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. 2008 Oct 15;68(20):8419-28.
doi: 10.1158/0008-5472.CAN-08-1242.

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 Staquicini  1 , Anita Tandle, Steven K Libutti, Jessica Sun, Maya Zigler, Menashe Bar-Eli, Fabiana Aliperti, Elizabeth C Pérez, Jeffrey E Gershenwald, Mario Mariano, Renata Pasqualini, Wadih Arap, José Daniel Lopes
Affiliations

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 Staquicini et al. Cancer Res. .

Abstract

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.

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Figures

Figure 1
Figure 1
Effects of B-1 lymphocytes in B16 melanoma progression. A, Left panel: effect of radiation-induced B-1 lymphocyte depletion on primary tumor growth. Right panel: 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 (X-linked immunosuppression, Xid) relative to otherwise isogenic control mice (wt). *, p
Figure 2
Figure 2
Screening of a phage display random peptide library on B16 melanoma cells post co-culture with B-1 lymphocytes yields MUC18 as a candidate target molecule. A, Enrichment of phage binding to malignant melanoma cells after co-culture with B-1 lymphocytes. B, Binding of individual MUC18-mimic phage clones to B16 malignant melanoma cells pre and post co-culture with B-1 lymphocytes. A phage clone displaying the peptide motif Arg-Met-Phe-Leu (mouse MUC18 residues A114-L117) had a marked increase in binding to B16 melanoma cells post co-culture with B-1 lymphocytes relative to control insertless phage (∼20-fold) or to malignant melanoma cells without co-culture 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 malignant melanoma cells pre and post co-culture by Western blot analysis. D, Both anti- CLFMRLAWC and anti-MUC18 antibodies co-immunoprecipitate MUC18.
Figure 3
Figure 3
MUC18-derived phage binds to MUC18. A, Binding of MUC18-derived phage on melanoma cells pre and post co-culture with B-1 lymphocytes. Left panel: a phage clone displaying a mouse MUC18-derived peptide (H111-S120) targets B16 malignant melanoma cells relative to insertless phage or phage clones displaying various scrambled versions of the MUC18-derived peptide. Right panel: 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 demonstrates that phage binding recapitulates the different levels of MUC18 expression pre and post co-culture with B-1 lymphocytes. B, Left panel: Specific binding of mouse MUC18-derived (H111-S120) phage to immunocaptured MUC18 relative to negative controls (insertless and scrambled peptide phage). Right panel: in vivo homing of MUC18-like phage to tumors before and after co-culture with B-1 lymphocytes. C, Double label immunofluorescence of tumors derived from melanoma cells before and after co-culture with B-1 lymphocytes. DAPI (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. Co-culture of B-1 lymphocytes with MUC18-negative melanoma cells does not increase melanoma metastatic protential.
Figure 3
Figure 3
MUC18-derived phage binds to MUC18. A, Binding of MUC18-derived phage on melanoma cells pre and post co-culture with B-1 lymphocytes. Left panel: a phage clone displaying a mouse MUC18-derived peptide (H111-S120) targets B16 malignant melanoma cells relative to insertless phage or phage clones displaying various scrambled versions of the MUC18-derived peptide. Right panel: 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 demonstrates that phage binding recapitulates the different levels of MUC18 expression pre and post co-culture with B-1 lymphocytes. B, Left panel: Specific binding of mouse MUC18-derived (H111-S120) phage to immunocaptured MUC18 relative to negative controls (insertless and scrambled peptide phage). Right panel: in vivo homing of MUC18-like phage to tumors before and after co-culture with B-1 lymphocytes. C, Double label immunofluorescence of tumors derived from melanoma cells before and after co-culture with B-1 lymphocytes. DAPI (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. Co-culture of B-1 lymphocytes with MUC18-negative melanoma cells does not increase melanoma metastatic protential.
Figure 4
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 co-culture with malignant melanoma cells. (A) The black histogram represents B-1 lymphocytes. The gray dotted line histogram represents B-1 lymphocytes after co-culture with B16 malignant melanoma cells. (B) The gray histogram represents B16 malignant melanoma cells after co-culture with B-1 lymphocytes. A ∼7-fold enhancement in MUC18 cell surface expression is detected after co-culture with B-1 lymphocytes. C, MUC18 expression in B-1 lymphocytes by RT-PCR analysis. No differences in expression levels of the MUC18 transcript were observed in B-1 lymphocytes co-cultured with melanoma cells (left panel). In contrast, we clearly detected increased expression of MUC18 in melanoma cells post co-culture with B-1 lymphocytes (right panel). D, Cell signaling through MAPK pathways. Phosphorylation of ERK1/2 was investigated by immunoblotting in different time points after melanoma cell activation. We observed specific phophorylation of p44/42 after 2.5 min of cell stimulation with MUC18-like peptide (upper panel). Control unrelated peptide showed no effect in protein phophorylation (lower panel). Total p44/42 served as the loading control.
Figure 5
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 point to MUC18–expressing melanoma cells in the epidermis (top left), dermis and exocrine ducts (top right) and lymph node metastases (bottom right). Arrowhead point to MUC18 positive melanoma cells in vascular endothelial cells (top right and bottom left) (100-fold magnification). Negative control is shown in inset, bottom-right panel. B, Phage binding assay to human melanoma cell lines. A scrambled version of the peptide and insertless phage were used as negative control.
Figure 6
Figure 6
Correlation between number of B-1 lymphocytes and MUC18 expression in human melanoma. A, Flow cytometric 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, Histological analysis of representative samples stained for MUC18 (20-fold magnification).
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