Elizabeth Cristina Perez

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

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.

B‐1 lymphocytes increase metastatic behavior of melanoma cells through the extracellular signal‐regulated kinase pathway

Increasing evidence indicates that tumors require a constant influx of myelomonocytic cells to support their malignant behavior. This is caused by tumor‐derived factors, which recruit and induce functional differentiation of myelomonocytic cells, most of which are macrophages. Although myeloid lineages are the classical precursors of macrophages, B‐lymphoid lineages such as B‐1 cells, a subset of B‐lymphocytes found predominantly in pleural and peritoneal cavities, are also able to migrate to inflammatory sites and differentiate into mononuclear phagocytes exhibiting macrophage‐like phenotypes. Here we examined the interplay of B‐1 cells and tumor cells, and checked whether this interaction provides signals to influence melanoma cells metastases. Using in vitro coculture experiments we showed that B16, a murine melanoma cell line, and B‐1 cells physically interact. Moreover, interaction of B16 with B‐1 cells leads to up‐regulation of metastasis‐related gene expression (MMP‐9 and CXCR‐4), increasing its metastatic potential, as revealed by experimental metastases assays in vivo. We also provide evidence that B16 cells exhibit markedly up‐regulated phosphorylation of the extracellular signal–regulated kinase (ERK) when cocultured with B‐1 cells. Inhibition of ERK phosphorylation induced by B‐1 cells with inhibitors of MEK1/2 strongly suppressed the induction of MMP‐9 and CXCR‐4 mRNA expression and impaired the increased metastatic behavior of B16. In addition, constitutive levels of ERK1/2 phosphorylation in B‐1 cells are necessary for their commitment to affect the metastatic potential of B16 cells. Our findings show for the first time that B‐1 lymphocytes can contribute to tumor cell properties required for invasiveness during metastatic spread. (Cancer Sci 2008; 99: 920–928)

Cross talk between peritoneal macrophages and B-1 cells in vitro.

B-1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. They represent the main B cell population found in mouse peritoneal and pleural cavities. The communication between B-1 cells and peritoneal macrophages has been previously studied, and the effect this interaction has on macrophages has been previously described. Using an in vitro co-culture model, herein we demonstrated that peritoneal macrophages were able to increase survival rates and to stimulate proliferation of B-1 cells. IL-6 was also found to be important in B-1 cell survival; recombinant IL-6 increases the percentage of viable B-1 cells in culture. Furthermore, molecules involved in the IL-6 signaling pathway, such as STAT-3 and Bcl-2, were highly expressed in B-1 cells after co-culture with peritoneal macrophages. IL-6-deficient peritoneal macrophages were not able to increase B-1 cell survival, confirming the importance of this cytokine. Altogether, our results indicate a novel mechanism in which peritoneal macrophages are able to regulate the B-1 population via IL-6 secretion.

Crosstalk between B16 melanoma cells and B-1 lymphocytes induces global changes in tumor cell gene expression

The analysis of gene expression patterns in cancers has improved the understanding of the mechanisms underlying the process of metastatic progression. However, the acquisition of invasive behavior in melanoma is poorly understood. In melanoma, components of the immune system can contribute to tumor progression, and inflammatory cells can influence almost all aspects of cancer progression, including metastasis. Recent studies have attributed an important role to B-1 cells, a subset of B lymphocytes, in melanoma progression. In vitro interactions between B16 melanoma cells and B-1 lymphocytes lead to increased B16 cell metastatic potential, but the molecular changes induced by B-1 lymphocytes on B16 cells have not yet been elucidated. In this study, we used a microarray approach to assess the gene expression profile of B16 melanoma cells following contact with B-1 lymphocytes (B16B1). The microarray analysis identified upregulation in genes involved with metastatic progression, such as ctss, ccl5, cxcl2 and stat3. RT-qPCR confirmed this increase in mRNA expression in B16B1 samples. As previous studies have indicated that the ERK1/2 MAPK cascade is activated in melanoma cells following contact with B-1 lymphocytes, RT-qPCR was performed with RNA from melanoma cells before and after contacting B-1 cells and untreated or treated with ERK phosphorylation inhibitors. The results showed that the expression of stat3, ctss and cxcl2 increased in B16B1 but decreased following ERK1/2 MAPK inhibition. Ccl5 gene expression increased after contacting B-1 cells and was maintained at the same level following inhibitor treatment. Stat3 was verified and validated at the protein level by Western blot analysis. STAT3 expression was also significantly increased in B16B1, suggesting that this pathway can also contribute to the increased metastatic phenotype observed in our model. These results indicated that B-1 cells induce important global gene expression changes in B16 melanoma cells. We also evaluated the relationship of some of the genes identified as differentially expressed and the ERK1/2 MAPK cascade. This work may have important implications for understanding the role of B-1 lymphocytes and the ERK/MAPK cascade in the metastatic process.

B16 melanoma cells increase B-1 cell survival, IL-10 production and radioresistance in vitro.

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. The role of both cell populations in cancer progression is still controversial. Previous studies have indicated that direct contact between B-1 cells and B16 melanoma tumor cells (B16) increases the metastatic potential of the tumor cells. However, cellular changes that are induced in B-1 cells during the interaction between these two cell types have not been evaluated. In the present study, it is hypothesized that B-1 cells are modified after their interaction with tumor cells, leading to both increased cell viability and rate of proliferation. Additionally, soluble factors that were secreted by B16 cells were sufficient to augment B-1 cell viability and to modify the production of IL-10 by B-1 cells. Impressively, after direct or indirect contact with the B16 cells, B-1 cells became resistant to radiation-induced cell death. Thus, future studies that assess the importance of concomitant immunity and other conventional therapies in cancer treatment are needed.

High dilutions of antimony modulate cytokines production and macrophage – Leishmania (L.) amazonensis interaction in vitro

Background: In previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node. Aims: To know the mechanisms involved, a series of in vitro studies was done, using co‐cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times. Methodology: Spreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate. Results: Cells treated with AC 30 cH (10−58 M) and AC 200 cH (10−398 M) presented a temporary reduction of the spreading after 02 h of incubation, followed by increase after 48 h, being the most significant increase observed after the AC 200 cH treatment. However, the percentage of internalized parasites at 48, 96 and 120 h of incubation was also higher in cells treated with AC 200 cH. It is suggested that the AC 200 cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines‐chemokines panel corroborated these results. Both dilutions potentiated the parasite‐induced reduction of cytokines production, especially IL‐6, IL 12 p40 and &ggr;‐IFN, after 48 h of incubation. In addition, the production of MIP‐1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120 h. A specific effect of AC 30 cH was seen by the inhibition of two peaks of CCL2 (MCP‐1) observed in infected macrophages, at 24 and 120 h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30 cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF‐A, associated with increase of internalized parasites was observed after 120 h of treatment with AC 200 cH, which could be associated to the regulation of the chronic inflammation events by M1‐M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30 cH and 200 cH was seen, in function of time. Conclusions: Both dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion. HIGHLIGHTSHigh diluted antimony modulates macrophage – L amazonensis interaction in vitro.Different levels of dilutions can change the production of different peptides.The results explain the anti‐inflammatory effect obtained previously in vivo.The clinical and epidemiological implications of these data are under discussion.

B-1 cell decreases susceptibility to encephalitozoonosis in mice

Encephalitozoon cuniculi is an opportunist intracellular pathogen of mammals. The adaptive immune response is essential to eliminate E. cuniculi, but evidence is mounting that the response initiated by the innate immune response may ultimately define whether or not the parasite can survive. B-1 cells may act as antigen-presenting cells or differentiate into phagocytes, playing different roles in many infection models. However, the role of these cells in the dynamics of Encephalitozoon sp. infections is still unknown. To investigate the role of B-1 cells in E. cuniculi infection, BALB/c and BALB/c XID (B-1 cells deficient) mice were infected with E. cuniculi spores. Cytometric analyses of peritoneal cells showed that B-1 cells and macrophages increased significantly in infected BALB/c mice compared to uninfected controls. Despite the increase in the number of CD4+ and CD8+ lymphocytes in XID mice, these animals were more susceptible to infection as evidenced histologically with more prominent inflammatory lesions and parasite burden. Pro-inflammatory cytokines increased in both infected BALB/c and BALB/c XID mice. To confirm B-1 cells role in encephalitozoonosis, we adoptively transferred B-1 cells to BALB/c XID mice and this group showed few symptoms and microscopic lesions, associated with an increased in cytokines. Together, these results suggest that B-1 cells may increase the resistance of BALB/c mice to encephalitozoonosis, evidencing for the first time the important role of B-1 lymphocytes in the control of microsporidia infection.

Cyclophosphamide immunosuppressed Xid mice model clarify the protective role of B cells in experimental encephalitozoonosis

Encephalitozoon cuniculi is an intracellular pathogen that stablishes a balanced relationship with immunocompetent individuals, which is dependent of T lymphocytes activity. We previously showed X-linked immunodeficiency (XID – B cell deficient) mice are more susceptible to encephalitozoonosis and B-1 cells presence influences in the immune response. Because XID mice are deficient both in B-1 and B-2 cells, here we investigate the role of these cells against E. cuniculi infection using cyclophosphamide (Cy) immunosuppressed murine model to exacerbate the infection. XID mice presented lethargy and severe symptoms, associated with encephalitozoonosis and there was an increase in the peritoneal populations of CD8+ and CD4+ T lymphocytes and macrophages and also in the proinflammatory cytokines IFN-γ, TNF-α and IL-6. In BALB/c mice, no clinical signs were observed and there was an increase of T lymphocytes and macrophages in the spleen, showing an effective immune response. B-2 cells transfer to XID mice resulted in reduction of symptoms and lesion area with increase of B-2 and CD4+ T populations in the spleen. B-1 cells transfer increased the peritoneal populations of B-2 cells and macrophages and also reduced the symptoms. Therefore, the immunodeficiency of B cells associated to Cy immunosuppression condition leads to disseminated and severe encephalitozoonosis in XID mice with absence of splenic immune response and ineffective local immune response, evidencing the B-1 and B-2 cells role against microsporidiosis. Author summary The adaptive immune response plays a key role against Encephalitozoon cuniculi, an opportunistic fungus for T cells immunodeficient patients. The role of B cells and antibody play in natural resistance to Encephalitozoon cuniculi remains unresolved. Previously, we demonstrated that B-1 deficient mice (XID), an important component of innate immunity, were more susceptible to encephalitozoonosis, despite the increase in the number of CD4+ and CD8+ T lymphocytes. In order to better understand the role of B-1 and B-2 cells and the relationship with the other cells of the immune response in encephalitozoonosis, we infected with E. cuniculi in cyclophosphamide immunosuppressed mice. Here we demonstrate that infected XID mice showed reduction of T cells and macrophages and increase of proinflammatory cytokines associated with disseminated and severe encephalitozoonosis with presence of abdominal effusion and lesions in multiple organs. This pattern of infection observed in mice with genetic deficiency in T cells, so we suggest that the absence of B-1 cells affects the cytotoxic capacity of these lymphocytes. When we transfer B-2 cells to XID mice, the lesion areas caused by the fungus, the populations of T lymphocytes in the peritoneum and the proinflammatory cytokines decrease, indicating a better resolution of the infection. We speculate that B-1 and B-2 cells participate in the immune response against E. cuniculi, interacting with the other components effective in immunity. The results shown here indicate that B-1 cells as a constituent of the innate response to microsporidia.

The axis IL-10/claudin-10 is implicated in the modulation of aggressiveness of melanoma cells by B-1 lymphocytes

B-1 lymphocytes are known to increase the metastatic potential of B16F10 melanoma cells via the extracellular signal-regulated kinase (ERK) pathway. Since IL-10 is associated with B-1 cells performance, we hypothesized that IL-10 could be implicated in the progression of melanoma. In the present work, we found that the C57BL/6 mice, inoculated with B16F10 cells that were co-cultivated with B-1 lymphocytes from IL-10 knockout mice, developed fewer metastatic nodules than the ones which were injected with the melanoma cells that were cultivated in the presence of wild-type B-1 cells. The impairment of metastatic potential of the B16F10 cells was correlated with low activation of the ERK signaling pathway, supporting the idea that the production of IL-10 by B-1 cells influences the behavior of the tumor. A microarray analysis of the B-1 lymphocytes revealed that IL-10 deficiency is associated with down-regulation of the genes that code for claudin-10, a protein that is involved in cell-to-cell contact and that has been linked to lung adenocarcinoma. In order to determine the impact of claudin-10 in the cross-talk between B-1 lymphocytes and the B16F10 tumor cells, we took advantage of small interfering RNA. The silencing of claudin-10 gene in B-1 lymphocytes inhibited activation of the ERK pathway and abrogated the B-1-induced aggressive behavior of the B16F10 cells. Thus, our findings suggest that the axis IL-10/claudin-10 is a promising target for the development of therapeutic agents against aggressive melanoma.

Abstract 468: BRAF mutation analysis in cell free tumoral DNA (cfDNA) of melanoma patients: results from the prospective study GEM1304 (Spanish Melanoma Group)

Backgroud: Tumor-derived circulating cell-free DNA (cfDNA) is a dynamic source for determination of tumor mutation status. We have previously demonstrated the prognostic value of BRAFV600 mutation status in pretreatment cfDNA (BRAF pre-cfDNA) in advanced melanoma patients (p) treated with BRAF inhibitors (median overall survival [OS] 7 months [m] vs 22m for BRAF pre-cfDNA positive and negative p, respectively p = 0.017)1. Based on these results, the Spanish Melanoma Group conducted a prospective study in 13 centers to determine the prognostic value of BRAFV600 mutation in pre-cfDNA, the change in mutation status at time of first evaluation (BRAF early-cfDNA), and the correlation of BRAF cfDNA dynamics with clinical evolution (GEM1304) (ClinicalTrials.gov Identifier: NCT01960634). Methods: One hundred and fifty nine plasma and serum samples from 66 stage IV BRAF mutant melanoma p were collected before and during treatment, until disease progression. A quantitative 5’-nuclease PCR based assay was used to determine BRAFV600 mutation status in cfDNA. Results: Most p were stage M1c (62%), treated with BRAF inhibitors (53%), and not previously treated (67%). BRAF pre-cfDNA was positive in 42 p (64%). Median OS was 6.4 m (95% CI: 10.9-23.6) and 17 m (95% CI: 3.5-9.2) for p with positive and negative BRAF pre-cfDNA, respectively (p = 0.06). Significant differences in OS were observed according to BRAF early-cfDNA negativization: 4.7 m (95%CI: 1.2-8.1) in those with persistence of BRAF in cfDNA (12 p), not reached (NR) in p with BRAF early-cfDNA negativization (11 p), and 22 m (95%CI:0.6-43.9) in those who continued to be negative (17 p) (p Conclusions: Patients with early negativization of BRAFV600 in cfDNA have excellent prognosis, at least as good as those with negative BRAF in pre-cfDNA. Gonzalez-Cao et al. Mel Res 2015; 25:486 Citation Format: Maria Gonzalez Cao, Jose Luis Manzano, Virtudes Soriano, Teresa Puertolas, Ainara Soria, Clara Mayo, Margarita Magem, Miguel Angel Molina, Clara Montagut, Eva Munoz, Delvys Rodriguez, Elizabeth Perez, Almudena Garcia, Javier Cortes, Nuria Jordana, Jordi Rodon, Niki Karachaliou, Rafael Rosell. BRAF mutation analysis in cell free tumoral DNA (cfDNA) of melanoma patients: results from the prospective study GEM1304 (Spanish Melanoma Group). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 468.