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Immunology |
1 Departments of Otolaryngology and Immunology, and 2 Pathology, University of Pittsburgh School of Medicine, and 3 Departments of Surgery and 4 Biostatistics, 5 University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The predilection of metastases for specific organs may depend on a variety of factors (3, 4) . According to the one theory, all of the migrating tumor cells can enter any tissue but would form a metastasis only if all requirements for their growth were met (5) . The second suggests that tissue-specific adhesion molecules on endothelial cells select migrating cells able to attach and form a premetastatic nucleus of cells (6) . The most recent theory proposes that chemoattractants, produced by stromal or immune cells, lead invasive cancer cells to the tissue of their potential secondary growth (7) .
Chemokine molecules constitute a superfamily of inducible, secreted, proinflammatory proteins (8, 9, 10, 11) involved in a variety of immune responses, acting primarily as chemoattractants and activators of specific types of leukocytes (12, 13, 14) . Recent studies have shown the involvement of chemokine receptors in cancer metastasis (15, 16, 17) . Chemokine ligand (CCL19)/MIP3-ß and CCL21/SLC, two chemokines constitutively expressed by lymph nodes (LN) and other immune cells, share a common chemokine receptor, CCR7 (16) . This receptor is also expressed on mature dendritic cells (DC), naïve, and some memory T cells. A recent study reported high levels of expression of CCR7 and CXCR4 in breast cancer cells and linked the receptor expression to the metastatic destination of tumor cells (17) .
Because of the unique site of cervical metastasis seen in squamous cell carcinoma of the head and neck (SCCHN) without frequent distant metastases, we hypothesized that SCCHN cells might also use a chemokine-mediated mechanism during the process of LN metastasis of cancer cells, similar to those regulating lymphocyte trafficking. However, we found that CCR7 up-regulation alone did not appear sufficient, rather loss by tumor cells of surface CCR6 enabled their LN metastasis. Interestingly, these results show that SCCHN cell metastasis displays a similar receptor pattern as manifested by immune cells, during the normal maturation and trafficking to LN away from peripheral sites of inflammation. Metastatic tumor cells appear to down-regulate CCR6 expression and up-regulate CCR7, enabling migration to secondary lymphoid tissues. SCCHN is thought to metastasize through afferent lymphatic channels, as opposed to the hematogenous route. Our data suggest that CCR7-mediated metastasis, using established gradient expression of the cognate ligand on vascular channels infiltrating tumors, may enable SCCHN cells to access the cervical LN.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cell lines PCI-4T/M, PCI-6T/M, PCI-15T/M, PCI 37-T/M (T: primary tumor, M: metastatic LN) were derived from the patient lesions (see Table 1
) at the University of Pittsburgh (18, 19, 20)
. The panel of cell lines TU686, 686LN, derived from a SCCHN patient’s primary (TU) and metastatic (LN) tumor, and 686LN-M3a2, 686LN-M3b2, and 686LN-M3b3 (3 different clones derived from a highly metastatic derivative of 686LN cells passaged three times in nude mice; Refs. 20, 21, 22
), were used to compare CCR7 expression by quantitative reverse transcription-PCR (qRT-PCR).
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Immunohistochemistry.
Immunostaining was performed by the avidin-biotin-peroxidase method; color reaction was developed in diaminobenzidine solution, and counterstaining was performed with Mayer’s hematoxylin solution. Tissues were stained using 2H4 primary antibody (Ab), mouse IgM, antihuman CCR7 (BD Biosciences Inc., San Diego, CA), or 53103.111 mouse IgG2B, antihuman CCR6 (R&D Systems Inc., Minneapolis, MN). Ab titrations and isotype control Ab determined optimal staining conditions.
DC Cultures.
DCs were generated from human peripheral blood mononuclear cells (24)
. Briefly, peripheral blood mononuclear cells were isolated from leukocyte-enriched buffy coats by density-gradient centrifugation through Ficoll-Paque Plus (Amersham Biosciences AB, Uppsala, Sweden). Peripheral blood mononuclear cells (5 x 107) were resuspended in 10 ml of warm AIM-V medium. After 1 h at 37°C, nonadherent cells were removed, and adherent cells were cultured in 10 ml of DC medium supplemented with interleukin 4 (1 x 103 units/ml) and granulocyte macrophage colony-stimulating factor (1 x 103 units/ml). On day 3, additional interleukin 4 (1 x 103 units/ml) and granulocyte macrophage colony-stimulating factor (1 x 103 units/ml) were added. On day 6, immature DCs were harvested for use in some experiments. DC maturation was induced using AIM-V plus 10 ng/ml tumor necrosis factor 
, 10 ng/ml interleukin 1ß, and 10 ng/ml interleukin 6 for 18 h. Both immature DCs and mature DCs were phenotyped by flow cytometry, using Abs to CD80, CD14, CD86, HLA-DR, CD40, and CD83 for staining as described previously.
Isolation of RNA and Reverse Transcription PCR (RT-PCR) Analysis.
Total RNA was isolated from tumor lines and biopsies using RNeasy Mini kit (Qiagen Inc.) according to the manufacturer’s protocol. All of the RT-PCR was done by using Thermoscript One-step System kit (Invitrogen Corp.), using primers as described (25)
. Reverse transcription was performed at 65°C for 35 min and 95°C for 5 min. PCR was performed at 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s, total for 33 cycles.
Quantitative Real-Time RT-PCR.
RNA from normal cervical LN was used as positive control and a calibrator of relative CCR7 expression. This calibrator RNA sample was amplified in parallel on all of the RT-PCR plates to allow comparison of samples run at different times. Reverse transcription was performed with random hexamer primers and Superscript 
(Invitrogen Corp.) as described previously (26)
. qRT-PCR was then carried out on the Applied Biosystems 7700 Sequence Detection Instrument, using Taqman Pre-Developed Assay Reagent for human CCR7 (Applied Biosystems, Foster City, CA). qRT-PCR was performed at 95°C for 12 min, and PCR was performed at 95°C for 15 s and 60°C for 60 s. Relative expression of the target gene:endogenous control gene, ß-glucuronidase (GUS), was calculated using the 
CT method described previously: (Relative expression = 2-CT; where CT = CT (Target gene) - CT (GUS); Ref. 27
).
Flow Cytometric Analysis of Chemokine Receptor Expression.
Tumor cells (105 cells) were suspended in 25 µl of 1% (v/v) FCS in PBS (containing 0.1% NaN3) for 15 min at room temperature and incubated with 5 µl aliquots of fluorescein-conjugated mouse antihuman CCR6 antibody, IgG2b (R&D Systems Inc.) or rat antihuman CCR7 antibody, IgG2a (BD Biosciences Inc.) for 1 h at 4°C. After washing the cells twice in 4-ml aliquots of the same PBS buffer, cells were resuspended and fixed in 200 µl 1% (w/v) paraformaldehyde for analysis. A murine IgG2b and a rat IgG2a antibody were used as negative controls. Ten thousand cells were collected using Beckman-Coulter Epics XL (Beckman Coulter, Inc., Fullerton, CA) and EXPO32 analysis software.
Chemotaxis and CCR Blocking Assays.
Migration studies used disposable 96-well chemotaxis chambers (ChemoTx Neuroprobe, Gaithersburg, MD; Ref. 28
) with a 5-µm pore size and 6-mm width/well, in triplicate. Cells and chemoattractants were resuspended in DMEM (Invitrogen Corp.) supplemented with 0.5% (w/v) BSA (Invitrogen Corp.). Human immature DCs and mature DCs derived were used as controls. CCR6 ligand (MIP-3) and CCR7 ligands (MIP-3ß and 6Ckine/SLC; R&D Systems, Inc.) were used. Aliquots (29 µl) of the CCL were added to the wells at a concentration of 50 or 500 ng/ml, and 50 µl of the cell suspension (1 x 105 cells) were placed on top of the filter. After 4 h or 18 h of incubation at 37°C in the presence of 5% CO2, the cells were removed with a cell harvester, and the filter was washed with medium. A 50-µl aliquot of trypsin-EDTA (Invitrogen Corp.) was added to the filter for 3 min at 37°C, 5% CO2 before centrifuging at 1500 rpm for 10 min. The filter was carefully removed, and cells in each lower well were counted under a light microscope in at least five different fields (original magnification, x200).
For CCR-blocking experiments, CCR specific monoclonal Abs (mouse antihuman CCR6 or antihuman CCR7 antibody) were used. The cells were incubated with monoclonal Ab (4 µg/10 µl/106 cells) for 1 h at room temperature before loading onto the filter, using an isotype control, and the chemotaxis assay was performed as above.
Statistical Analysis.
Data are presented as mean ± SD of repeated assays. The signed rank test was used to test differences among samples that were matched by patient, cell line, or cell type.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Comparing the semiquantitative RT-PCR results of CCR expression in each pair of cells, a consistent pattern of CCR6 down-regulation compared with metastases and higher CCR7 expression was found in metastatic tumor cell lines (Fig. 1A)
. Importantly the same pattern of CCR6 down-regulation and CCR7 up-regulation in metastatic as compared with primary tumor tissues was confirmed by standard RT-PCR analysis in 14 sets of paired primary and metastatic biopsy specimens, obtained from the same patients (Fig. 1B)
. Fig. 1
shows data for the first 12 patients, whereas the T/M biopsies of 2 other patients demonstrated similar results (data not shown). Whereas other CCR genes were expressed to various extents in tumor cells and tissues, no other consistent expression pattern could be observed for the other CCRs or between individual patients.
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Significant Up-Regulation of CCR7 Expression in Metastatic Tumor Cells by qRT-PCR.
As described above, CCR7 mRNA was detected in both primary and metastatic tumor lines and fresh specimens by RT-PCR analysis (see Fig. 1
), raising the question as to whether levels of expression were similar between primary and metastatic tumor cells. Therefore, using real time qRT-PCR, we determined the relative CCR7 expression between primary tumors and metastases, in paired cell lines and fresh tumor tissues. We carefully dissected tumor from surrounding nodal architecture to remove contaminating immune cells. Because CCR6 expression was not detected in metastases, it was not analyzed using qRT-PCR. These experiments showed up to 50-fold higher expression level (P < 0.001) of CCR7 in metastases than in primary tumors (Fig. 2)
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, CCR7 up-regulation was determined quantitatively to be significantly associated with increased metastatic potential when comparing TU686 with 686LN (P < 0.05), as well as comparing TU686 with any of the three subcloned, highly metastatic cells (P < 0.005). The three subcloned, highly metastatic cells were randomly selected to ensure that this up-regulation was a generally observed phenomenon. Because of the homology between murine and human CCR7 ligands and cross-reactivity with human CCR7 (30
, 31)
, functionally relevant signaling can explain this significant CCR7 up-regulation.
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. On the other hand, metastatic tumors expressed surface CCR7, but not CCR6 (see Fig. 4A
). Increased expression of CCR7 in 686LN and 686LN-M3 cells compared with TU686 cells was also observed using this technique (data not shown). Fig. 4B
also shows CCR7 responses after treatment with its ligand, MIP-3ß, at a concentration of 500 ng/ml for 4 h by flow cytometry. Metastatic (M) cells manifest the expected CCR7 down-regulation only after treatment with the appropriate ligand (MIP-3ß). However, down-regulation of CCR6, after treatment of primary (T) tumor cells, was observed after MIP3- treatment (data not shown). Treatment of each M cell line with the CCR6 ligand, MIP-3, as a control did not result in CCR7 down-regulation, as shown in Fig. 4B
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. Using immunohistochemistry, differential CCR6 and CCR7 expression in primary versus metastatic head and neck tumors (n = 4 patients) was confirmed (P1-P4 in Table 1
) from patient tumors studied using qRT-PCR. Immunostained tissue sections indicated that primary tumors expressed high levels of CCR6, but metastatic tumors expressed high levels of CCR7 (Fig. 5, A–F)
. These results rule out the possibility that high CCR7 expression in metastatic tumors was due to lymphoid cells in tumor-metastatic LN specimens, and is consistent with the retention of different migratory responses of primary (T) and metastatic (M) tumor cell lines, even after culture in vitro for months.
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(Fig. 6A)
. Enhanced responsiveness to both CCR7 ligands, MIP3ß and SLC (Fig. 6B)
, was observed in three of three cell lines tested, consistent with CCR7 up-regulation seen by qRT-PCR.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The consistent loss of expression of CCR6 in our system is reminiscent of the response of immune cells to inflammatory stimuli. For example, after antigen uptake or other stimuli that activate their progression to mature DCs, CCR6 expression is lost, whereas CCR7 is significantly up-regulated. Emigration from peripheral sites and trafficking to regional LNs usually leads to interaction with other immune cells. Whereas CCR7 is also used by circulating naïve and memory T cells, we would expect that metastatic SCCHN cells are likely to access cervical LNs using afferent lymphatics, similar to DCs. However, our data do not currently distinguish between vascular pathways used for this phenomenon, as our studies only used DCs as controls in qRT-PCR and migration studies. Lymphangiogenesis is not well understood currently, including the ligand-mediated signals related to embolism and implantation into regional LN.
The abundant expression of the homeostatic chemokine SLC (a ligand of CCR7) in LNs makes it a likely candidate to attract CCR7+ tumor cells (32, 33, 34, 35) . On the other hand, CCR6 down-regulation in metastatic tumor cells indicates that the coordinate expression of CCR6 and CCR7 may be important in orchestrating SCCHN metastasis (31) . Remarkably, our observations in fresh tumor specimens were maintained in cell lines cultured in vitro for months at a time. This also helps rule out that, in fresh metastatic tumor biopsies, CCR7 up-regulation was simply the result of immune cell contamination of our tumor RNA. In addition, the pattern of this coordinate CCR expression pattern in metastases indicates that the up-regulation of CCR7 in these tumor cells is not due to the LN microenvironment in fresh tissues, because even in metastatic tumor cell lines, this effect is maintained over long periods in culture. The finding that increasingly metastatic tumor derivatives in a metastatic mouse model showed quantitatively increased CCR7 expression demonstrates the in vivo importance of this phenomenon, in a completely separate experimental system.
Thus, a specific and differential expression pattern of CCR6, CCR7 is manifested by metastatic tumor cells, as their migration appears to be regulated by the loss of CCR6 expression, acting as the brake, and gain of expression of CCR7, acting as the accelerator, leading to migration of tumor cells to proximal lymphoid tissues. Although the organ predilection of metastatic cells might ultimately depend on multiple chemokine receptor-ligand interactions as we encountered here, rather than a single interaction, our data suggest that inhibition of CCR7 signaling could be a clinically useful target for therapy in the prevention of SCCHN metastasis. Additional work is under way to determine metastatic pathways responsible for tumor cell chemokine receptor-associated metastasis, in proinflammatory, nuclear factor 
B-mediated signaling pathways (36, 37, 38)
. Preventing such a poor prognostic feature in this disease would likely enable more successful locoregional tumor control and improve survival, even with current surgical and/or chemoradiotherapeutic modalities.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Robert L. Ferris, Departments of Otolaryngology and Immunology, University of Pittsburgh Cancer Institute, Hillman Cancer Center - Research Wing, Room 1.19d, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: (412) 623-7738; Fax: (412) 623-1415; E-mail: ferrisrl{at}upmc.edu
Received 9/19/03. Revised 12/18/03. Accepted 12/22/03.
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REFERENCES |
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B pathway. J. Biol. Chem., 278: 4675-4686, 2003. B and induces proinflammatory cytokine and chemokine production via a C-terminal signaling determinant. J. Immunol., 167: 505-513, 2001.This article has been cited by other articles:
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