..................................................................................................................................................................................................
J. Dellacasagrande,* O. J. F. Schreurs, y P. O. Hofgaard,* H. Omholt,* S. Steinsvoll, y K. Schenck, y B. Bogen* & Z. Dembic*
*Institute of Immunology; and y Department of
Oral Biology, University of Oslo, The National
Hospital, Oslo, Norway
Received 25 February 2003; Accepted in revised form 10 March 2003
Correspondence to: Dr Z. Dembic, Institute of
Oral Biology, Dental Faculty, University of Oslo,
Sognsvannsveien 10, Oslo, Norway. E-mail: zlatko.dembic@labmed.uio.no
When injected subcutaneously, mouse plasmacytoma (MOPC315) grew rapidly in situ , and metastatic cells became detectable first in the lymph nodes (LNs) and bone marrow, and later in the liver and lungs. We studied MOPC315 cell migration by tracking metastatic cells labelled with green fluorescent protein
(GFP). We measured the levels of their chemokine receptor mRNA (by semiquantitative and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), because chemokines can regulate organ predilection of metastasis. Freshly sorted metastatic cells and tumour cell lines derived from the liver of BALB/ c mice overexpressed functional CCR6 and CCR7 molecules compared with primary tumour. Preincubation with the CCR6 ligand (CCL20) induced liver-sorted tumour cells to preferentially colonize the liver, demonstrating an association between liver metastasis and CCR6 expression in the mouse.
Because the liver is a common site for metastasis, second only to draining LNs, we wished to ascertain whether this finding could be generalized, i.e. whether other cancers can use the similar mechanism of metastasis to the liver, and whether it holds true for humans. We found that CCR6 is overexpressed in small liver metastases of colon, thyroid and ovarian carcinomas compared with normal liver. Because human liver constitutively expresses CCL20, it could attract and select CCR6
þ cancer cells. We suggest that chemotaxis via CCR6 might be a common mechanism by which malignant cancers metastasize to the liver. As metastasis in patients with cancer poses the biggest peril for survival, inhibition of CCR6 signalling, either during or after medical or surgical treatment, might be useful in preventing liver metastasis.
Genetic progression and selection in malignant tumours may cause haphazard dislodging and emigration of cells via lymphatics and/or blood. The organ predilection of metastases may depend on a variety of factors, and three theories have been put forward as explanations [1]. According to the first hypothesis, all migrants can enter any tissue, but would form a metastasis only if all requirements for their growth were met [2, 3]. The second suggests that tissuespecific adhesion molecules on endothelial cells select migrants to attach and form a premetastatic nucleus [4].
The most recent hypothesis proposes that chemoattractants would lead invasive cancer cells to the tissue of their potential secondary growth [5]. Perhaps an interplay between all proposed mechanisms might operate to a different extent across various cancer types. For example, some blood-borne spreading cancer cells may resemble leucocytes in rolling and adhesion to intrahepatic endothelium [6], but other cancers [7] may not. Similarly, some chemokines may promote growth of metastasis in addition to their chemoattractant roles [8].
Selectins, chemokines and integrins, together with their ligands are molecular substrates for leucocyte tethering and rolling on endothelial cells and anchoring in small postcapillary blood vessels, promoting diapedesis of arrested cells. The adhesion molecules in such a process are integrin molecules. For anchoring, they can temporarily increase the ligand affinity almost 10-fold through a process that requires energy and the activation of leucocytes by chemokine exposure [9]. Recent evidence indicated that some murine and human cancer cells might metastasize to lymph nodes (LNs) using L-selectin [10], CCR7 [11–13],
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Liver Metastasis and CCR6 535
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CXCR4 [14] or a 4 integrin [13] molecules. Likewise, lung metastasis was associated with CXCR4 [14] or b 4 integrin
[15] molecules, whereas skin metastasis of melanoma involved CCR10 [14]. Here, we analysed the homing stage of organ-specific metastasis by tracking tumour migrants and profiling their chemokine receptors. We focused on liver as it is a common site of metastasis second only to draining LNs for a variety of cancers [16], and because E-selectin, integrin ( a 5) and integrin ligands
(ICAM-1 and VCAM-1) have been implicated in the adhesion of murine melanoma cells to hepatic microvasculature [6]. However, it is unknown whether specific chemokine(s)/chemokine receptor(s) is associated with metastasis of cancer to the liver. We used mouse extramedullary plasmocytoma MOPC315 tumour labelled with green fluorescent protein (GFP) to track the migration of metastatic cells. We measured the levels of mouse chemokine receptor mRNA by multiplex semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR in primary and metastatic tumour cells, and ascertained their function in vitro and in vivo . We then checked a selection of human liver samples for the presence of related chemokine receptor mRNAs and their proteins in metastatic cancer cells.
Thymus
A
Draining LN
Other LN
Bone marrow
Spleen
10
–8
10
–6
10
–4
10
–2
Frequency
1
C
D
GFP
GFP
Lungs
B
E
CD138-gated
Liver
Bone marrow
Spleen
F
GFP
GFP-gated
10
–8
10
–6
10
–4
Frequency
10
–2 1
CD138
Figure 1 Frequency of metastasis and characterization of mouse plasmacytoma (MOPC315) and MOPC315-green fluorescent protein
(GFP) tumours. (A) Frequency of MOPC315 clones isolated by in vitro limiting dilution from various organs of BALB/ c mice as a function of tumour size ( & , > 0.5 cm and & , > 1.5 cm); (B) frequency of metastasis in large MOPC315 ( & ) or MOPC315-GFP ( ) tumours of equal sizes;
(C) flow cytometric detection of GFP in cultured MOPC315-GFP cells;
(D–F) flow cytometric detection of GFP in subcutaneous MOPC315-
GFP tumour. GFP expression and CD11b specific staining (D), GFP expression of CD138-gated cells (E), and cell-surface CD138 expression of GFP-gated cells (- - -, staining with isotype-matched control) (F). LN, lymph node.
In vivo tumour growth and mice.
MOPC315 cells (American Type Culture Collection, Manassas, VA, USA) were expanded in RPMI medium containing 10% fetal calf serum (FCS). A subline, MOPC315.4, was established that grows well in vitro and in vivo [17], and was used for subcutaneous (s.c.) injections (10
5
, without FCS) in
6–18 weeks old BALB/ c or BALB/ c Rag2
–/– mice (M&B,
Ry, Denmark). In some experiments, M315 myeloma protein concentration in serum was assayed by enzymelinked immunosorbent assay to follow tumour growth, as previously described [18]. Mice were kept under specific pathogen-free conditions and were killed before tumour weight reached 10% of body weight. All experiments with mice were approved by the committee for experiments on animals, The National Hospital, Oslo, Norway.
Establishment of mouse plasmacytoma-green fluorescent protein cell clones.
MOPC315.4 cells were transduced with a retroviral vector [19] containing EGFP and neo
R genes
(BD Biosciences Clontech, Palo Alto, CA, USA). Clones obtained by limiting dilution were selected for the highest
GFP luminescence by flow cytometry. After several rounds of in vivo passages and limiting dilution and selection in vitro , the clone MOPC315-7.13 (MOPC315-GFP) was chosen (Fig. 1B,C).
Metastatic cell lines.
Primary tumour, liver and lungs were minced and incubated with collagenase/DNAse
(Sigma-Aldrich, St. Louis, MO, USA) for 30 min at
37 C. All tissues were squashed and passed through a steel mesh to obtain single-cell suspensions. Cells were washed and cultured in 96-well plates under limiting dilution conditions in RPMI medium for 10 days. The clones were then expanded in flasks for several days before being used in experiments.
Flow cytometry and cell sorting.
Cell suspensions from primary tumour or the liver were stained, as previously described [20, 21]. CD11b-allophycocyanin, CD138phycoerythrin antibodies (Abs) and the corresponding isotype-matched Abs were from BD Pharmingen (San Diego,
CA, USA). Cells were analysed on a FACSCalibur or sorted using a FACSVantage (Becton Dickinson, San
Jose, CA, USA) flow cytometer. For intracardiac injection experiments, the GFP
þ cells were sorted once (Fig. 2A and C).
For RNA isolation, cells were doubly sorted (Fig. 2B and D).
Immunohistochemistry.
Tumour sections were stained with biotinylated or fluorochrome-conjugated MoAb specific for mouse CD138 (Becton Dickinson), GFP or their isotype-matched controls (R&D, Oxon, UK), as previously described [20]. Frozen sections were cut from biopsies from metastatic liver tumours and fixed in 4%
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536 Liver Metastasis and CCR6 J. Dellacasagrande et al.
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paraformaldehyde for 10 min. Endogenous peroxidase activity was quenched with 0.3% H
2
O
2
. The preparations were consecutively blocked with 5% normal horse serum, avidin and biotin. Between incubations, sections were washed with phosphate-buffered saline. Further incubations were with either anti-CCR6 (R&D) or antihepatocyte (Dako, Glostrup, Denmark) Abs, biotinylated horse antimouse Abs (Vector, Burlingame, CA, USA) and avidin–biotin–peroxidase complex (Vector). Staining was developed by incubation with diaminobenzidine tetrahydrochloride. Counterstaining was with haematoxylin.
Semiquantitative multiplex reverse transcriptase-polymerase chain reaction.
Total RNA was extracted from 5 10
5 cells using Trizol (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized, as previously described [21]. The levels of mRNA for multiple chemokine receptors were measured using commercial kits (CytoXpress, Biosource, Camarillo,
CA, USA). PCR products were electrophoresed in ethidium bromide-containing agarose gel, and product intensity was determined by Gel Doc 2000 transilluminator using
QUANTITY ONE software (Bio-Rad, Hercules, CA, USA). All results were normalized to the intensity of glutaraldehyde-
3-phosphate dehydrogenase (GAPDH) product, which was also used to check the quality of RNA.
Real-time quantitative reverse transcriptase-polymerase chain reaction.
Tissue samples were frozen by standard protocol and kept frozen in liquid nitrogen. Frozen human liver biopsies were then crushed in liquid nitrogen, whereas MOPC315-GFP cells were pelleted, prior to their dissolution in Trizol and subsequent RNA extraction. Human liver total RNA was purchased from
BD Biosciences Clontech. cDNA was synthesized, as previously described [21]. Specific primers and probes for mouse and human CCR6, CCR7 and b -actin were purchased from Applied Biosystems (Foster City, CA,
USA), primers and probe for mouse GAPDH (sense:
5
0
-TGTGTCCGTCGTGGATCTGA; antisense: 5
0
CCTGCTTCACCACCTTCT; and probe: 5
0
-TG-
-CCTGGA-
GAAACCTGCCAAGTATGATGACA) were designed using
PRIMER EXPRESS software (Applied Biosystems) and synthesized by Sigma Genosys (Cambridge, UK). PCR products were quantitated by an ABI PRISM 7700
Sequence Detection System. For human genes, the results were normalized to b -actin expression and then shown in relation to normal liver counterparts using the cycle threshold ( C t
) comparative method, according to instructions (Applied Biosystems). For quantitation of mouse transcripts, a calibration curve was established using serial dilutions of genomic DNA. All RNA samples were pretreated with RNase-free DNAse, prior to cDNA synthesis.
Actin polymerization.
The actin polymerization in response to chemokines was measured, as described [22].
Briefly, cells were incubated with 500 ng/ml of CCL21 or CCL20 chemokine (R&D systems) at 37 C for various periods of time. The reaction was stopped, and the cells were simultaneously stained by the addition of a solution of RPMI containing 3.6% paraformaldehyde,
80 m
M phalloidin-fluorescein isothiocyanate and 100 m g/ml of lysophosphatidylcholine (Sigma-Aldrich). The intensity of green fluorescence associated with the polymerized actin was measured by flow cytometry. For each time point, the background level of fluorescence was measured in cells pretreated for 2 h at 37 C with 1 m g/ml of pertussis toxin (Sigma-Aldrich) before the addition of chemokines. The results were normalized to the mean fluorescence intensity before incubation with chemokines.
In vivo homing of tumour cells.
Sorted MOPC315-GFP cells from primary tumour or the liver (Fig. 2) were mixed with 6 m m diameter CaliBRITE-allophycocyanin (APC) beads (cell/bead ratio ¼ 1 : 20; Becton Dickinson) and injected intracardially, according to a previously described method [23]. In some experiments, sorted cells were incubated with 1 m g/ml of CCL20 for 20 min at 37 C before the injections. Thirty to 40 min after the injections, the mice were killed and single-cell suspensions were prepared from the livers and lungs. The number of MOPC-GFP cells and CaliBRITE-APC beads were measured by flow cytometry, according to experimentally established gates
(Fig. 3). The results are expressed as the number of
MOPC315-GFP/10
4
CaliBRITE-APC beads.
A
C
10
0
10
1
10
2
B
10
3
10
4
10
0
D
10
1
10
2
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3
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4
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0
10
1
10
2
GFP
10
3
10
4
10
0
10
1
10
2
GFP
10
3
10
4
Figure 2 Cell suspensions from primary mouse plasmacytoma-green fluorescent protein (MOPC315-GFP) tumour (A and B) and the liver
(C and D), before (A and C) and after (B and D) enrichment of GFP
þ cells by fluorescence-activated cell sorter.
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J. Dellacasagrande et al.
Liver Metastasis and CCR6 537
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A
300 30 3
116 21 2
B
10
0
10
1
10
2
GFP
10
3
10
4
10
0
10
1
10
2
GFP
10
3
10
4
10
0
10
1
10
2
GFP
10
3
10
4
Lungs Liver
R3
R2
R3
R2
Figure 3 Establishment of settings for flow cytometric quantification of re-injected mouse plasmacytoma-green fluorescent protein
(MOPC315-GFP) cells. (A) Gates for detecting MOPC315-GFP cells in liver
(number of added MOPC315-GFP cells to liver cell suspension). (B) Gates for detecting
CaliBRITE-APC beads in the liver of
MOPC315-GFP-injected mice (5 10 5 events)(R3). Gates for allophycocyaninlabelled beads from the experiment shown in
Fig. 6 and Table 2 (experiment 1). Upper panel: sorted primary tumour (PT þ CCL20) cells
(Fig. 6). Lower panel: sorted liver metastatic
(Li þ CCL20) cells (Fig. 6). R2: GFP
þ cells (this gate was not used for detection).
10
0
10
1
10
2
GFP
Liver
10
3
10
4
R3
R2
10
0
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1
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2
GFP
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Lungs
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R2
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10
2
GFP
10
3
10
4
Mouse plasmacytoma can form micrometastases in the liver
MOPC315 originally arose in the ileo-coecal region of mouse colon [24]. Subcutaneous injection of in vitro derived MOPC315 cells induced tumours in BALB/ c mice within 2–3 weeks [20, 21]. Metastases were detectable with primary tumours of less than 5 mm in diameter
(blood M315 < 50 m g/ml, [18]) by an in vitro limiting dilution method of single-cell suspensions from various organs and tissues [17]. Metastatic lines were predominantly found in cultures from bone marrow, draining
(LNs) and spleen, and their frequency increased with the size of the primary tumour (Fig. 1A). In mice with large tumours, metastatic MOPC315 cells were found in the liver and lungs at frequencies as high as 10
4
, similar to those observed in bone marrow (Fig. 1B). Therefore, liver metastasis could be easily studied in such mice.
GFP has often been used in tumour models to track cells metastasizing to various tissues. Thus, in order to purify and analyse migrating tumour cells, we transduced
MOPC315 cells with the GFP gene [19]. For further experiments, we selected MOPC315-7.13
clone
(MOPC315-GFP), because of high luminescence (Figs 1C and 4A) and similar metastatic propensity as that of the parent tumour (Fig. 1B).
Cell suspension obtained from primary s.c. MOPC315 contains only 10–20% of tumour cells identified by their forward and side scatter and CD138 staining (data not shown), the remaining cells being mainly of fibroblast phenotype. In the primary s.c. MOPC315-GFP tumour,
5–20% of the total cells appeared to have green luminescence by flow cytometry (Fig. 1D). The plasma cell marker
CD138, which was expressed on all cultured MOPC315-
GFP cells, was found on up to 45% of total tumour cells, and less than half of CD138
þ cells were GFP
þ
(Fig. 1E).
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538 Liver Metastasis and CCR6 J. Dellacasagrande et al.
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Figure 4 Detection of mouse plasmacytoma-green fluorescent protein (MOPC315-GFP) tumour cells, and immunohistochemistry of mouse and human liver sections. (A) Fluorescence of cultured MOPC315-GFP cells; (B–C) sections from the primary MOPC315-GFP tumour stained with GFP-specific antibodies (green) (B) and in combination with CD138-specific (red) antibodies (C); (D–G) sections of liver tissue from two different sites, stained with
GFP-specific antibodies (green) (D and E) with (F) or without CD138-specific (red) (G) antibodies; (H–J) sections of human liver biopsies with metastases of colon (H), ovarian (I) and thyroid medullary (J) carcinomas, stained with CCR6-specific monoclonal antibody. Isotype-matched antibody
(mouse IgG2b) gave no staining in parallel sections. Nuclei were stained with either 4 1 , 6 1 -diamidino-2-phenylindole (DAPI) (blue) (B–G) or with haematoxylin (H–J).
This was confirmed by immunohistochemical staining, as there were less GFP
þ than CD138
þ cells in frozen sections of MOPC315-GFP tumours (Fig. 4B,C). We considered that the low abundance ( < 50%) of GFP
þ cells in MOPC315-
GFP tumours is perhaps because of the cell-cycle related changes in the GFP levels (assuming that two adjacent cells in Fig. 4A had recently divided) and/or because of the loss of transduced GFP gene. Almost all GFP
þ cells expressed CD138 (Fig. 1F) and were clearly negative for macrophage/dendritic cell marker CD11b (Fig. 1D), indicating their suitability for tracking purposes.
The number of metastatic cells that were isolated from any organ in our model was generally very low. In addition, because the s.c. tumour rapidly reached the size when we were obliged to end the experiment, no metastases could be observed macroscopically in peripheral organs.
To obtain sufficient quantities of cells for analysis, we cultured tumour cell lines from metastases as well as primary tumours by limiting dilution in vitro . This assay also showed that MOPC315-GFP cells in various organs were indeed viable tumour cells and that they were not passively carried there, for example, by phagocytosing cells. In liver
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J. Dellacasagrande et al.
Liver Metastasis and CCR6 539
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sections, clusters of GFP
þ
CD138
þ cells were found surrounded by normal parenchyma (Fig. 4D–G) closely resembling melanoma micrometastases [25], unlike the intravessel cells observed in a carcinoma model [7].
Liver micrometastases overexpress CCR6 and CCR7 chemokine receptors
Molecular analyses of chemokine receptors showed that
MOPC315 and MOPC315-GFP cell lines derived from the liver, lungs and draining LNs had a higher expression of CCR2, CCR6, CCR7 and CXCR4 transcripts compared with those from the primary tumour of a representative BALB/ c mouse (semiquantitative RT-PCR, Fig. 5A).
This was not the case for spleen- and bone marrow-derived lines (Fig. 5A). In the BALB/ c Rag2
–/– mouse, which lacks
B and T cells, MOPC315 lines also had the CCR2, CCR6 and CCR7 upregulated pattern, suggesting that their phenotype was not dependent on the presence of specific immunocytes (Fig. 5B). The upregulated CXCR4 transcript phenotype as well as the expression of CCR3/5/9,
CXCR1-3 and CX3CR1 transcripts (Fig. 5A) could not be reproduced in Rag2
–/– mouse (Fig. 5B), suggesting that these might be affected by the existence of B and T cells.
We also tested CCR10 expression, but the results varied greatly and most lines lacked its expression. When the data from BALB/ c and Rag2
–/– mice were pooled, the liver, lung and draining LN lines showed a consistent, beyond
50%, increase in CCR2, CCR6 and CCR7 transcripts over the primary tumour cell lines, although there was a smaller increase in CXCR4 (Fig. 5C). We focused on
CCR6 and CCR7 molecules, because their transcripts were detected at almost 10-fold higher level than those from CCR2 in all tested cell lines (Fig. 5C), perhaps indicating their pathophysiologic importance. In addition, as we detected no expression of other chemokine receptors in Rag2
–/– mouse (or unchanging, in the case of CXCR4), it was unlikely that other receptors were important in liver metastasis.
Representative samples of metastatic tumour cell lines were further checked by real-time quantitative RT-PCR using GAPDH as the control for cDNA input. The quantitative RT-PCR tests (Table 1) confirmed that the levels of CCR6 and CCR7 transcripts were higher in the liver- and lung-derived MOPC315 metastatic lines than in primary tumour lines.
Next, we wished to ascertain whether the increased
CCR6 and CCR7 transcript levels in liver metastatic lines were paralleled at the protein level, as to cause a functional difference. Indeed, the lines from liver metastases polymerized actin [22] in response to CCL20 and CCL21 chemokines (ligands to CCR6 and CCR7, respectively) to a greater extent than those from the primary tumour (Fig. 5D,E). The functional response of lung-derived metastatic lines was somewhat lower than that found in metastatic MOPC315 lines from the liver.
As high in vitro functional expression of CCR6 and
CCR7 was found with lines derived from liver metastases, both receptors might have potential roles in in vivo processes. We sought in particular to evaluate the role of
CCR6 in colonization of metastatic cells to the liver, because its ligand – the liver and activation-related chemokine (LARC, CCL20) – transcripts were produced by the liver in response to various inflammatory stimuli (lipopolysaccharide (LPS), cytokines) [26, 27], whereas no CCR7 ligand mRNAs were ever detected in the liver [28, 29]. For this, we sorted the tumour cells from the liver by flow cytometry using the GFP marker (Fig. 2B and D), and additionally ascertained their identity by staining with
CD138-specific Abs. Such freshly double-sorted liver
GFP
þ cells showed increased levels of CCR6 and CCR7 transcripts compared with the counterparts from the primary tumour by real-time RT-PCR analysis (Table 1).
Sorted metastatic cells from the liver have in vivo functional
CCR6 receptors
Next, we wished to determine whether sorted MOPC315-
GFP metastatic cells from the liver could home into the organ of their origin [23]. Sorted cells, 20,000 in number
(Fig. 2A and C), were re-injected into the heart of each mouse together with a 20-fold higher amount of red
(allophycocyanin) fluorescent beads. The beads served not only as a control for the injection (Fig. 3), but also to normalize results against variations in blood perfusion of organs. In order to assess the number of re-injected
GFP
þ cells, we analysed complete cell suspensions of livers and lungs by flow cytometry. Both, liver-sorted and primary tumour-sorted GFP
þ cells gave a low-level colonization of the liver after half an hour with no apparent differences between them (Table 2 and Fig. 6A,C). Similarly, lodging to the lungs of the same animals was at an even lower level
(Table 2). At the first glance, this result would support the view that random seeding of tissues by haematogenically spreading tumour cells could cause metastasis. However, the finding that liver MOPC315-GFP metastatic cells and lines had increased CCR6 levels would remain unexplained. The ligands for CCR6, CCL20 and b 2-defensins are induced upon inflammatory stimuli in various tissues in mice [27, 30, 31]. Furthermore, CCL20 is constitutively expressed in the small intestine and colon in mice
[27] and LPS [27]/inflammation [26] can induce its expression in the liver and lungs. Thus, it is conceivable that chemokine-induced adhesive forces might have played a role in the colonization of the liver by MOPC315 cells.
In order to test this assumption, we wished to activate chemokine receptors on sorted tumour cells and analyse their homing efficiency. Indeed, preincubation with
CCL20 caused a marked (3.5–4.7 times) increase in homing
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540 Liver Metastasis and CCR6 J. Dellacasagrande et al.
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A
CCR1
CCR2
CCR3
CCR4
CCR5
CCR6
CCR7
CCR8
CCR9
CX3CR1
CXCR1&2
CXCR3
CXCR4
CXCR5
Tumour
Liver
Lung
Bone marrow
Spleen
C
0 20 40 60 80 100
% GAPDH intensity
50
B
CCR1
CCR2
CCR3
CCR4
CCR5
CCR6
CCR7
CCR8
CCR9
CX3CR1
CXCR1&2
CXCR3
CXCR4
CXCR5
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Liver
Lung
Bone marrow
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Draining LN
0 20 40 60 80 100
% GAPDH intensity
40
30
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Draining LN
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10
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0
0
CCR2
30 60
Time (s)
90 120
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E
130
CCR7
120
110
100
90
0 30 60
Time (s)
90 120
Figure 5 Differential chemokine receptor expression and function of metastatic mouse plasmacytoma (MOPC315) cells cloned in vitro . (A–C) The mean level of chemokine receptor transcripts determined by semiquantitative reverse transcriptasepolymerase chain reaction (RT-PCR) in metastatic cell clones ( n ¼ 3–7) derived from various organs of a single BALB/ c
(A), or BALB/ c Rag2 –/– mouse (B) is shown in relation to their glutaraldehyde-3phosphate dehydrogenase (GAPDH) levels.
The pooled mean level ( standard error of the mean) of metastatic cell line-derived transcripts from four mice is shown for each selected chemokine receptor (C); (D and E) functional in vitro analysis: actin polymerization of tumour cell lines derived from primary tumour ( * ), lungs ( & ) or liver ( n ) in response to CCL21 (D) or
CCL20 (E).
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Table 1 Relative amount of cDNA encoding CCR6 and CCR7 measured by quantitative real-time reverse transcriptase-polymerase chain reaction
Mouse cells
CCR6* CCR7*
Cell lines from y
Tumour (Primary)
Liver (Metastatic)
Lung
Cell-sorted MOPC315-GFP from z
Tumour (primary)
Liver (metastatic cells)
Human tissues
1
6.67
5
1
6.02
1
14
3.95
(0)§
(20)§
Normal liver
Liver metastases of**
Colon (sigmoid) carcinoma ( < 2 cm)
Colorectal carcinoma (9 cm)
Colorectal carcinoma (9.5 cm)
Ovarian carcinoma
CCR6 {
1
31.35
0.26
0.42
128.34
CCR7
1
ND
0.13
0.1
24.25
{
ND, not detected.
*CCR6 and CCR7 levels of mRNA are normalized to glutaraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels and related to the value obtained for the tumour.
y Origin of the tissue from which the cell lines were derived by the limiting dilution.
z Origin of the tissue from which mouse plasmacytoma-green fluorescent protein (MOPC315-GFP) cells were sorted by flow cytometry.
§In brackets is the percentage of CCR7 level of expression normalized to the level of GAPDH mRNA.
{ CCR6 and CCR7 levels of mRNA are normalized to -actin transcript levels related to the value obtained for normal liver tissue.
**Origin of the primary tumour, and approximate size in diameter of metastases are shown in brackets.
frequency to the liver, but not to the lungs, of liversorted MOPC315-GFP cells as compared with primary tumour counterparts (Table 2 and Fig. 6B,D). Unfortunately, the number of sorted metastatic cells from lungs was too low to perform similar homing experiments.
Nevertheless, these results demonstrated that ligation of
CCR6 molecules facilitated homing of MOPC-GFP tumour cells to the liver.
tumour cells of colon (Fig. 4H), ovarian (Fig. 4I) and thyroid (Fig. 4J) carcinomas, supporting our previous quantitative RT-PCR findings. In order to discriminate with certitude between tumour cells and hepatocytes, adjacent sections were stained with a hepatocyte-specific MoAb, which, as well as isotype-matched control MoAbs, did not reveal any staining of tumour cells (data not shown).
In conclusion, human liver metastases of various cancers express elevated levels of CCR6 in RNA and Protein.
Small liver metastases of human cancers express CCR6 molecules
To find out whether CCR6 molecules might play a role in metastases of human cancer, we analysed liver biopsies containing metastases of various carcinomas. They were analysed by quantitative real-time RT-PCR and compared with normal liver tissue. Others have previously found low, but equal levels of CCR6 transcripts in normal liver as well as primary hepatocellular carcinoma [32]. By contrast, our results showed higher than normal levels of
CCR6 transcripts in liver metastases of colon, thyroid and ovarian carcinomas compared with normal tissue
(Table 1). CCR7 transcripts were usually not detectable.
Interestingly, elevated CCR6 transcripts were found in small metastasis of colon and ovarian carcinomas, but not in larger liver metastases of colon carcinomas
(Table 1). We explain this by hypothesizing that genetic progression of liver metastasis could obviate the necessity of cancer cells to express CCR6 after they migrated to the liver. Moreover, CCR6-specific Abs stained metastatic
Our results establish a link between the expression of
CCR6 on tumour cells and the metastasis to the liver.
The sequence of events leading to metastasis could be the following: in the primary MOPC315-GFP tumour, probably because of the genetic instability and progression,
CCR6 and CCR7 molecules might have been upregulated on some tumour cells, thereby facilitating their emigration.
CCR7
þ tumour cells might depart via afferent lymph because lymphatic endothelium expresses the CCR7 ligand
CCL21 [28], as suggested previously for metastasis of melanoma [12], chronic lymphocytic [13] and adult
T-cell [11] leukemias. This could explain the present finding of high CCR7/CCR6 transcript MOPC315-GFP lines in LNs (Fig. 5B). In addition, MOPC315-GFP cells might have directly gained access to the blood by invading tumour mosaic vessels. The endothelium of these vessels has a shared blood–lymph makeup [33], and could attract high CCR7 MOPC315-GFP cells perhaps by secreting
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Scandinavian Journal of Immunology 57, 534–544

542 Liver Metastasis and CCR6 J. Dellacasagrande et al.
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A
C
10
0
10
1
B
10
2
10
3
10
4
D
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
GFP
10
3
10
4
10
0
10
1
10
2
GFP
10
3
10
4
Figure 6 Homing of sorted mouse plasmacytoma-green fluorescent protein (MOPC315-GFP) cells to the liver. (A–D) Sorted MOPC315-
GFP cells from primary tumour (A, B; Fig. 2A,B) or from the liver (C, D;
Fig. 2C,D) were incubated with (B and D) or without (A and C) CCL20, mixed with CaliBRITE-APC beads, and injected into the hearts of
BALB/ c mice. Homing was analysed 30–40 min after injections.
The number of MOPC315-GFP cells and CaliBRITE-APC beads in the liver was assessed by flow cytometry using experimentally determined gates (Fig. 3) to exclude autofluorescence (FL2). The acquisition threshold was set at 1.8
10
2 in FL1 channel to eliminate unlabelled liver cells
(20–30 10
6 events).
CCL21.
This would explain why blood-borne
MOPC315-GFP cells of such phenotype were found at a concentration of about 1500/ml in mice with large tumours. Supposedly, blood-borne migrating tumour cells can be randomly arrested in small vasculature. However, mechanical capture in capillaries is clearly not a sufficient explanation for increased homing to the liver of liver-sorted MOPC315-GFP cells after the treatment with
CCL20, because untreated counterparts should have been arrested at a similar rate (Fig. 6C,D and Table 2). It has been shown on T cells [34] that the interaction between
CCR6 and its ligand resulted in CD18-mediated adhesion to inflamed endothelium. Similarly, the activation of unknown adhesion molecule(s) on migrating MOPC315-
GFP cells might occur via CCL20–CCR6 interaction during their passage through the gut and/or the liver
(resembling the preactivation step of cell-sorted liver
GFP
þ cells, Fig. 6D). This would in turn promote their attachment to endothelium of blood vessels in the liver
(similar to the homing of sorted MOPC315-GFP cells in vivo , Fig. 6D). The reason why untreated sorted liver
MOPC315-GFP cells were not activated in such a way might be because not all injected cells have passed through areas with CCL20 secretion on the way to the liver. Then, the arrested tumour cells in the liver might diapedese and form micrometastases in the parenchyma (Fig. 4D–G).
The survival of liver micrometastases probably depends on processes including genetic progression and selection for the best growing phenotype, because, in another tumour model, 99% of micrometastases disappeared within a short period of time after colonization [25]. It follows that, with genetic progression, some metastases might lose their original (CCR6-hi) homing phenotype.
In our model, the frequency of metastasis was similar in bone marrow and in liver and lungs when the tumour was
> 1.5 cm. In humans, liver metastasis, compared with bone marrow metastasis, is rare in patients with multiple myeloma and, therefore, we examined liver metastasis that originated from other primary tumours. Our preliminary results suggest that firstly, cancer metastasis to human liver might be influenced by the CCR6 expression. Secondly, in human liver, which in contrast to the mouse constitutively expresses CCL20 [35], migrating CCR6
þ cancer cells might be arrested by the CCR6–CCL20 interaction.
Thirdly, CCR6 might not be necessary for the survival
Table 2 Homing of sorted mouse plasmacytoma-green fluorescent protein (MOPC315-GFP) cells to the liver and lungs
PT Li PT þ CCL20 Li þ CCL20
Homing to liver*
Experiment 1 y
Experiment 2 z
Homing to lungs*
Experiment 1
Experiment 2 y z
130
135 z
9 z
18
100
107 z
35 z
23
63§
83
143§
20
219§
393
18§
60
PT, sorted primary tumour GFP
þ cells; Li, sorted liver GFP
þ
*Values denote MOPC315-GFP/10
4
CaliBRITE-APC beads.
cells; y Flow cytometric results of data in this row are shown in Fig. 4.
z Right-heart injection.
þ CCL20, incubation in vitro with CCL20.
§Left-heart injection. We assumed that the right-heart injection occurred when lungs : liver ratio of CaliBRITE-APC beads was in the range from 10 to 30, and for the left-heart injection, from 0.3 to 0.2.
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J. Dellacasagrande et al.
Liver Metastasis and CCR6 543
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and growth of metastasis, because some large metastases of colon carcinomas lacked its transcript. It would therefore be necessary to further investigate whether all known types of cancer can use a similar pathway to metastasize into the liver. We propose that constitutive expression of CCL20 by the liver attracts CCR6-expressing migrating cancer cells.
The liver may then select attracted cells to attach and form micrometastases, perhaps by binding putative integrin-like adhesion molecule that was possibly either induced or activated on cancer cells via CCL20–CCR6 interaction.
Although the organ predilection of metastatic cells might ultimately depend on multiple chemokine receptor–ligand interactions rather than a single interaction as we encountered here, we suggest that inhibition of CCR6 signalling could already be beneficial as therapy in the prevention of liver metastasis.
We thank Gøril Olsen for excellent technical help in cell sorting, Bradley D. Howard for GFP transduction of
MOPC315 cells, Dag Sørensen for help with intracardiac injections, Kristin Bjørnland and Jahn M. Nesland for providing human liver biopsies with metastatic cancer and Hilde Tveitan and Per Arne Andresen for help with
AB17700. We thank Drs Guttorm Haraldsen and David
Fraser for a critical analysis of the manuscript.
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