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. 2016 Jan;65(1):1-11.
doi: 10.1007/s00262-015-1768-3. Epub 2015 Nov 5.

Metastatic spread in patients with non-small cell lung cancer is associated with a reduced density of tumor-infiltrating T cells

Philipp Müller  1 , Sacha I Rothschild  2   3 , Walter Arnold  4 , Petra Hirschmann  5 , Lukas Horvath  2 , Lukas Bubendorf  5 , Spasenija Savic  5 , Alfred Zippelius  6   7
Affiliations

Metastatic spread in patients with non-small cell lung cancer is associated with a reduced density of tumor-infiltrating T cells

Philipp Müller et al. Cancer Immunol Immunother. 2016 Jan.

Abstract

Tumor-infiltrating lymphocytes play an important role in cell-mediated immune destruction of cancer cells and tumor growth control. We investigated the heterogeneity of immune cell infiltrates between primary non-small cell lung carcinomas (NSCLC) and corresponding metastases. Formalin-fixed, paraffin-embedded primary tumors and corresponding metastases from 34 NSCLC patients were analyzed by immunohistochemistry for CD4, CD8, CD11c, CD68, CD163 and PD-L1. The percentage of positively stained cells within the stroma and tumor cell clusters was recorded and compared between primary tumors and metastases. We found significantly fewer CD4(+) and CD8(+) T cells within tumor cell clusters as compared with the stromal compartment, both in primary tumors and corresponding metastases. CD8(+) T cell counts were significantly lower in metastatic lesions than in the corresponding primary tumors, both in the stroma and the tumor cell islets. Of note, the CD8/CD4 ratio was significantly reduced in metastatic lesions compared with the corresponding primary tumors in tumor cell islets, but not in the stroma. We noted significantly fewer CD11c(+) cells and CD68(+) as well as CD163(+) macrophages in tumor cell islets compared with the tumor stroma, but no difference between primary and metastatic lesions. Furthermore, the CD8/CD68 ratio was higher in primary tumors than in the corresponding metastases. We demonstrate a differential pattern of immune cell infiltration in matched primary and metastatic NSCLC lesions, with a significantly lower density of CD8(+) T cells in metastatic lesions compared with the primary tumors. The lower CD8/CD4 and CD8/CD68 ratios observed in metastases indicate a rather tolerogenic and tumor-promoting microenvironment at the metastatic site.

Keywords: Anti-tumor immunity; Immune cells; Metastasis; Non-small cell lung cancer; Primary tumor.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Representative images of immunohistochemical staining results. NSCLC tissue sections from the primary tumor as well as the corresponding metastases were stained for CD4 (a) or CD8 (b). Tumor sections from two independent patients with CD4+ and CD8+ T cell infiltrates are shown (original magnification ×10 and ×40)
Fig. 2
Fig. 2
Quantitative comparison of tumor-infiltrating CD4+ and CD8+ T cells on large tumor sections as depicted in Fig. 1 showing relationships in individual patients. A paired analysis of CD4-positive cells (a), CD8-positive cells (b), and the CD8-to-CD4 ratio (c) was performed. From left to right: paired stroma [comparison of infiltrates within the tumor stroma between primary tumor (PT) and metastatic site (MET)]; paired tumor (comparison of infiltrates within the tumor islets between primary tumors and metastatic sites); PT (stroma:tumor) (primary tumor: comparison of infiltrates between the tumor stroma and the tumor islets); MET (stroma:tumor) (metastatic site: comparison of infiltrates between the tumor stroma and the tumor islets). IHC positive events have been depicted as % of total nucleated cells; paired two-sample t test was used to compare paired samples (n = 34)
Fig. 3
Fig. 3
Representative images of immunohistochemical staining results. NSCLC tissue sections from the primary tumor as well as the corresponding metastatic site were stained for CD11c (a), CD68 (b) or CD163 (c). Tumor sections from two independent patients with CD11c+ dendritic cell (DC) and CD68+ and CD163+ macrophage infiltrates are shown (original magnification ×10 and ×40)
Fig. 4
Fig. 4
Quantitative comparison of tumor-infiltrating CD11c+ dendritic cells and CD68+ and CD163+ macrophages, respectively, on large tumor sections as depicted in Fig. 3. A paired analysis of CD11c-positive cells (a), CD68-positive cells (b), the CD8-to-CD68 ratio (c), and CD163-positive cells (d) was performed. From left to right: paired stroma [comparison of infiltrates within the tumor stroma between primary tumor (PT) and metastatic site (MET)]; paired tumor (comparison of infiltrates within the tumor islets between primary tumors and metastatic sites); PT (stroma:tumor) (primary tumor: comparison of infiltrates between the tumor stroma and the tumor islets); MET (stroma:tumor) (metastatic site: comparison of infiltrates between the tumor stroma and the tumor islets). IHC positive events have been depicted as % of total nucleated cells; paired two-sample t test was used to compare paired samples (n = 34 for CD8 and CD68 staining; n = 14 for CD11c staining)
Fig. 5
Fig. 5
a Representative images of immunohistochemical staining results. NSCLC tissue sections from the primary tumor as well as the corresponding metastatic site were stained for PD-L1. Tumor sections from two independent patients with PD-L1-positive tumor cells and PD-L1-positive tumor-infiltrating myeloid cells are shown (original magnification ×10 and ×40). b Quantitative comparison of PD-L1 expression on tumor and myeloid cells on large tumor sections as depicted in a of this figure. A paired analysis of PD-L1-positive tumor cells and PD-L1-positive myeloid cells in primary tumors and metastatic lesions was performed. IHC positive events have been depicted as % of total nucleated cells; paired two-sample t test was used to compare paired samples (n = 34)
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