200 original magnification) of PCSK9^lo (A) or PCSK9^hi (B) tissue adjudicated by immunohistochemistry (upper left panel, ×200 magnification). (C) Typical views of other tumors from the PCSK9^lo or PCSK9^hi groups (×200 magnification). (D) Quantitative estimates of the density of CD8⁺ T cells: proportion of CD8 = CD8 number/DAPI number. **P < 0.01.

Figure 4

Tumor growth was attenuated with PCSK9 inhibition in combination with CD137 costimulation in syngeneic mice. (A) Experimental protocol. n = 5 mice per group. Intratumoral. (B) Tumor growth curve of each group (P-value was calculated by two-way ANOVA). (C) Overall survival (log-rank test). (D) Tumor growth curve of each mouse. (E) Percent change in tumor volume on day 21 compared with day 5 (baseline) after inoculation (unpaired t-test). (F) Tumors harvested on day 21. Data shown as mean ± SEM. *P < 0.05, **P < 0.01. LLC, Lewis lung carcinoma cells; PCSK9i, PCSK9 inhibitor; aCD137, anti-CD137 agonist; Doublet, the group treated with the PCSK9 inhibitor and the anti-CD137 agonist.

Figure 5

The proportion of CD8⁺ and GzmB⁺ CD8⁺ T cells increased significantly in the tumors of the doublet group with a reduction of Tregs. (A) Frequency of tumor-infiltrating CD4⁺ and CD8⁺ T cells of CD3⁺CD45⁺ leukocytes in LLC tumors of each group on day 21 (n = 3 per group). (B–G) Quantitative estimate of various immune effector cells in 1 * 10⁶ immune cells of tissue in each group determined by flow cytometry. n = 3 tumors per group (P-value was calculated by unpaired t-test or the Mann–Whitney test). (H–J) Quantitative estimate of CD4⁺, CD8⁺, and GzmB⁺ CD8⁺ T cells in 0.1 * 10⁶ immune cells of the spleen in each group determined by flow cytometry. n = 3 tumors per group (P-value was calculated by unpaired t-test or the Mann–Whitney test). *ns, No significant difference, *P < 0.05, **P < 0.01.

Figure 6

Body weight and serological changes of the mouse models after the treatments. (A) Body weight change of mice in each group (n = 5 per group, two-way ANOVA). (B–E) Serum ALT, TG, TCHO, and LDL-C levels of mice in each group on day 21 (n = 5 per group, unpaired t-test or the Mann–Whitney test). *ns, No significant difference, *P < 0.05, **P < 0.01.