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. 2007 Nov;171(5):1608-18.
doi: 10.2353/ajpath.2007.060661. Epub 2007 Oct 4.

Stromal fibroblasts in colorectal liver metastases originate from resident fibroblasts and generate an inflammatory microenvironment

Lars Mueller  1 , Freya A Goumas, Marianne Affeldt, Susanne Sandtner, Ursula M Gehling, Silke Brilloff, Jessica Walter, Nadia Karnatz, Katrin Lamszus, Xavier Rogiers, Dieter C Broering
Affiliations

Stromal fibroblasts in colorectal liver metastases originate from resident fibroblasts and generate an inflammatory microenvironment

Lars Mueller et al. Am J Pathol. 2007 Nov.

Abstract

Cancer-associated stromal fibroblasts (CAFs) are the main cellular constituents of reactive stroma in primary and metastatic cancer. We analyzed phenotypical characteristics of CAFs from human colorectal liver metastases (CLMs) and their role in inflammation and cancer progression. CAFs displayed a vimentin(+), alpha-smooth-muscle actin(+), and Thy-1(+) phenotype similar to resident portal-located liver fibroblasts (LFs). We demonstrated that CLMs are inflammatory sites showing stromal expression of interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis. In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-alpha (TNF-alpha). The effect of TNF-alpha on CAFs is inhibited by the nuclear factor-kappaB inhibitor parthenolide. Conditioned medium of CAFs and LFs similarly stimulated the migration of DLD-1, Colo-678, HuH7 carcinoma cells, and human umbilical vein endothelial cells in vitro. Pretreatment of CAFs with TNF-alpha increased the chemotaxis of Colo-678 colon carcinoma cells by conditioned medium of CAFs; however, blockage of IL-8 activity showed no inhibitory effect. In conclusion, these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-alpha-induced up-regulation of IL-8 via nuclear factor-kappaB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target.

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Figures

Figure 1
Figure 1
Phenotype of CAFs and fibroblastic hepatic cell populations in situ. Immunohistochemical analyses of vimentin, α-SMA, Thy-1, ICAM-1, desmin, and laminin. Representative images of cryosections from metastatic tissue (A) and liver tissue (B) of 20 patients. Original magnification, ×400 (P, portal area; CV, central vein). The stromal compartment of the metastatic tissue displays intense immunoreactivity for vimentin, α-SMA, Thy-1, ICAM-1, and laminin. Desmin was detected in the walls of larger vessels. The arrowheads in the vimentin staining of B indicate vimentin-positive hepatic stellate cells or Kupffer cells that are located along the sinusoid linings. In portal and pericentral regions there are vimentin+, α-SMA+, and Thy-1+ cells (LFs), whereas the HSCs residing in perisinusoidal linings showed no immunoreactivity for α-SMA and Thy-1.
Figure 2
Figure 2
Representative images of cultured CAFs (A) and LFs (B) stained for vimentin, α-SMA, Thy-1, ICAM-1, cytokeratin (CK)-7, laminin, CK-19, and neural cell adhesion molecule (NCAM), indicating that the myofibroblastic phenotype of CAFs and LFs was stable in vitro (original magnification, ×400; positive staining, red).
Figure 3
Figure 3
The metastatic site is associated with leukocyte infiltration. A: Representative immunohistochemical staining of serial sections from colorectal liver metastases and their margins for CD45 and laminin (original magnification, ×400). B: Mean numbers of CD45-positive cells within tumor, tumor margin, and liver tissue (n = 14; mean ± SEM; *P < 0.05, **P < 0.01).
Figure 4
Figure 4
CAFs in situ stain positive for IL-8, and IL-8 mRNA is increased in metastatic tissue. A: Representative immunohistochemical analysis of IL-8 in colorectal liver metastases and the tumor margin (original magnification, ×400). B: Representative Northern blot analysis showing IL-8 mRNA expression in tumor tissue (I), adjacent, peritumor tissue (II), and distant liver tissue (III) in tissue samples from two different patients. Rehybridization for glyceraldehyde-3-phosphate dehydrogenase for normalization. In the diagram below (C), mean results of Northern analyses for IL-8 mRNA expressions in tumor tissue (I), adjacent, peritumor tissue (II), and distant liver tissue (III) are quantified (n = 10; mean ± SEM; **P < 0.01 versus peritumor tissue (II) and distant liver tissue (III)).
Figure 5
Figure 5
TNF-α induces expression of IL-8 by human CAFs and LFs in vitro. A: Representative Northern blot analysis showing the effect of cytokines [transforming growth factor-β (TGF-β), 5 ng/ml; PDGF-BB, 10 ng/ml; and TNF-α, 50 ng/ml) and increased concentration of FBS (10%) on IL-8 mRNA expression by CAFs and liver fibroblasts. Controls (CON) are nonstimulated CAFs or LFs (0.5% FBS) (ST, molecular RNA weight marker). The lower bands show glyceraldehyde-3-phosphate dehydrogenase mRNA expression after rehybridization for normalization. Note that the IL-8-specific band is still abundant. B: Results of densitometric band analysis of IL-8 mRNA expression by CAFs and LFs (n = 4; mean ± SEM; ns, not significant; * and #P < 0.01 versus respective control). C: Protein expression of IL-8 by CAFs and LFs in vitro, determined by ELISA measurements (n = 5; mean ± SEM; * and #P < 0.01 versus respective control).
Figure 6
Figure 6
The NF-κB inhibitor parthenolide inhibits the effect of TNF-α on IL-8 expression by CAFs. A: Representative Northern blot analysis of IL-8 mRNA expression by the CAFs in vitro under incubation with or without TNF-α in the presence or absence of increasing concentrations of parthenolide. B: Results of densitometric band analyses of IL-8 mRNA expression by CAFs that were exposed to TNF-α in either the presence or absence of increasing concentrations of parthenolide (n = 4; mean ± SEM; *P < 0.05, **P < 0.001). C: The combined effect of increasing concentrations parthenolide and TNF-α on IL-8 protein expression released by CAFs that were incubated for 24 hours was measured by ELISA and quantified for statistical analysis (n = 4; mean ± SEM; *P < 0.05, **P < 0.001).
Figure 7
Figure 7
Boyden chamber analyses testing chemotaxis of HuH7 hepatoma cells (A), DLD1 colon carcinoma cells (B), Colo-678 colon carcinoma cells (C), and HUVECs (D) induced by conditioned medium from CAFs and LFs. Increasing concentrations of the conditioned media were placed in the lower chamber of a modified Boyden chamber system as described in Materials and Methods. Tumor cells (15 × 103) or HUVECs in 50-μl medium containing 2% bovine serum albumin were seeded in the upper wells. Each experimental condition was examined by quadruplicate measurements. Quantification results from five different CAF cultures and corresponding LF cultures are shown (n = 5; mean ± SEM; * and #P < 0.001 versus respective control).
Figure 8
Figure 8
Effect of TNF-α on chemotactic properties of CAFs. The migration of HuH7 hepatoma cells (A), DLD1 colon carcinoma cells (B), Colo-678 colon carcinoma cells (C), and HUVECs (D) toward conditioned medium of CAFs that were incubated for 24 hours in 10 ng/ml TNF-α [CAF-CM (TNF)] and toward CM of nonstimulated CAFs (CAF-CM) were compared. In the CM of nonstimulated CAFs, TNF-α was added after the incubation time to exclude that differences in migration were caused by the presence of exogenous TNF-α. The effect of IL-8 blockage was tested by preincubation with an IL-8-neutralizing antibody (10 μg/ml; R&D Systems). The experiments were repeated four times using CAF cultures from different patients (n = 4; mean ± SEM; *P < 0.05 versus CAF-CM).
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