Spatial and temporal heterogeneity of tumor immune microenvironment between primary tumor and brain metastases in NSCLC

Abstract

Background: Brain metastasis is a common outcome in non-small cell lung cancer, and despite aggressive treatment, its clinical outcome is still frustrating. In recent years, immunotherapy has been developing rapidly, however, its therapeutic outcomes for primary lung cancer and brain metastases are not the same, suggesting that there may be differences in the immune microenvironment of primary lung cancer and brain metastases, however, we currently know little about these differences.

Methods: Seventeen paired samples of NSCLC and their brain metastases and 45 other unpaired brain metastases samples were collected for the current study. Immunohistochemical staining was performed on all samples for the following markers: immune checkpoints CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, IDO1, and EphA2; tumor-infiltrating lymphocytes (TILs) CD3, CD4, CD8, and CD20; tumor-associated microglia/macrophages (TAMs) CD68 and CD163; and tumor proliferation index Ki-67. The differences in expression of these markers were compared in 17 paired samples, and the effect of the expression level of these markers on the prognosis of patients was analyzed in lung adenocarcinoma brain metastases samples. Subsequently, multiplex immunofluorescence staining was performed in a typical lung-brain paired sample based on the aforementioned results. The multiplex immunofluorescence staining results revealed the difference in tumor immune microenvironment between primary NSCLC and brain metastases.

Results: In 17 paired lesions, the infiltration of CTLA-4⁺ (P = 0.461), PD-1⁺ (P = 0.106), CD3⁺ (P = 0.045), CD4⁺ (P = 0.037), CD8⁺ (P = 0.008), and CD20⁺ (P = 0.029) TILs in brain metastases were significantly decreased compared with primary tumors. No statistically significant difference was observed in the CD68 (P = 0.954) and CD163 (P = 0.654) TAM infiltration between primary NSCLC and paired brain metastases. In all the brain metastases lesions, the expression of PD-L1 is related to the time interval of brain metastases in NSCLC. In addition, the Cox proportional hazards regression models showed high expression of B7-H4 (hazard ratio [HR] = 3.276, 95% confidence interval [CI] 1.335-8.041, P = 0.010) and CD68 TAM infiltration (HR = 3.775, 95% CI 1.419-10.044, P = 0.008) were independent prognosis factors for lung adenocarcinoma brain metastases patients.

Conclusions: Both temporal and spatial heterogeneity is present between the primary tumor and brain metastases of NCSLC. Brain metastases lesions exhibit a more immunosuppressive tumor immune microenvironment. B7-H4 and CD68⁺ TAMs may have potential therapeutic value for lung adenocarcinoma brain metastases patients.

Keywords: Brain metastases; Immune checkpoint; Immunotherapy; Non-small cell lung cancer; Tumor microenvironment.

Fig. 1

Representative immunohistochemical staining image of NSCLC and its paired brain metastases (not from the same patients). A–M list the typical immunohistochemical images of CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, IDO1, EphA2, CD3, CD4, CD8, CD20, CD68, and CD163, respectively. Scale bars, 100 μm, Original magnification 200×

Fig. 2

Comparison of TILs and TAMs between primary NSCLC and brain metastases lesions. Significant differences were observed in CD3, CD4, CD8, and CD20 expression levels (Numbers/HPF) between the primary NSCLC and paired brain metastases (P = 0.045, P = 0.037, P = 0.008, and P = 0.029, respectively). No significant differences were observed in the PD-1, CTLA-4, CD68 and CD163 expression levels (Numbers/HPF) between the primary NSCLC and paired brain metastases (P = 0.106, P = 0.461, P = 0.954 and P = 0.654, respectively). Differences of TILs and TAMs between primary lung cancer and brain metastasis were analyzed by the Paired Student’s t-test or Wilcoxon matched-pairs signed rank test on normality or non-normality variables, respectively. *, P < 0.05; **, P < 0.01; ns, not significant. HPF high power field

Fig. 3

Representative images of multiplex immunofluorescence in primary lung cancer and brain metastasis tissues. Scale bars, 50 μm

Fig. 4

Infiltration differences of TILs in synchronous and metachronous brain metastases patients. The infiltration of CD8⁺ lymphocytes were remarkably decreased in synchronously metastatic brain lesions (P = 0.034). No significant difference was observed between the paired lesions of synchronous and metachronous metastases among the other markers. *, P < 0.05; ns, not significant. The significance in difference was analyzed by Mann-Whitney U Test

Fig. 5

The expression difference of PD-L1 (P = 0.006), B7-H3 (P = 0.471), B7-H4 (P = 0.956), IDO1 (P = 0.866) and EphA2 (P = 0.928) in synchronous and metachronous brain metastases. **, P < 0.01; ns, not significant. The significance in difference was analyzed by Chi-Square test

Fig. 6

Kaplan–Meier survival curves of CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, IDO1, EphA2, CD68, and Ki-67 in lung adenocarcinoma brain metastases patients. (A-D) CTLA-4 (P = 0.441), PD-1 (P = 0.162), PD-L1 (P = 0.347), and B7-H3 (P = 0.630) expression was not correlated with patient survival. (E and F) High expression of B7-H4 and IDO1 in tumor cells was associated with worse survival (P = 0.004 and P = 0.011, respectively). (G) EphA2 expression in tumor cells was not associated with patient survival (P = 0.482). (H) High CD68⁺ cells in the stroma were associated with worse survival (P = 0.024). (I) Ki-67 index was not associated with patient survival (P = 0.116). The significance in survival differences was analyzed by log-rank test

Fig. 7

Representative pictures of different immune infiltration types and corresponding Kaplan–Meier survival curves. The significance in survival differences was analyzed by log-rank test. Scale bars, 100 μm, Original magnification 200×