Heriberto Prado-Garcia

Lactate Contribution to the Tumor Microenvironment: Mechanisms, Effects on Immune Cells and Therapeutic Relevance

Malignant transformation of cells leads to enhanced glucose uptake and the conversion of a larger fraction of pyruvate into lactate, even under normoxic conditions; this phenomenon of aerobic glycolysis is largely known as the Warburg effect. This metabolic reprograming serves to generate biosynthetic precursors, thus facilitating the survival of rapidly proliferating malignant cells. Extracellular lactate directs the metabolic reprograming of tumor cells, thereby serving as an additional selective pressure. Besides tumor cells, stromal cells are another source of lactate production in the tumor microenvironment, whose role in both tumor growth and the antitumor immune response is the subject of intense research. In this review, we provide an integral perspective of the relationship between lactate and the overall tumor microenvironment, from lactate structure to metabolic pathways for its synthesis, receptors, signaling pathways, lactate-producing cells, lactate-responding cells, and how all contribute to the tumor outcome. We discuss the role of lactate as an immunosuppressor molecule that contributes to tumor evasion and we explore the possibility of targeting lactate metabolism for cancer treatment, as well as of using lactate as a prognostic biomarker.

Tumor cell metabolism An integral view

Cancer is a genetic disease that is caused by mutations in oncogenes, tumor suppressor genes and stability genes. The fact that the metabolism of tumor cells is altered has been known for many years. However, the mechanisms and consequences of metabolic reprogramming have just begun to be understood. In this review, an integral view of tumor cell metabolism is presented, showing how metabolic pathways are reprogrammed to satisfy tumor cell proliferation and survival requirements. In tumor cells, glycolysis is strongly enhanced to fulfill the high ATP demands of these cells; glucose carbons are the main building blocks in fatty acid and nucleotide biosynthesis. Glutaminolysis is also increased to satisfy NADPH regeneration, whereas glutamine carbons replenish the Krebs cycle, which produces metabolites that are constantly used for macromolecular biosynthesis. A characteristic feature of the tumor microenvironment is acidosis, which results from the local increase in lactic acid production by tumor cells. This phenomenon is attributed to the carbons from glutamine and glucose, which are also used for lactic acid production. Lactic acidosis also directs the metabolic reprogramming of tumor cells and serves as an additional selective pressure. Finally, we also discuss the role of mitochondria in supporting tumor cell metabolism.

Tumor-induced CD8+ T-cell dysfunction in lung cancer patients.

Lung cancer is the leading cause of cancer deaths worldwide and one of the most common types of cancers. The limited success of chemotherapy and radiotherapy regimes have highlighted the need to develop new therapies like antitumor immunotherapy. CD8+ T-cells represent a major arm of the cell-mediated anti-tumor response and a promising target for developing T-cell-based immunotherapies against lung cancer. Lung tumors, however, have been considered to possess poor immunogenicity; even so, lung tumor-specific CD8+ T-cell clones can be established that possess cytotoxicity against autologous tumor cells. This paper will focus on the alterations induced in CD8+ T-cells by lung cancer. Although memory CD8+ T-cells infiltrate lung tumors, in both tumor-infiltrating lymphocytes (TILs) and malignant pleural effusions, these cells are dysfunctional and the effector subset is reduced. We propose that chronic presence of lung tumors induces dysfunctions in CD8+ T-cells and sensitizes them to activation-induced cell death, which may be associated with the poor clinical responses observed in immunotherapeutic trials. Getting a deeper knowledge of the evasion mechanisms lung cancer induce in CD8+ T-cells should lead to further understanding of lung cancer biology, overcome tumor evasion mechanisms, and design improved immunotherapeutic treatments for lung cancer.

Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression

BackgroundNon-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3ζ expression. However, the hypothesis of tumor counterattack is still controversial.MethodsWe analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG1 peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3ζ, CD3ε, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3ζ and CD3ε in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied.ResultsOur results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3ε but not CD3ζ chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3ε, but not CD3ζ, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors. ConclusionsOur data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3ε in T-cells induced by NSCLC cells might lead to T-cell dysfunction.

Lung squamous cell carcinoma and adenocarcinoma cell lines use different mediators to induce comparable phenotypic and functional changes in human monocyte-derived dendritic cells

Tumor-derived immunosuppressive factors contribute to the evasion of malignant cells from the immune response, partially by hampering dendritic cell (DC) differentiation. Here, we analyze whether soluble mediators released by the most frequent histological types of non-small cell lung carcinoma, squamous cell carcinoma (SCC), and adenocarcinoma (AD) cells, affect the development and functionality of DC. Monocytes from healthy donors were differentiated in vitro into DC with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, in the absence or presence of soluble factors (SF) from SCC or AD cell lines. Monocytes were differentiated in parallel into macrophages (MΦ s) with macrophage colony-stimulating factor (M-CSF). SF-treated DC were phenotypically and functionally more similar to MΦ s than to untreated DC [control DC (Ctrl-DC)]. Both tumors increased myelomonocytic markers (CD14, CD16, CD32, and CD163) and impaired CD1a expression on DC. SF-treated DC increased their endocytic capacity, and released higher levels of IL-6, IL-10, and lower levels of IL-12, compared to Ctrl-DC. SF-treated DC were poor stimulators in mixed lymphocyte reactions, and naïve CD4+ T lymphocytes stimulated by SF-treated DC secreted lower levels of interferon (IFN)-γ and higher amounts of IL-10 than controls. In contrast to AD, the effects caused by SCC were mostly abolished by IL-6 neutralization during monocyte differentiation. However, tumor-derived prostanoid blockade recovered the IFN-γ levels secreted by lymphocytes stimulated with SF-treated DC, whereas prostanoid/IL-6 or prostanoid/IL-10 blockade decreased IL-10 production only by SCC-DC-stimulated lymphocytes. Thus, we provide evidence that lung SCC and AD cause comparable deficiencies on DC in vitro, skewing monocyte differentiation from DC to MΦ -like cells, but most of these changes occurred via different mediators.

Lack of correlation between growth inhibition by TGF-β and the percentage of cells expressing type II TGF-β receptor in human non-small cell lung carcinoma cell lines

Abstract To determine the mechanisms involved in the evasion from TGF-β growth regulation in the small cell lung carcinoma (SCLC) cell lines and the non-small cell lung carcinoma (NSCLC) cell lines, we studied: (a) production of TGF-β1 and TGF-β2; (b) percentage of cells expressing TGF-β RII; (c) responsiveness of the tumour cell lines to exogenous TGF-β1 or TGF-β2; and (d) presence of mRNA transcripts of the three TGF-β isoforms and of the TGF-β RII. Our results indicate that the SCLC cell lines do not synthesize the isoforms TGF-β1 and TGF-β2 nor the TGF-β RII, thus avoiding inhibitory autocrine and paracrine TGF-β actions. However, NSCLC cell lines express not only TGF-β1, TGF-β2 and TGF-β RII mRNA transcripts, but also synthesize both isoforms and the TGF-β RII. Although approximately 50% of the cells from the studied cell lines expressed the TGF-β RII, different cell lines varied greatly in the sensitivity to the inhibitory action of TGF-β. This could result from alterations in: (i) the structure of TGF-β RII; (ii) the phosphorylation motif of TGF-β RI; (iii) the molecules involved in the intracellular signalling pathway of TGF-β; and (iv) cell cycle regulation.

Activation-induced cell death of memory CD8+ T cells from pleural effusion of lung cancer patients is mediated by the type II Fas-induced apoptotic pathway

Lung cancer is the second most common form of cancer and the leading cause of cancer death worldwide. Pleural effusions, containing high numbers of mononuclear and tumor cells, are frequent in patients with advanced stages of lung cancer. We reported that in pleural effusions from primary lung cancer, the CD8+ T cell subpopulation, and particularly the terminally differentiated subset, is reduced compared to that of non-malignant effusions. We analyzed the participation of activation-induced cell death (AICD) and extrinsic pathways (type I or II) as mechanisms for the decrease in pleural effusion CD8+ T cell subpopulation. Pleural effusion or peripheral blood CD4+ and CD8+ T cells, from lung cancer patients, were stimulated with anti-CD3 antibody and analyzed for (a) apoptosis by annexin-V-binding and TUNEL assay, (b) transcript levels of Fas ligand (FasL) and TRAIL by real-time RT–PCR, (c) expression of FasL and TRAIL, measured as integrated mean fluorescence intensities (iMFI) by flow cytometry, (d) expression of Bcl-2 and BIM molecules, measured as MFI, and (e) apoptosis inhibition using caspase-8 and -9 inhibitors. Pleural effusion CD8+ T cells, but not CD4+ T cells, from cancer patients underwent AICD. Blocking FasL/Fas pathway protected from AICD. Upregulation of FasL and TRAIL expressions was found in pleural effusion CD8+ T cells, which also showed a subset of Bcl-2 low cells. In memory CD8+ T cells, AICD depended on both extrinsic and intrinsic apoptotic pathways. Hence, in the pleural space of lung cancer patients, AICD might compromise the antitumor function of CD8+ T cells.

Relationship of dendritic cell density, HMGB1 expression, and tumor-infiltrating lymphocytes in non-small cell lung carcinomas.

Lung cancer is the leading cause of cancer death worldwide and non–small cell lung carcinoma (NSCLC) is the most common type of lung carcinomas. In adenocarcinomas, the most frequent histologic type of NSCLC, dendritic cells (DCs) are localized in close contact with tumor cells, and tumor-infiltrating lymphocytes (TILs) are observed in the peritumoral zones. In NSCLC, no studies investigating the density of intratumoral DCs and their impact on the density of TILs have been performed. In addition, the role of the alarmin high-mobility group box1 (HMGB1) in intratumoral DCs recruitment has not been analyzed. In the present study, a total of 82 cases of advanced stages of NSCLC were included. Tissue samples were obtained from biopsies and autopsies. DCs in biopsies or combinations of DCs and NK cells, CD3+ T lymphocytes, or CD8+ T lymphocytes from autopsy specimens were quantified in high power fields. Also, distribution of HMGB1 in tumor cells was detected. In lung adenocarcinomas, irrespective of subclassification, high densities of infiltrating DCs directly associated to high densities of peritumoral TILs. A 2.5-fold increase in TILs was found in specimens with high densities of infiltrating DCs compared with TILs from adenocarcinomas with low densities of infiltrating DCs. High densities of infiltrating DCs were associated with lung adenocarcinomas expressing cytoplasmic or nuclear-cytoplasmic HMGB1. Our results suggest that in adenocarcinoma patients, HMGB1 produced by tumor cells recruits DCs, which associate to an increase of TILs. Encouraging tumor-DCs-T lymphocytes interactions should improve the quality of life and survival of NSCLC patients.

The PD-L1/PD-1 pathway promotes dysfunction, but not “exhaustion”, in tumor-responding T cells from pleural effusions in lung cancer patients

Malignant pleural effusions are frequent in patients with advanced stages of lung cancer and are commonly infiltrated by lymphocytes and tumor cells. CD8+ T cells from these effusions have reduced effector functions. The programmed death receptor 1(PD-1)/programmed death ligand 1 (PD-L1) pathway is involved in T-cell exhaustion, and it might be responsible for T-cell dysfunction in lung cancer patients. Here, we show that PD-L1 is expressed on tumor cell samples from malignant effusions, on lung cancer cell lines, and, interestingly, on MRC-5 lung fibroblasts. PD-L1 was up-regulated in lung cancer cell lines upon treatment with IFN-gamma, but not under hypoxic conditions, as detected by RT-qPCR and flow cytometry. Blockade of PD-L1 on tumor cells restored granzyme-B expression in allogenic CD8+ T cells in vitro. Remarkably, pleural effusion CD8+ T cells that responded to the tumor antigens MAGE-3A and WT-1 (identified as CD137+ cells) were lower in frequency than CMV pp65-responding CD8+ T cells and did not have an exhausted phenotype (PD-1+ TIM-3+). Nonetheless, tumor-responding CD8+ T cells had a memory phenotype and expressed higher levels of PD-1. A PD-L1 blocking antibody increased the expression of granzyme-B and perforin on polyclonal- and tumor-stimulated CD8+ T cells. Taken together, our data show that rather than being exhausted, tumor-responding CD8+ T cells are not completely differentiated into effector cells and are prone to negative regulation by PD-L1. Hence, our study provides evidence that lung cancer patients respond to immunotherapy due to blockade of the PD-L1/PD-1 pathway.

LAP TGF-Beta Subset of CD4+CD25+CD127− Treg Cells is Increased and Overexpresses LAP TGF-Beta in Lung Adenocarcinoma Patients

Lung cancer is the leading cause of cancer death worldwide. Adenocarcinoma, the most commonly diagnosed histologic type of lung cancer, is associated with smoking. Cigarette smoke promotes inflammation on the airways, which might be mediated by Th17 cells. This inflammatory environment may contribute to tumor development. In contrast, some reports indicate that tumors may induce immunosuppressive Treg cells to dampen immune reactivity, supporting tumor growth and progression. Thus, we aimed to analyze whether chronic inflammation or immunosuppression predominates at the systemic level in lung adenocarcinoma patients, and several cytokines and Th17 and Treg cells were studied. Higher proportions of IL-17-producing CD4+ T-cells were found in smoking control subjects and in lung adenocarcinoma patients compared to nonsmoking control subjects. In addition, lung adenocarcinoma patients increased both plasma concentrations of IL-2, IL-4, IL-6, and IL-10, and proportions of Latency Associated Peptide (LAP) TGF-β subset of CD4+CD25+CD127− Treg cells, which overexpressed LAP TGF-β. This knowledge may lead to the development of immunotherapies that could inhibit the suppressor activity mediated by the LAP TGF-β subset of CD4+CD25+CD127− Treg cells to promote reactivity of immune cells against lung adenocarcinoma cells.