Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis

Abstract

Metastatic progression depends on genetic alterations intrinsic to cancer cells as well as the inflammatory microenvironment of advanced tumours. To understand how cancer cells affect the inflammatory microenvironment, we conducted a biochemical screen for macrophage-activating factors secreted by metastatic carcinomas. Here we show that, among the cell lines screened, Lewis lung carcinoma (LLC) were the most potent macrophage activators leading to production of interleukin-6 (IL-6) and tumour-necrosis factor-alpha (TNF-alpha) through activation of the Toll-like receptor (TLR) family members TLR2 and TLR6. Both TNF-alpha and TLR2 were found to be required for LLC metastasis. Biochemical purification of LLC-conditioned medium (LCM) led to identification of the extracellular matrix proteoglycan versican, which is upregulated in many human tumours including lung cancer, as a macrophage activator that acts through TLR2 and its co-receptors TLR6 and CD14. By activating TLR2:TLR6 complexes and inducing TNF-alpha secretion by myeloid cells, versican strongly enhances LLC metastatic growth. These results explain how advanced cancer cells usurp components of the host innate immune system, including bone-marrow-derived myeloid progenitors, to generate an inflammatory microenvironment hospitable for metastatic growth.

Figure 1. Metastatic carcinomas secrete factors that induce macrophage production of TNF-α needed for lung metastasis

A. BMDM were cultured with serum-free medium (SFM) or SFM conditioned by mouse carcinoma cells (CM) and cytokine production was measured (averages ± s.d., n=3). B. BMDM were cultured with SFM or LCM and cytokine mRNAs were measured by quantitative (Q)-RT-PCR. Fold-induction above SFM-treated cells was determined (averages ± s.d., n=3; *, p<0.001 by Student’s t test). C. Survival of WT (n=22) and Tnfα^−/− (n=15) mice inoculated with LLC (1×10⁶ cells via the tail vein (p<0.001; Log-rank test for significance). D. Lungs of WT and Tnfα^−/− mice 47 days after LLC inoculation (2×10⁵ cells). Tumor multiplicities (TM) are shown underneath (averages ± s.d., n=11, p<0.001 by Student’s t test).

Figure 2. LLC-secreted factor activates TLR2 to induce lung inflammation

A. BMDM from indicated mouse strains were cultured with SFM or LCM and IL-6 production was measured (m: mutant allele; averages ± s.d., n=3, presented as % of WT LCM-stimulated value). B. BMDM were treated with LCM or LPS (100 ng ml⁻¹). Cell lysates were examined for kinase phosphorylation (P) and IκBα degradation by immunoblotting. Total ERK and HSP90 are loading controls. C. BMDM from indicated mouse strains were cultured with LCM or Pam3CSK4 (1 ng ml⁻¹), and IL-6 production was measured (averages ± s.d., n=4). D. RNA was extracted from lungs of WT or Tlr2^−/− mice at indicated times after LLC inoculation (2×10⁵ cells). mRNAs were quantitated as above and the amounts in non-inoculated WT or Tlr2^−/− lungs were given a value of 1.0 [averages ± s.e.m., n=3; *, p<0.05; **, p<0.005 (compared to WT) by Student’s t test].

Figure 3. TLR2 is required for metastatic growth

A. WT and Tlr2^−/− lungs were analyzed 9 days after inoculation of DsRed-LLC (2×10⁵ cells) for DsRed and myeloid cells markers (CD11b, CD11c) using fluorescence microscopy (magnification: 200x). B. Survival of LLC inoculated (2×10⁵ cells) WT (n=8) and Tlr2^−/− (n=10) mice (p<0.02; Log-rank test for significance). C. Lungs and H&E-stained lung sections (magnification: 25×) 20 days after LLC inoculation. Tumor multiplicity is shown on the left (averages ± s.e.m., n=8, p<0.001 by Student’s t test). D. Tumor multiplicities of lung and liver metastatic nodules and incidence of adrenal metastasis (met) 17 days after primary tumor removal (averages ± s.e.m., WT, n=9 and Tlr2^−/−, n=6; *, p<0.05 by Student’s t test). E. Survival of WT/WT or WT/Tlr2^−/− chimeric mice inoculated with LLC (2×10⁵ cells) 6–7 weeks after BM reconstitution (p<0.04; Log-rank test for significance; n=6). F. Lung tumor multiplicity (left); size (middle); and liver tumor multiplicity (right) in chimeric mice, 27 (lung) or 48 (liver) days after LLC injection (2×10⁵ cells) together with SFM or LCM (averages ± s.e.m., n=8; *, p<0.05 by Student’s t test).

Figure 4. Versican is a TLR2 agonist and metastasis enhancing factor

A. Identification of candidate LCM macrophage activators by mass spectrometry. Probability based Mowse scores (upper) and tryptic peptides corresponding to the indicated proteins (lower). B. LLC were transduced with indicated shRNA lentiviruses. After selection, expression of the indicated proteins was analyzed. C. (left) LLC transduced with indicated lentiviruses were injected (2×10⁵ cells) into mice (n=8). Lung tumor multiplicity was enumerated at 20 days (averages ± s.e.m., p<0.001 by Student’s t test). (right) Survival of mice inoculated with LLC transduced with control or Vers shRNAs (n=9; *, p<0.05; Log-rank test for significance). D. Lung and liver tumor multiplicities and incidence of adrenal metastasis 17 days after primary tumor removal (averages ± s.e.m., n=8 and n=5 for mice injected with control or Vers shRNA transduced cells, respectively; *, p<0.05 by Student’s t test). E. (upper) Non-transfected and human Versican (hVers) transfected P29-LLC cells were analyzed for versican expression. (lower) hVers non-expressing and expressing P29-LLC cells were tail vein injected (n=7). After 27 days, lung metastatic nodules were enumerated (*, p<0.05 by Student’s t test). F. (left) Silver staining of purified 6xHis-hVers. (right) BMDM were incubated without or with His-hVers for 20–24 hrs, and cytokine secretion was measured (averages ± s.d., n=3). G. Lysates of Raw264.7 cells incubated with LCM or SFM for 1 hr were immunoprecipitated with versican-specific or control antibody and analyzed by immunoblotting with the indicated antibodies.