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Cancer Research 68, 8419, October 15, 2008. doi: 10.1158/0008-5472.CAN-08-1242
© 2008 American Association for Cancer Research
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Immunology

A Subset of Host B Lymphocytes Controls Melanoma Metastasis through a Melanoma Cell Adhesion Molecule/MUC18-Dependent Interaction: Evidence from Mice and Humans

Fernanda I. Staquicini1, Anita Tandle2, Steven K. Libutti2, Jessica Sun1, Maya Zigler1, Menashe Bar-Eli1, Fabiana Aliperti3, Elizabeth C. Pérez3, Jeffrey E. Gershenwald1, Mario Mariano3, Renata Pasqualini1, Wadih Arap1 and José Daniel Lopes3

1 The University of Texas M. D. Anderson Cancer Center, Houston, Texas; 2 Surgery Branch, National Cancer Institute, Bethesda, Maryland; and 3 São Paulo Federal University, São Paulo, Brazil

Requests for reprints: Wadih Arap and Renata Pasqualini, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3873; Fax: 713-745-2999; E-mail: warap{at}mdanderson.org and rpasqual{at}mdanderson.org or José Daniel Lopes, Federal University of São Paulo, Rua Botucatu 862, São Paulo 04023-062, Brazil. Phone: 55-011-5576-4532; Fax: 55-011-5571-1095; E-mail: jdlopes{at}unifesp.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Host immunity affects tumor metastasis but the corresponding cellular and molecular mechanisms are not entirely clear. Here, we show that a subset of B lymphocytes (termed B-1 population), but not other lymphocytes, has prometastatic effects on melanoma cells in vivo through a direct heterotypic cell-cell interaction. In the classic B16 mouse melanoma model, one mechanism underlying this phenomenon is a specific up-regulation and subsequent homophilic interaction mediated by the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule). Presence of B-1 lymphocytes in a panel of tumor samples from melanoma patients directly correlates with MUC18 expression in melanoma cells, indicating that the same protein interaction exists in humans. These results suggest a new but as yet unrecognized functional role for host B-1 lymphocytes in tumor metastasis and establish a biochemical basis for such observations. Our findings support the counterintuitive central hypothesis in which a primitive layer of the immune system actually contributes to tumor progression and metastasis in a mouse model and in melanoma patients. Given that monoclonal antibodies against MUC18 are in preclinical development but the reason for their antitumor activity is not well understood, these translational results are relevant in the setting of human melanoma and perhaps of other cancers. [Cancer Res 2008;68(20):8419–28]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Studies addressing the role of the immune system in tumor growth and metastasis have yielded conflicting and often counterintuitive results. Over the 1970s, Prehn and colleagues proposed that the immune response mediated by lymphoid cells could paradoxically lead to tumor cell stimulation (1–3). To date, the interplay of immunity, inflammation, and cancer is still not entirely understood (4, 5). To add a further level of complexity—depending on the experimental model used—it is evident that host immunity can actually lead to enhancement, suppression, or even no effect at all on the metastatic potential of tumor cells, so that no global generalizations can be easily made (6).

Specifically in the B16 mouse melanoma model, previous reports show that melanoma cells can be stimulated by lymphocytes (7) and that melanoma progression can indeed be delayed if tumor-bearing mice are rendered immunosuppressed (8). However, the basis for these intriguing experimental observations remains elusive. In particular, the relevance of cell subpopulations from the more primitive layers of the immune system such as B-1 lymphocytes (9–12) on tumor phenotype has not been fully elucidated, although clues for such a role have recently emerged (13, 14).

Here, we have evaluated the cellular and molecular cross-talk by which B-1 lymphocytes affect melanoma growth and metastasis. First, we used the classic B16 mouse melanoma model to show that one mechanism accounting for this observation is the up-regulation and subsequent homophilic interaction of the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule). Next, we show that B-1 lymphocytes are also present in human tumors and directly correlate with MUC18 expression in melanoma cells, indicating that the same functional mechanism is conserved across species and likely active in human disease. Together, our results strongly suggest an important role for host B-1 lymphocytes in melanoma-derived metastasis and its corresponding biochemical basis in tumor-bearing mice and in patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Animals. Female mice were purchased and housed in the animal facilities of the University of Texas M. D. Anderson Cancer Center, Federal University of São Paulo, or University of Campinas. All animal procedures were approved by the respective Institutional Animal Care and Use Committee.

Human specimens. Incidental human melanoma samples were obtained, through written informed consent, from patients treated at the Surgery Branch of the National Cancer Institute (NCI) or at The University of Texas M. D. Anderson Cancer Center.

Reagents. Anti-MUC18 (mouse and human) antibodies were purchased from Santa Cruz Biotechnology and Zymed. Antibacteriophage (Sigma) and FITC-conjugated anti-human IgM, allophycocyanin-conjugated anti-human CD5, and phycoerythrin (PE)-conjugated anti-human MUC18 (BD Biosciences) were commercially obtained. MART-1 antibody was purchased from BioGenex and labeled with FITC by using EZ-Label FITC Protein Labeling kit (Pierce) and Zeba desalt spin columns (Pierce). Horseradish peroxidase (HRP)-conjugated anti-rabbit, PE-conjugated anti-mouse (Pharmingen), Cy3-conjugated anti-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories. Keyhole limpet hemocyanin–conjugated peptides and synthetic peptides were synthesized and conjugated to our specifications (AnaSpec).

Cell culture and coculture of B-1 lymphocytes and B16 melanoma cells. B16-derived melanoma cells (The Jackson Laboratory) were cultured in RPMI-1640 (Sigma) containing 10% of fetal bovine serum (Cultilab), antibiotics, and supplements. Purified B-1 lymphocytes were obtained as described (15). Only samples showing >95% purity were used.

Tumor growth and experimental metastasis assays. We used a standard model (16, 17) to deplete B-1 lymphocytes in mice. Untreated, radiated, or reconstituted cohorts of C57BL/6 mice received B16 cells i.v. (105 per mouse). Mice were sacrificed and the number of colonies on the surface of lungs was determined on day 15 after administration. Primary tumor growth into the mouse footpad was measured daily.

Phage display screening and binding assays. We used a random phage library displaying the insert CX7C (C, cysteine; X, any residue) for selection of peptides binding to melanoma cells after coculture with B-1 lymphocytes (18). As a preclearing step, 106 B16 cells without exposure to B-1 lymphocytes were detached, washed, and resuspended in RPMI-1640 containing 2% bovine serum albumin (BSA) plus 109 transducing units (TU) of unselected phage library. Cells and phage were transferred to the top of a nonmiscible organic lower phase [dibutyl phthalate/cyclohexane, 9:1 (v:v)] and centrifuged at 10,000 x g for 10 min. The unbound phage population remaining in the aqueous upper phase (precleared library) was collected into a fresh Eppendorf tube and incubated with 106 B16 cells isolated after coculture with B-1 lymphocytes. Phage in the organic lower phase was recovered from the cell pellet by bacterial host infection (19–24).

For phage binding assays to B16 melanoma, 106 cells before and after coculture with B-1 lymphocytes were incubated with each specific phage clone (109 TU) or negative controls. Melanoma cells and phage were centrifuged through the organic phase and the cell-bound phage clones were recovered by bacterial infection (18).

Immunocapture assays. Immunocapture experiments were with anti-MUC18 or IgG control antibodies, as described (19). ELISA with anti-IgG confirmed equal molar concentration of IgG on each of the wells. After blocking with PBS containing 3% BSA, 30 µg of protein from cell membrane extracts were added onto the wells for overnight incubation. Following washes, phage (2 x 109 TU) was added to each well. Bound phage were recovered by bacterial infection.

In vivo phage display. Homing of phage to s.c. tumors was performed as described (25). Animals received 1 x 1010 TU of phage diluted in DMEM. Tumors and control organs were collected after 6 h of circulation. Bound phage was recovered by bacterial infection (25).

Immunofluorescence and flow cytometry. B16 melanoma cells before and after coculture with B-1 lymphocytes were seeded in an eight-chamber slide (Nalge Nunc International) and incubated with phage (109 TU). Cells were washed, fixed, and incubated with an antibacteriophage antibody followed by secondary antibody. For flow cytometry, melanoma cells or purified B-1 lymphocytes were incubated with primary antibody anti-MUC18 followed by PE-conjugated secondary antibody. To investigate the presence of B-1 lymphocytes in human melanoma samples, cells were isolated, washed, fixed, and permeabilized with BD Cytofix/Cytoperm (BD Biosciences). Cells were stained either with isotype control or with specific antibody. Cells were analyzed with a FACSCalibur machine (BD Biosciences) equipped with CellQuest software.

Immunofluorescence and immunohistochemical staining for MUC18 detection in tissue specimens. Tissue specimens were sectioned, mounted, and air dried for 24 h. Antigen retrieval was performed with 0.1 mol/L citrate buffer (pH 6). Sections were stained with the UltraVision Plus Detection System Anti-Polyvalent, HRP/AEC kit (LabVision Corp.) and counterstained with Gill's hematoxylin (Sigma). For immunofluorescence, sections were washed, blocked, and incubated with specific antibodies and Cy3-conjugated secondary antibodies.

Western blot and immunoprecipitation assays. Cells were lysed by using PBS containing 250 mmol/L sucrose, 50 mmol/L octylglucoside, 1 mmol/L EDTA, and protease inhibitors, resolved in a 4% to 20% gradient SDS-PAGE gel, transferred to nitrocellulose membranes, and developed with the enhanced chemiluminescence reagent (Amersham Pharmacia). For detection of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), total proteins were extracted as described (14).

Reverse transcription-PCR. RNA was purified by using the Perfect RNA Mini kit extraction method (Eppendorf). First-strand cDNA synthesis was performed by using the SuperScript II Reverse Transcriptase kit (Invitrogen). For the mouse MUC18 transcript amplification, we used the primers 5'-GGATCCTTGGCTTGCGCCCTCCGTCGG-3' and 5'-CTAATGCCTCAGATCGATGTATTTCTCTCC-3' under the same conditions for template denaturation and elongation but with the annealing temperature of 60°C. As a loading control, we used primers for the mouse glyceraldehyde-3-phosphate dehydrogenase: 5'-CGCCTGGTCACCAGGGCTGC-3' and 5'-CACCACCCTGTTGCTGTAGCC-3'.

Design of small hairpin RNA and lentivirus production. Mouse MUC18 small interfering RNA (siRNA) sequences 5'-GGAGAGAAATACATCGATC-3' and 5'-GATCGATGTATTTCTCTCC-3' were obtained from Dharmacon (On-Target Plus, NM_023061). Nonspecific control siRNA [nontargeting short hairpin RNA (shRNA)] sequences were 5'-TAAGGCTATGAAGAGATAC-3' and 5'-GTATCTCTTCATAGCCTTA-3'. shRNA sequences for both targeting and nontargeting were ligated into a lentiviral vector pLVTHM, which drives the expression of the green fluorescent protein (GFP; a gift from Dr. Didier Trono, University of Geneva, Geneva, Switzerland; ref. 26). The restriction enzymes Cla1 and Mlu1 were used. The lentiviruses were produced by infecting human embryonic kidney cells (293FT) with the sequence-verified pLVTHM, the packing plasmid (MD2G), and the envelope plasmid (PAX2), required for viral production. GFP-positive cells were enriched to 100% by fluorescence-activated cell sorting (FACS).

Statistical analysis. Graphical analyses (balloon plots) were used to depict protein expression levels based on flow cytometry results. Spearman's rank correlation test was used to analyze the correlation between number of B-1 lymphocytes and MUC18 expression profile on patients. Statistical analysis of in vivo experiments was carried out by using Student's t tests as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
B-1 lymphocytes influence malignant melanoma metastasis in vivo. We first evaluated the role of B-1 lymphocytes in melanoma growth and metastasis in vivo by selectively depleting the predominantly B-1 lymphocyte population from peritoneal and pleural surfaces of mice (11, 12, 15, 27). We used external beam ionizing radiation to deplete B-1 lymphocytes with no detectable effect on other cell types (16, 17). We confirmed the depletion by flow cytometry analysis of cell surface markers: we observed a severe reduction in the B-1 lymphocyte population (typically over 80% cell depletion) by using this procedure (Supplementary Figs. S1 and S2). Next, we compared s.c. melanoma growth and experimental metastasis in radiated versus nonradiated (control) mice (Fig. 1A ). In the radiated cohorts, we observed tumor growth suppression (Fig. 1A, left) and marked reduction in melanoma metastasis (Fig. 1A, right). In either case, reconstitution with total peritoneal cells reverted tumor growth and metastasis to levels undistinguishable from those observed in control nonradiated mice. To evaluate which depleted cell population mediates this phenomenon, we reconstituted radiated mice with either B-1 lymphocytes (Fig. 1B) or all other resident peritoneal cells but B-1 lymphocytes (Fig. 1C). We show that B-1 lymphocytes are necessary and sufficient to revert the radiation-induced metastasis suppression of melanoma. Finally, by using an unrelated genetic model of immunosuppression [X-linked immunodeficiency (Xid)], we also observed melanoma metastasis inhibition when mutant mice (constitutively B-1 lymphocyte deficient; refs. 28, 29) were compared with their otherwise isogenic wild-type counterparts (Fig. 1D). These observations in Xid mutant mice are consistent with the results obtained from radiation-induced B-1 lymphocyte depletion. Together, these data from two independent experimental systems confirm that B-1 lymphocytes can control experimental metastasis derived from B16 melanoma cells.


Figure 1
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  		Figure 1. Effects of B-1 lymphocytes in B16 melanoma progression. A, left, effect of radiation-induced B-1 lymphocyte depletion on primary tumor growth; right, effect of radiation-induced B-1 lymphocyte depletion on metastases. Representative lungs containing melanoma metastases from control, radiated, and reconstituted mice. B and C, effect of reconstitution with B-1 lymphocytes or other cell types on metastases. B-1 lymphocyte reconstitution (B), but not all other peritoneal cell types (C), reverts the radiation-induced metastases suppression. D, effects of constitutive B-1 lymphocyte depletion on metastases. Suppression of metastases from B16 melanoma in mice with constitutive B-1 lymphocyte depletion (Xid) relative to otherwise isogenic control mice (wt). *, P < 0.05; **, P < 0.01.

 
Next, we cultured melanoma cells either in a Transwell system or with B-1 lymphocytes in coculture. Surprisingly, coculture enhanced melanoma metastasis. No effects on melanoma metastatic potential were observed when cells were cultured in shared medium (13, 14). Moreover, we observed cell clusters forming between B16 melanoma cells and B-1 lymphocytes by 48 to 72 h of coculture but not before; such heterotypic cell clusters contained 1 B16 melanoma cell plus 5 to 10 B-1 lymphocytes (Supplementary Fig. S3A–C). We confirmed an intimate physical membrane interaction between the two cell types by transmission electron microscopy (13). In sum, these data show that a direct and "prolonged" (defined as ≥72 h) cell-cell contact between B-1 lymphocytes and B16 melanoma cells renders the tumor cells more metastatic.

A MUC18-MUC18 homophilic interaction mediates the physical contact between melanoma cells and B-1 lymphocytes. We hypothesized that adhesion molecules expressed on B16 melanoma cell surfaces after contact with host B-1 lymphocytes would mediate the cell-cell interaction. To identify such molecules, we used a phage display-based combinatorial approach (18). We designed a two-step aqueous-to-organic phase separation strategy to select ligands to melanoma cells with enhanced metastatic potential. First, we precleared the phage library on B16 melanoma cells before coculture with B-1 lymphocytes. Next, we selected the unbound bulk phage population (precleared library) on isolated B16 melanoma cells after 72 h of coculture with B-1 lymphocytes and obtained strong serial enrichment (Fig. 2A ). We then proceeded to evaluate the binding of phage selected from the enriched population and found that 7 of 10 (70%) individual clones tested preferentially bound to melanoma cells after coculture with B-1 lymphocytes (range, 2- to 7-fold; median, 3-fold) relative to an insertless phage that served as negative control. Protein similarity searches revealed that several peptides displayed by the phage showing preferential binding to melanoma after coculture were reminiscent of the sequence of the glycoprotein MUC18 (30, 31).


Figure 2
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  		Figure 2. Screening of a phage display random peptide library on B16 melanoma cells after coculture with B-1 lymphocytes yields MUC18 as a candidate target molecule. A, enrichment of phage binding to melanoma cells after coculture with B-1 lymphocytes. B, binding of individual MUC18-mimic phage clones to B16 melanoma cells before and after coculture with B-1 lymphocytes. A phage clone displaying the peptide motif Arg-Met-Phe-Leu (mouse MUC18 residues R114-L117) had a marked increase in binding to B16 melanoma cells after coculture with B-1 lymphocytes relative to control insertless phage (~20-fold) or to melanoma cells without coculture with B-1 lymphocytes (~12-fold). Experiments were performed three times with similar results; a representative binding experiment is shown. C, the selected CLFMRLAWC phage is a mimic of MUC18. Anti-MUC18 and anti-CLFMRLAWC antibodies were used to detect MUC18 on the membrane of melanoma cells before and after coculture by Western blot analysis. D, both anti-CLFMRLAWC and anti-MUC18 antibodies coimmunoprecipitate MUC18.

 
We then searched whether additional selected ligand peptides had homologous sequences to that protein. In total, we found 48 motifs sharing sequence homology to the extracellular domain of MUC18 (Supplementary Fig. S4). Of these, we evaluated a panel of phage clones displaying peptides with homology to MUC18. We found that 10 of 15 (67%) individual clones bound preferentially to B16 melanoma cells after coculture with B-1 lymphocytes (range, 2.5- to 20-fold; median 4-fold). In particular, phage displaying the cyclic peptide CLFMRLAWC, which contains an embedded MUC18-like motif in reverse (sequence Arg-Met-Phe-Leu present in the extracellular portion of IgG1 domain; mouse MUC18 residues R114-L117), showed marked enrichment in phage binding relative to the negative control insertless phage (Fig. 2B). We then set out to functionally characterize the CLFMRLAWC-displaying phage and the corresponding homologous region within MUC18. We developed antibodies against the melanoma-targeting CLFMRLAWC peptide to evaluate whether they recognize MUC18. Proteins from cell membrane extracts of melanoma cells cocultured with or without B-1 lymphocytes were then probed with anti-MUC18 or anti-CLFMRLAWC peptide antibodies. Both antibodies against the native mouse MUC18 or against the CLFMRLAWC synthetic peptide detected undistinguishable protein bands by Western blot (Fig. 2C), suggesting that both recognize MUC18; reciprocal coimmunoprecipitation experiments were also consistent with such interpretation (Fig. 2D). Western blotting (Fig. 2C), immunofluorescence (Fig. 3A ), and flow cytometry (Fig. 4B ) analysis showed an increase in MUC18 expression in melanoma cells after coculture with B-1 lymphocytes, which directly correlated with the increase in MUC18 targeting.


Figure 3
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  		Figure 3. MUC18-derived phage binds to MUC18. A, binding of MUC18-derived phage on melanoma cells before and after coculture with B-1 lymphocytes. Left, a phage clone displaying a mouse MUC18-derived peptide (H111-S120) targets B16 melanoma cells relative to insertless phage or phage clones displaying various scrambled versions of the MUC18-derived peptide; right, immunostaining of melanoma cells with an anti-MUC18 antibody reveals a differential pattern of MUC18 expression on the cell surface. Staining with anti-phage antibody shows that phage binding recapitulates the different levels of MUC18 expression before and after coculture with B-1 lymphocytes. B, left, specific binding of mouse MUC18-derived (H111-S120) phage to immunocaptured MUC18 relative to negative controls (insertless and scrambled peptide phage); right, in vivo homing of MUC18-like phage to tumors before and after coculture with B-1 lymphocytes. C, double-label immunofluorescence of tumors derived from melanoma cells before and after coculture with B-1 lymphocytes. 4',6-Diamidino-2-phenylindole (blue) was used for nuclei staining. D, silencing of MUC18 expression in melanoma cells with small hairpin RNA. Decrease in protein expression was confirmed by immunoblotting. Coculture of B-1 lymphocytes with MUC18-negative melanoma cells does not increase melanoma metastatic potential.

 

Figure 4
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  		Figure 4. A and B, FACS analysis shows that B-1 lymphocytes express high levels of cell surface MUC18. In contrast to B16 melanoma cells (B), we observed lack of MUC18 up-regulation on B-1 lymphocytes after coculture with melanoma cells. A, black histogram, B-1 lymphocytes; gray dotted line histogram, B-1 lymphocytes after coculture with B16 melanoma cells. B, gray histogram, B16 melanoma cells after coculture with B-1 lymphocytes. A ~7-fold enhancement in MUC18 cell surface expression is detected after coculture with B-1 lymphocytes. C, MUC18 expression in B-1 lymphocytes by RT-PCR analysis. Left, no differences in expression levels of the MUC18 transcript were observed in B-1 lymphocytes cocultured with melanoma cells; right, in contrast, we clearly detected increased expression of MUC18 in melanoma cells after coculture with B-1 lymphocytes. D, cell signaling through MAPK pathways. Phosphorylation of ERK1/2 was investigated by immunoblotting in different time points after melanoma cell activation. Top, we observed specific phosphorylation of p44/42 after 2.5 min of cell stimulation with MUC18-like peptide; bottom, control unrelated peptide showed no effect in protein phosphorylation. Total p44/42 served as the loading control.

 
Based on the experiments described above, we predicted that the native MUC18 protein sequence would recapitulate the phage binding mediated by the peptide CLFMRLAWC. To experimentally test such prediction, we designed and constructed a phage clone displaying a peptide that encompasses the corresponding native MUC18 protein sequence (residues H111-S120) for use in binding assays on melanoma cells before and after coculture with B-1 lymphocytes. Consistent with our hypothesis, we observed that the display of a MUC18-derived (H111-S120) peptide sequence on phage promotes preferential binding to the surface of B16 melanoma cells after coculture with B-1 lymphocytes (2.5-fold relative to the baseline binding to malignant melanoma cells before coculture with B-1 lymphocytes); in contrast, several negative controls (a series of phage clones engineered to display scrambled versions of the peptide insert) had their binding to melanoma cells abolished to background levels regardless of coculture with B-1 lymphocytes (Fig. 3A, left). Moreover, to evaluate whether MUC18 might indeed be responsible for the differential phage binding, we compared the magnitude of phage binding to cell membrane expression of that molecule (Fig. 3A, right). We observed (a) that MUC18 expression increases after coculture with B-1 lymphocytes and (b) that there is a direct correlation between targeted phage binding and cell surface expression of MUC18 relative to controls. Together, these data not only show that a MUC18-derived ligand peptide mediates binding to B16 melanoma cells but also establish the overexpression of the cell surface receptor MUC18 itself in the melanoma cells after B-1 lymphocyte coculture.

To confirm that the H111-S120 peptide can functionally behave as MUC18 in the phage context as well, we evaluated the binding of H111-S120 phage to immunocaptured MUC18. We show that H111-S120 phage but not negative controls (including insertless or scrambled insert phage) binds to immunocaptured MUC18; no binding was observed when immunocapture was carried out by using an irrelevant IgG isotype control (Fig. 3B, left). We also show that anti-MUC18 antibodies specifically inhibit phage binding mediated by the MUC18-derived peptide H111-S120 relative to controls (Supplementary Figs. S5A and B). Furthermore, to study the specificity of the MUC18-targeted phage in vivo, we evaluated phage homing in mice s.c. implanted with melanoma cells before and after coculture with B-1 lymphocytes (Fig. 3B). We observed marked binding of MUC18-targeted phage to tumors derived from melanoma after coculture with B-1 lymphocytes compared with melanoma and control organ (Fig. 3B, right). Phage binding is accompanied by increased expression of MUC18 in tumors from melanoma cocultured with B-1 lymphocytes (Fig. 3C).

Next, we used shRNA to silence the expression of MUC18 in melanoma cells and to determine whether presence of MUC18 on the cell surface is required for the biological phenomenon to occur. Decrease in expression of MUC18 was confirmed by immunoblotting (Fig. 3D). MUC18-depleted cells were cocultured with B-1 lymphocytes for 72 h and injected i.v. into mice. We used B16 and B16 transduced with nontargeting shRNA as controls. As previously observed, coculture of MUC18-expressing melanoma cells (parental B16 or B16 transduced with nontargeting shRNA) with B-1 lymphocytes increases melanoma metastasis. However, such prometastatic effect is abrogated when MUC18-negative cells are used, a result consistent with our hypothesis that a MUC18-MUC18-mediated cell interaction renders melanoma cells more metastatic. Furthermore, a marked decrease in the number of lung colonies was observed in animals inoculated with MUC18-negative cells, again supporting the importance of this molecule in metastasis.

Taken together, these results confirm the specificity of the interaction and support the concept that a MUC18-MUC18 homophilic interaction mediates the physical contact between B16 cells and B-1 lymphocytes. To gain insight into the molecular basis of such interaction, we generated a panel of phage to combine alanine scanning site-directed mutagenesis and binding assays. Compared with wild-type H111-S120 phage, we identified four key residues (Arg114, Cys118, Lys119, and Ser120) in MUC18 whose mutation abolished phage binding to melanoma cells regardless of coculture with B-1 lymphocytes (Supplementary Fig. S5C). Results of these mutational studies again indicate binding specificity.

B-1 lymphocytes express MUC18. Given that phage selected to mimic a ligand expressed on the surface of B-1 lymphocytes resembled MUC18 and bound specifically to MUC18 on the surface of melanoma cells, we evaluated whether B-1 lymphocytes would also express MUC18 on their own cell surfaces. Flow cytometry analysis revealed cell surface expression of MUC18 in B-1 lymphocytes (Fig. 4A). However, in contrast to the MUC18 overexpression clearly observed in B16 melanoma cells after coculture with B-1 lymphocytes (Fig. 4B), no change in MUC18 expression was detected on the cell surfaces of B-1 lymphocytes themselves after coculture. We next used reverse transcription-PCR (RT-PCR) analysis to confirm changes in MUC18 expression after cell-cell contact. Consistent with the previous findings, we again observed an up-regulation of MUC18 transcripts in melanoma cells (but not in B-1 lymphocytes) after coculture (Fig. 4C), suggesting that MUC18 transcriptional control is differentially regulated in each cell type. Indeed, binding of B-1 lymphocytes to melanoma cells in vitro induces activation of the mitogen-activated protein kinase (MAPK) signaling pathway only in melanoma cells, whereas ERK1/2 phosphorylation seems to be constitutive in B-1 lymphocytes (14). Thus, to further investigate the role of MUC18 in this cell-cell interaction, we evaluated the effect of MUC18-like peptide in the activation of the MAPK pathway in melanoma cells. Cells were treated with MUC18-like synthetic peptide and protein phosphorylation was analyzed by immunoblotting (Fig. 4D). We show phosphorylation of ERK1/2 at 2.5 min after cell activation. In contrast, an unrelated control peptide did not induce ERK1/2 phosphorylation. Collectively, these data suggest (a) that MUC18 is expressed in both cell types but differentially regulated and (b) that a homophilic MUC18-MUC18 ligand-receptor system on the cell surface of B16 melanoma cells and B-1 lymphocytes mediates a heterotypic cell-cell interaction that ultimately leads to ERK1/2 phosphorylation and increase in melanoma metastasis.

A potential functional role for B-1 lymphocytes in human malignant melanoma. To investigate the relevance of our findings in human disease, we first evaluated the expression of MUC18 in patient-derived melanoma primary tumors and metastases: immunohistochemical analysis of MUC18 expression in skin, "in-transit," and lymph nodes (Fig. 5A ) showed marked MUC18 expression in both melanoma cells and vascular endothelial cells, consistent with other descriptive reports (32, 33). Of note, only melanoma cells but not lymphocytes stained positive for MUC18 within lymph nodes, a result again consistent with our observation that B-1 lymphocytes are the only MUC18-expressing B cells in both mice and humans. Moreover, we also examined the binding capacity of the MUC18-targeted phage to a panel of eight well-established human melanoma cell lines (Fig. 5B). We observed specific binding of the H111-S120 phage to all cell lines relative to negative controls.


Figure 5
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  		Figure 5. Expression of MUC18 in human melanoma. A, samples of skin, in-transit, and lymph node metastases were immunostained with anti-human MUC18 antibody. Arrows, MUC18-expressing melanoma cells in the epidermis (top left), dermis and exocrine ducts (top right), and lymph node metastases (bottom right). Arrowheads, MUC18-positive melanoma cells in vascular endothelial cells (top right and bottom left; 100-fold magnification). Bottom right, inset, negative control. B, phage binding assay to human melanoma cell lines. A scrambled version of the peptide and insertless phage were used as negative control.

 
Next, we asked (a) whether the human counterpart (34, 35) of murine B-1 lymphocytes (heretofore termed "human B-1 lymphocytes") are present in sites of human melanoma metastasis and (b) whether this B-cell population would recapitulate the functional behavior of mouse B-1 lymphocytes in the context of malignant melanoma. We used flow cytometry analysis to evaluate patient-derived samples (obtained from surgically removed metastatic melanoma cases; n = 16) for the presence of human B-1 lymphocytes (Fig. 6A ). B-1 cells were distinguished among other lymphocytes by CD5/IgM double expression, whereas human MUC18-expressing tumor cells were identified within MART-1+ melanoma cells. Expression of surface markers was graphically represented as a "balloon plot" (Fig. 6A). We observed a direct correlation between increasing number of human B-1 lymphocytes within the tumors and increasing expression of MUC18 on melanoma cells (r = 0.6; P < 0.05). Histologic analysis of representative melanomas with mild (patients 1 and 3), moderate (patients 8 and 9), and marked (patient 13) levels of MUC18 expression on melanoma cells illustrates differential expression of the protein (Fig. 6B). Together, these results establish that human B-1 lymphocytes are present in melanoma metastases and that such presence accounts for increased expression of MUC18 in human melanoma cells. As such, the MUC18 homophilic mechanism of heterotypic cell interaction seems clearly preserved across species and it is likely functional in human melanoma as well.


Figure 6
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  		Figure 6. Correlation between number of B-1 lymphocytes and MUC18 expression in human melanoma. A, flow cytometry analysis of melanoma samples is graphically represented as a balloon plot. A positive correlation is observed between increasing number of B-1 lymphocytes within the tumors and increasing expression of MUC18 on melanoma cells. B, histologic analysis of representative samples stained for MUC18 (20-fold magnification).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
It is often assumed that host immunity assembles its components to curb disease; however, the functional relationship between cellular immune system and tumor cells is far from clear. In mice, B-1 lymphocytes arise very early during the ontogeny of the immune system compared with the more evolved layers. Such cell lineage share several features with primitive B lymphocytes such as neonatal development, limited antibody repertoire, self-renewal capacity, and regulatory feedback in adults. B-1 lymphocytes can be readily identified by their cell surface phenotype (IgMhigh, IgDlow, CD23–, B220low, and CD11b+) and can be further subdivided in B-1a (CD5+) and B-1b (CD5–) subsets (9–12).

We evaluated whether B-1 lymphocytes influence tumor progression in the well-established B16 melanoma mouse model. We found that B-1 lymphocyte depletion markedly decreases lung metastasis from melanoma cells. B-1 lymphocyte reconstitution specifically inhibits this phenomenon and restores metastasis to baseline levels. These results show that B-1 lymphocytes have a key role in melanoma growth and metastasis.

Given that B-1 lymphocytes are known to produce and release high amounts of cytokines, we originally designed experiments to evaluate whether a soluble factor such as, for instance, interleukin-10 (36) might account for the observed metastasis enhancement. Unexpectedly, we found instead that a direct physical interaction between B-1 lymphocytes and melanoma cells is needed for metastasis. These findings led to the hypothesis that cell surface molecules are likely to mediate the phenomenon.

To gain insight into the molecular mechanism of this cell-cell interaction and the resulting biological effects, we attempted to identify the functionally active molecules expressed at the surface of each cell type. To that end, we selected a combinatorial peptide library on melanoma cells after coculture with B-1 lymphocytes. By comparing sequences of selected peptides with those available in protein databases, we found that ligand motifs were similar to the cell surface glycoprotein MUC18. Because MUC18 is an adhesion molecule that correlates with tumor growth and metastasis (32, 37, 38), we next evaluated whether a MUC18-dependent cell interaction would influence melanoma metastasis. First, we established that MUC18 serves as the partner on the surface of B16 melanoma cells ("receptor"). Because MUC18-like peptides also bind to MUC18 itself, we reasoned that MUC18 could also serve as the partner on the surface of B-1 lymphocytes ("ligand"). If so, one would predict that the ligand-receptor system affecting B16 melanoma cells through B-1 lymphocytes is actually a MUC18-MUC18 interaction. Our data show that this is indeed the case because (a) MUC18-like peptides enhance phage binding to melanoma cells after coculture, (b) antibodies against MUC18 specifically inhibit phage targeting, and (c) MUC18 is also abundant on the surface of B-1 lymphocytes. Together, our findings indicate that an unrecognized biochemical interaction between MUC18 expressed on the surface of B16 melanoma cells and B-1 lymphocytes regulates metastatic potential. Although the identification of a MUC18-like motif "in reverse" may originally have suggested an antiparallel MUC18-MUC18 interaction, in vitro and in vivo experiments performed with a phage clone designed to display the corresponding native MUC18 sequence indicate that this particular molecular mimicry is not affected by peptide orientation. However, the same may not necessarily be true for the native MUC18 protein itself (due to potential steric hindrance). Therefore, whether this protein-protein interaction is influenced by orientation of the native protein expressed on the surface of melanoma and B-1 cells remains uncertain; a full understanding of the structural requirements for protein-protein or protein-peptide will likely have to wait for the elucidation of the X-ray crystal structures of MUC18-MUC18 and of MUC18-CLFMRLAWC complexes.

To evaluate whether these observations are relevant in human disease, we investigated the distribution of B-1 lymphocytes in patients with melanoma. Although the origin and characteristics of the "human B-1 lymphocyte" counterpart are still poorly defined, a considerable proportion of IgM+ B cells in the human peritoneal cavity are CD5+, a phenotypic hallmark of mouse B-1 lymphocytes. Mice and human B-1 lymphocytes are both largely responsible for the production of autoreactive IgM antibodies in patients with certain autoimmune diseases (34, 35).

We detected IgM+/CD5+ B cells, presumably human B-1 lymphocytes, in 100% of the analyzed samples from a cohort of patients with metastatic melanoma (n = 16), suggesting that B-1 lymphocytes likely play a functional role in human cancer. Importantly, the presence of B-1 lymphocytes within tumors directly correlates with increased expression of MUC18 in melanoma cells (38), again supporting the relevance of a functional and active MUC18-dependent cross-talk between human B-1 lymphocytes and melanoma.

Notably, disruption of MUC18-dependent cell interactions may be of therapeutic value. In fact, (a) anti-MUC18 antibodies have shown promise in preclinical models (37, 39) and (b) overexpression of MUC18 occurs in human melanoma among other tumor types (40, 41). One might speculate that if a MUC18-dependent interaction accounts for malignant melanoma homing to lung vasculature for instance, then anti-MUC18 antibodies and/or MUC18-based peptidomimetics might simultaneously block the functional protein-protein interaction from both partners. Considering that reattachment of circulating tumor cells is a rate-limiting step in metastasis, our observations might help explain the difference of magnitude of effect we observed between the (mild to moderate) B16 tumor growth suppression relative to lung (marked) metastasis inhibition in B-1 lymphocyte-deficient mice.

In summary, we show that human and mouse melanoma cells can subvert a putatively defensive function of the immune system through a heterotypic cell-cell interaction with primitive B cells and that this molecular cross-talk can influence tumor progression with adverse net effects to the host. New mechanism-based strategies, such as physical elimination or functional modulation of human B-1 lymphocytes (through radiation or specific antibodies), may be considered as an experimental therapy against human melanomas; also, blocking MUC18 with antibodies or targeting MUC18 for ligand-directed delivery of agents in patients with melanoma may have translational potential.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Fundação de Amparo à Pesquisa do Estado de São Paulo (J.D. Lopes), Department of Defense (F.I. Staquicini, R. Pasqualini, and W. Arap), NCI (R. Pasqualini and W. Arap), Gillson-Longenbaugh Foundation, and AngelWorks. F.I. Staquicini received a predoctoral fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo.

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.

We thank Dr. Richmond Prehn, Dr. Isaiah J. Fidler, Dr. Leonore A. Herzenberg, and Dr. Olivera J. Finn for critical reading of the manuscript and helpful advice, Dr. Renato Mortara for confocal microscopy assistance, Dr. Alexander Lazar for immunohistochemistry, and Dr. John Wunderlich from the TIL Laboratory (NCI Surgery Branch) for human melanoma samples.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/ 2/08. Revised 7/31/08. Accepted 8/11/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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