Tumor Microenvironment: Cellular Interaction and Metabolic Adaptations.

The tumor microenvironment (TME) plays a critical role in cancerogenesis [...].

Bisphenol-A in Drinking Water Accelerates Mammary Cancerogenesis and Favors an Immunosuppressive Tumor Microenvironment in BALB-neuT Mice.

Bisphenol-A (BPA), a synthetic compound ubiquitously present in the environment, can act as an endocrine disruptor by binding to both canonical and non-canonical estrogen receptors (ERs). Exposure to BPA has been linked to various cancers, in particular, those arising in hormone-targeted tissues such as the breast. In this study, we evaluated the effect of BPA intake through drinking water on ErbB2/neu-driven cancerogenesis in BALB-neuT mice, transgenic for a mutated ErbB2/neu receptor gene, which reproducibly develop carcinomas in all mammary glands. In this model, BPA accelerated mammary cancerogenesis with an increase in the number of tumors per mouse and a concurrent decrease in tumor-free and overall survival. As assessed by immunohistochemistry, BALB-neuT tumors were ER-negative but expressed high levels of the alternative estrogen receptor GPR30, regardless of BPA exposure. On the other hand, BPA exposure resulted in a marked upregulation of progesterone receptors in preinvasive tumors and of Ki67, CD31, and phosphorylated Akt in invasive tumors. Moreover, based on several infiltration markers of immune cells, BPA favored an immunosuppressive tumor microenvironment. Finally, in vitro cell survival studies performed on a cell line established from a BALB-neuT breast carcinoma confirmed that BPA's impact on cancer progression can be particularly relevant after chronic, low-dose exposure.

Curcumin potentiates the ErbB receptors inhibitor Afatinib for enhanced antitumor activity in malignant mesothelioma.

Several attempts have been made to develop targeted therapies for malignant mesothelioma (MM), an aggressive tumour with a poor prognosis. In this study we evaluated whether Curcumin (CUR) potentiated the antitumor activity of the ErbB receptors inhibitor Afatinib (AFA) on MM, employing cell lines cultured in vitro and mice bearing intraperitoneally transplanted, syngeneic MM cells. The rationale behind this hypothesis was that CUR could counteract mechanisms of acquired resistance to AFA. We analysed CUR and AFA effects on MM cell growth, cell cycle, autophagy, and on the modulation of tumour-supporting signalling pathways.This study demonstrated that, as compared to the individual compounds, the combination of AFA + CUR had a stronger effect on MM progression which can be ascribed either to increased tumour cell growth inhibition or to an enhanced pro-apoptotic effect. These results warrant future studies aimed at further exploring the therapeutic potential of AFA + CUR-based combination regimens for MM treatment.

The Combination of Bioavailable Concentrations of Curcumin and Resveratrol Shapes Immune Responses While Retaining the Ability to Reduce Cancer Cell Survival.

The polyphenols Curcumin (CUR) and Resveratrol (RES) are widely described for their antitumoral effects. However, their low bioavailability is a drawback for their use in therapy. The aim of this study was to explore whether CUR and RES, used at a bioavailable concentration, could modulate immune responses while retaining antitumor activity and to determine whether CUR and RES effects on the immune responses of peripheral blood mononuclear cells (PBMCs) and tumor growth inhibition could be improved by their combination. We demonstrate that the low-dose combination of CUR and RES reduced the survival of cancer cell lines but had no effect on the viability of PBMCs. Although following CUR + RES treatment T lymphocytes showed an enhanced activated state, RES counteracted the increased IFN-γ expression induced by CUR in T cells and the polyphenol combination increased IL-10 production by T regulatory cells. On the other hand, the combined treatment enhanced NK cell activity through the up- and downregulation of activating and inhibitory receptors and increased CD68 expression levels on monocytes/macrophages. Overall, our results indicate that the combination of CUR and RES at low doses differentially shapes immune cells while retaining antitumor activity, support the use of this polyphenol combinations in anticancer therapy and suggest its possible application as adjuvant for NK cell-based immunotherapies.

Tumor antigens heterogeneity and immune response-targeting neoantigens in breast cancer.

Breast cancer is both the most common type of cancer and the most frequent cause of cancer mortality in women, mainly because of its heterogeneity and limited immunogenicity. The aim of specific active cancer immunotherapy is to stimulate the host's immune response against cancer cells directly using a vaccine platform carrying one or more tumor antigens. In particular, the ideal tumor antigen should be able to elicit T cell and B cell responses, be specific for the tumor and be expressed at high levels on cancer cells. Neoantigens are ideal targets for immunotherapy because they are exclusive to individual patient's tumors, are absent in healthy tissues and are not subject to immune tolerance mechanisms. Thus, neoantigens should generate a specific reaction towards tumors since they constitute the largest fraction of targets of tumor-infiltrating T cells. In this review, we describe the technologies used for neoantigen discovery, the heterogeneity of neoantigens in breast cancer and recent studies of breast cancer immunotherapy targeting neoantigens.

Combined treatment with inhibitors of ErbB Receptors and Hh signaling pathways is more effective than single treatment in reducing the growth of malignant mesothelioma both in vitro and in vivo.

Malignant mesothelioma (MM) is a rare orphan aggressive neoplasia with low survival rates. Among the other signaling pathways, ErbB receptors and Hh signaling are deregulated in MM. Thus, molecules involved in these signaling pathways could be used for targeted therapy approaches. The aim of this study was to evaluate the effects of inhibitors of Hh- (GANT-61) and ErbB receptors (Afatinib)-mediated signaling pathways, when used alone or in combination, on growth, cell cycle, cell death and autophagy, modulation of molecules involved in transduction pathways, in three human MM cell lines of different histotypes. The efficacy of the combined treatment was also evaluated in a murine epithelioid MM cell line both in vitro and in vivo. This study demonstrated that combined treatment with two inhibitors counteracting the activation of two different signaling pathways involved in neoplastic transformation and progression, such as those activated by ErbB and Hh signaling, is more effective than the single treatments in reducing MM growth in vitro and in vivo. This study may have clinical implications for the development of targeted therapy approaches for MM.

Fatty acid metabolism complements glycolysis in the selective regulatory T cell expansion during tumor growth.

The tumor microenvironment restrains conventional T cell (Tconv) activation while facilitating the expansion of Tregs. Here we showed that Tregs' advantage in the tumor milieu relies on supplemental energetic routes involving lipid metabolism. In murine models, tumor-infiltrating Tregs displayed intracellular lipid accumulation, which was attributable to an increased rate of fatty acid (FA) synthesis. Since the relative advantage in glucose uptake may fuel FA synthesis in intratumoral Tregs, we demonstrated that both glycolytic and oxidative metabolism contribute to Tregs' expansion. We corroborated our data in human tumors showing that Tregs displayed a gene signature oriented toward glycolysis and lipid synthesis. Our data support a model in which signals from the tumor microenvironment induce a circuitry of glycolysis, FA synthesis, and oxidation that confers a preferential proliferative advantage to Tregs, whose targeting might represent a strategy for cancer treatment.

Genetically driven CD39 expression shapes human tumor-infiltrating CD8+ T-cell functions.

In our study, we investigated the role of CD39 on tumor-infiltrating CD8+ T lymphocytes (CD8+ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39+ CD8+ TILs. On the one hand, CD39+ CD8+ TILs (as compared to their CD39- counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39+ CD8+ TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8+ TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39+ CD8+ T-cell effector function ex vivo, and that CD39+ CD8+ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39+ CD8+ T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8+ TILs.

PlGF Immunological Impact during Pregnancy.

During pregnancy, the mother's immune system has to tolerate the persistence of paternal alloantigens without affecting the anti-infectious immune response. Consequently, several mechanisms aimed at preventing allograft rejection, occur during a pregnancy. In fact, the early stages of pregnancy are characterized by the correct balance between inflammation and immune tolerance, in which proinflammatory cytokines contribute to both the remodeling of tissues and to neo-angiogenesis, thus, favoring the correct embryo implantation. In addition to the creation of a microenvironment able to support both immunological privilege and angiogenesis, the trophoblast invades normal tissues by sharing the same behavior of invasive tumors. Next, the activation of an immunosuppressive phase, characterized by an increase in the number of regulatory T (Treg) cells prevents excessive inflammation and avoids fetal immuno-mediated rejection. When these changes do not occur or occur incompletely, early pregnancy failure follows. All these events are characterized by an increase in different growth factors and cytokines, among which one of the most important is the angiogenic growth factor, namely placental growth factor (PlGF). PlGF is initially isolated from the human placenta. It is upregulated during both pregnancy and inflammation. In this review, we summarize current knowledge on the immunomodulatory effects of PlGF during pregnancy, warranting that both innate and adaptive immune cells properly support the early events of implantation and placental development. Furthermore, we highlight how an alteration of the immune response, associated with PlGF imbalance, can induce a hypertensive state and lead to the pre-eclampsia (PE).

Polyphenol-Mediated Autophagy in Cancer: Evidence of In Vitro and In Vivo Studies.

One of the hallmarks of cellular transformation is the altered mechanism of cell death. There are three main types of cell death, characterized by different morphological and biochemical features, namely apoptosis (type I), autophagic cell death (type II) and necrosis (type III). Autophagy, or self-eating, is a tightly regulated process involved in stress responses, and it is a lysosomal degradation process. The role of autophagy in cancer is controversial and has been associated with both the induction and the inhibition of tumor growth. Autophagy can exert tumor suppression through the degradation of oncogenic proteins, suppression of inflammation, chronic tissue damage and ultimately by preventing mutations and genetic instability. On the other hand, tumor cells activate autophagy for survival in cellular stress conditions. Thus, autophagy modulation could represent a promising therapeutic strategy for cancer. Several studies have shown that polyphenols, natural compounds found in foods and beverages of plant origin, can efficiently modulate autophagy in several types of cancer. In this review, we summarize the current knowledge on the effects of polyphenols on autophagy, highlighting the conceptual benefits or drawbacks and subtle cell-specific effects of polyphenols for envisioning future therapies employing polyphenols as chemoadjuvants.

Polyphenols as Immunomodulatory Compounds in the Tumor Microenvironment: Friends or Foes?

Polyphenols are natural antioxidant compounds ubiquitously found in plants and, thus, ever present in human nutrition (tea, wine, chocolate, fruits and vegetables are typical examples of polyphenol-rich foods). Widespread evidence indicate that polyphenols exert strong antioxidant, anti-inflammatory, anti-microbial and anti-cancer activities, and thus, they are generally regarded to as all-purpose beneficial nutraceuticals or supplements whose use can only have a positive influence on the body. A closer look to the large body of results of years of investigations, however, present a more complex scenario where polyphenols exert different and, sometimes, paradoxical effects depending on dose, target system and cell type and the biological status of the target cell. Particularly, the immunomodulatory potential of polyphenols presents two opposite faces to researchers trying to evaluate their usability in future cancer therapies: on one hand, these compounds could be beneficial suppressors of peri-tumoral inflammation that fuels cancer growth. On the other hand, they might suppress immunotherapeutic approaches and give rise to immunosuppressive cell clones that, in turn, would aid tumor growth and dissemination. In this review, we summarize knowledge of the immunomodulatory effects of polyphenols with a particular focus on cancer microenvironment and immunotherapy, highlighting conceptual pitfalls and delicate cell-specific effects in order to aid the design of future therapies involving polyphenols as chemoadjuvants.

Discovery of chemotherapy-associated ovarian cancer antigens by interrogating memory T cells.

According to the immunogenic cell death hypothesis, clinical chemotherapy treatments may result in CD8(+) and CD4(+) T-cell responses against tumor cells. To discover chemotherapy-associated antigens (CAAs), T cells derived from ovarian cancer (OC) patients (who had been treated with appropriate chemotherapy protocols) were interrogated with proteins isolated from primary OC cells. We screened for immunogenicity using two-dimensional electrophoresis gel-eluted OC proteins. Only the selected immunogenic antigens were molecularly characterized by mass-spectrometry-based analysis. Memory T cells that recognized antigens associated with apoptotic (but not live) OC cells were correlated with prolonged survival in response to chemotherapy, supporting the model of chemotherapy-induced apoptosis as an adjuvant of anti-tumor immunity. The strength of both memory CD4(+) and CD8(+) T cells producing either IFN-γ or IL-17 in response to apoptotic OC antigens was also significantly greater in Responders to chemotherapy than in nonresponders. Immunogenicity of some of these antigens was confirmed using recombinant proteins in an independent set of patients. The T-cell interrogation system represents a strategy of reverse tumor immunology that proposes to identify CAAs, which may then be validated as possible prognostic tumor biomarkers or cancer vaccines.

Dietary Polyphenols Effects on Focal Adhesion Plaques and Metalloproteinases in Cancer Invasiveness.

Focal adhesion plaques (FAPs) play an important role in the communication between cells and the extracellular matrix (ECM) and in cells' migration. FAPs are macromolecular complexes made by different proteins which also interact with matrix metalloproteinases (MMPs). Because of these fundamental properties, FAPs and MMPs are also involved in cancer cells' invasion and in the metastatic cascade. The most important proteins involved in FAP formation and activity are (i) integrins, (ii) a complex of intracellular proteins and (iii) cytoskeleton proteins. The latter, together with MMPs, are involved in the formation of filopodia and invadopodia needed for cell movement and ECM degradation. Due to their key role in cancer cell migration and invasion, MMPs and components of FAPs are often upregulated in cancer and are thus potential targets for cancer therapy. Polyphenols, a large group of organic compounds found in plant-based food and beverages, are reported to have many beneficial healthy effects, including anticancer and anti-inflammatory effects. In this review, we discuss the growing evidence which demonstrates that polyphenols can interact with the different components of FAPs and MMPs, inhibit various pathways like PI3K/Akt, lower focal adhesion kinase (FAK) phosphorylation and decrease cancer cells' invasiveness, leading to an overall antitumoral effect. Finally, here we highlight that polyphenols could hold potential as adjunctive therapies to conventional cancer treatments due to their ability to target key mechanisms involved in cancer progression.

DNAM-1 chimeric receptor-engineered NK cells: a new frontier for CAR-NK cell-based immunotherapy.

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy.

Tumor Microenvironment Remodeling in Gastrointestinal Cancer: Role of miRNAs as Biomarkers of Tumor Invasion.

Gastrointestinal (GI) cancers are the most frequent neoplasm, responsible for half of all cancer-related deaths. Metastasis is the leading cause of death from GI cancer; thus, studying the processes that regulate cancer cell migration is of paramount importance for the development of new therapeutic strategies. In this review, we summarize the mechanisms adopted by cancer cells to promote cell migration and the subsequent metastasis formation by highlighting the key role that tumor microenvironment components play in deregulating cellular pathways involved in these processes. We, therefore, provide an overview of the role of different microRNAs in promoting tumor metastasis and their role as potential biomarkers for the prognosis, monitoring, and diagnosis of GI cancer patients. Finally, we relate the possible use of nutraceuticals as a new strategy for targeting numerous microRNAs and different pathways involved in GI tumor invasiveness.

Recent findings on the impact of ErbB receptors status on prognosis and therapy of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type, has often an aggressive course and is poorly responsive to current therapeutic approaches, so that 5-year survival rates for patients diagnosed with advanced disease is lower than 50%. The Epidermal Growth Factor Receptor (EGFR) has emerged as an established oncogene in HNSCC. Indeed, although HNSCCs are a heterogeneous group of cancers which differ for histological, molecular and clinical features, EGFR is overexpressed or mutated in a percentage of cases up to about 90%. Moreover, aberrant expression of the other members of the ErbB receptor family, ErbB2, ErbB3 and ErbB4, has also been reported in variable proportions of HNSCCs. Therefore, an increased expression/activity of one or multiple ErbB receptors is found in the vast majority of patients with HNSCC. While aberrant ErbB signaling has long been known to play a critical role in tumor growth, angiogenesis, invasion, metastatization and resistance to therapy, more recent evidence has revealed its impact on other features of cancer cells' biology, such as the ability to evade antitumor immunity. In this paper we will review recent findings on how ErbB receptors expression and activity, including that associated with non-canonical signaling mechanisms, impacts on prognosis and therapy of HNSCC.

Antitumoral effects of Bortezomib in malignant mesothelioma: evidence of mild endoplasmic reticulum stress in vitro and activation of T cell response in vivo.

BACKGROUND: Malignant mesothelioma (MM) is a rare tumor with a dismal prognosis. The low efficacy of current treatment options highlights the urge to identify more effective therapies aimed at improving MM patients' survival. Bortezomib (Bor) is a specific and reversible inhibitor of the chymotrypsin-like activity of the 20S core of the proteasome, currently approved for the treatment of multiple myeloma and mantle cell lymphoma. On the other hand, Bor appears to have limited clinical effects on solid tumors, because of its low penetration and accumulation into tumor tissues following intravenous administration. These limitations could be overcome in MM through intracavitary delivery, with the advantage of increasing local drug concentration and decreasing systemic toxicity. METHODS: In this study, we investigated the effects of Bor on cell survival, cell cycle distribution and modulation of apoptotic and pro-survival pathways in human MM cell lines of different histotypes cultured in vitro. Further, using a mouse MM cell line that reproducibly forms ascites when intraperitoneally injected in syngeneic C57BL/6 mice, we investigated the effects of intraperitoneal Bor administration in vivo on both tumor growth and the modulation of the tumor immune microenvironment. RESULTS: We demonstrate that Bor inhibited MM cell growth and induced apoptosis. Further, Bor activated the Unfolded Protein Response, which however appeared to participate in lowering cells' sensitivity to the drug's cytotoxic effects. Bor also affected the expression of EGFR and ErbB2 and the activation of downstream pro-survival signaling effectors, including ERK1/2 and AKT. In vivo, Bor was able to suppress MM growth and extend mice survival. The Bor-mediated delay of tumor progression was sustained by increased activation of T lymphocytes recruited to the tumor microenvironment. CONCLUSIONS: The results presented herein support the use of Bor in MM and advocate future studies aimed at defining the therapeutic potential of Bor and Bor-based combination regimens for this treatment-resistant, aggressive tumor.

DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study.

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.