Lactate secreted by cervical cancer cells modulates macrophage phenotype.

Cervical cancer continues to be a public health problem in developing countries. Previous studies have shown that cervical cancer cells display markers of aerobic glycolysis, indicating that these tumors are likely to secrete lactate. Mostly, lactate is recognized as a molecule capable of suppressing immune responses, through inhibition of T cells, Mϕs, and dendritic cells. We and others have previously shown that Mϕs are frequent cells infiltrating cervical cancers with the ability to inhibit antitumor immune responses and promote tumor growth through angiogenesis. Here, we have tested the hypothesis that lactate, secreted by cervical cancer cells, can modulate Mϕ phenotype. First, we showed higher lactate plasma concentrations in patients with increasing cervical lesion grades, with maximum concentration in the plasma of cancer patients, which supported our hypothesis. We then inhibited lactate production in tumor cell spheroids established from cervical cancer derived cell lines, using the lactate dehydrogenase inhibitor, oxamate, prior to co-culture with monocytes. Lactate mediated part of the crosstalk between tumor cells and Mϕs, promoting secretion of IL-1ß, IL-10, IL-6, and up-regulation of hypoxia induced factor-1α expression, and down-regulation of p65-NFκB phosphorylation in Mϕs. We also showed that Mϕs from co-cultures treated with oxamate were better inducers of T cell activation. Of note, experiments performed with inhibition of the monocarboxylate transporters rendered similar results. Our data confirms the hypothesis that lactate, secreted by cervical tumor cells, influences the phenotype of tumor Mϕs, promoting a suppressive phenotype.

Modulation of Tumor-Associated Macrophages (TAM) Phenotype by Platelet-Activating Factor (PAF) Receptor.

Platelet-activating factor (PAF) plays an important role in the pathogenesis of several types of tumors. The biological effects of PAF are mediated by the PAF receptor (PAFR), which can be expressed by tumor cells and host cells that infiltrate the tumor microenvironment. In the present study, we investigated the role of PAFR expressed by leukocytes that infiltrate two types of tumors, one that expresses PAFR (TC-1 carcinoma) and another that does not express the receptor (B16F10 melanoma) implanted in mice that express the receptor or not (PAFR KO). It was found that both tumors grew significantly less in PAFR KO than in wild-type (WT) mice. Analysis of the leukocyte infiltration shown in PAFR KO increased the frequency of neutrophils (Gr1+) and of CD8+ lymphocytes in B16F10 tumors and of CD4+ lymphocytes in TC-1 tumors. PAFR KO also had a higher frequency of M1-like (CD11c+) and lower M2-like (CD206+) macrophages infiltrated in both tumors. This was confirmed in macrophages isolated from the tumors that showed higher iNOS, lower arginase activity, and lower IL10 expression in PAFR KO tumors than WT mice. These data suggest that in the tumor microenvironment, endogenous PAF-like activity molecules bind PAFR in macrophages which acquire an M2-like profile and this promotes tumor growth.

HPV associated tumor cells control tumor microenvironment and leukocytosis in experimental models.

Human papillomavirus (HPV) is the main etiological factor for cervical cancer development. HPV is also associated with other anogenital and oropharyngeal tumors. HPV associated tumors are frequent and constitute a public health problem, mainly in developing countries. Therapy against such tumors is usually excisional, causing iatrogenic morbidity. Therefore, development of strategies for new therapies is desirable. The tumor microenvironment is essential for tumor growth, where inflammation is an important component, displaying a central role in tumor progression. Inflammation may be a causal agent, suppressor of anti-tumor T cell responses, or may have a role in angiogenesis, drug resistance, and metastasis. The aim of this work was to investigate the role of HPV transformed cells in the tumor microenvironment and tumor effects on myeloid populations in lymphoid organs in the host. We used experimental models, where we injected cervical cancer derived cell lines in immunodeficient mice, comparing HPV positive, SiHa, and HeLa cells (HPV 16 and HPV18, respectively), with HPV negative cell line, C33A. Our data shows that HPV positive cell lines were more efficient than the HPV negative cell line in leukocyte recruitment to the tumor microenvironment and increase in myeloid cell proliferation in the bone marrow and spleen. We also observed that HPV positive cells lines expressed significantly higher levels of IL-6 and IL-8, while C33A expressed significantly higher levels of IL-16 and IL-17. Finally, in spite of cytokine secretion by tumor cells, leukocytes infiltrating SiHa and HeLa tumors displayed almost negligible STAT3 and no NFκB phosphorylation. Only the inflammatory infiltrate of C33A tumors had NFκB and STAT3 activated isoforms. Our results indicate that, although from the same anatomical site, the uterine cervix, these cell lines display important differences regarding inflammation. These results are important for the design of immunotherapies against cervical cancer, and possibly against HPV associated tumors in other anatomical sites.

HPV16-associated tumors control myeloid cell homeostasis in lymphoid organs, generating a suppressor environment for T cells.

Tumors are complex structures containing different types of cells and molecules. The importance of the tumor microenvironment in tumor progression, growth, and maintenance is well-established. However, tumor effects are not restricted to the tumor microenvironment. Molecules secreted by, as well as cells that migrate from tumors, may circulate and reach other tissues. This may cause a series of systemic effects, including modulation of immune responses, and in some cases, leukocytosis and metastasis promotion. Leukocytosis has been described as a poor prognostic factor in patients with cervical cancer. The main etiological factor for cervical cancer development is persistent infection with high oncogenic risk HPV. Our laboratory has been exploring the effects of high oncogenic risk, HPV-associated tumors on lymphoid organs of the host. In the present study, we observed an increase in myeloid cell proliferation and alteration in cell signaling in APCs in the spleen of tumor-bearing mice. In parallel, we characterized the cytokines secreted in the inflammatory and tumor cell compartments in the tumor microenvironment and in the spleen of tumor-bearing mice. We show evidence of constitutive activation of the IL-6/STAT3 signaling pathway in the tumor, including TAMs, and in APCs in the spleen. We also observed that IL-10 is a central molecule in the tolerance toward tumor antigens through control of NF-κB activation, costimulatory molecule expression, and T cell proliferation. These systemic effects over myeloid cells are robust and likely an important problem to be addressed when considering strategies to improve anti-tumor T cell responses.

Generation and characterization of new HER2 monoclonal antibodies.

Antibodies are among the most commonly used research and diagnostic tools. Antibody type and clonality are important in any assay as they can influence epitope detection. HER2 oncoprotein is overexpressed or undergoes gene amplification in approximately 30% of invasive breast carcinomas and 20% of gastric adenocarcinomas. Overexpression of HER2 is primarily detected by immunohistochemistry (IHC) on neoplastic tissue sections. We produced five murine hybridoma clones secreting monoclonal antibodies (MAbs) against HER2 protein. For hybridoma production, spleen cells from BALB/c mice immunized with a recombinant fragment of the extracellular portion of HER2 (rHER2) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened by indirect ELISA. MAbs secreted were characterized according to isotypes, functional affinity constants, reaction with the native protein in MCF-7 cells by indirect immunofluorescence and in tissue sections from HER2 positive breast cancer specimens by IHC. Two MAbs were IgG2b and three were IgG1, and their affinity constants ranged from 6×10(7) to 1×10(9)M(-1). All MAbs reacted with the native protein and two stained strongly the membrane of neoplastic cells overexpressing HER2. These two MAbs could be useful in assaying HER2 overexpression in human tissues for research and possibly diagnostic purposes after a proper large-scale validation study.