Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma.

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1ß inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.

Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma.

OBJECTIVE: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. DESIGN: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. RESULTS: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. CONCLUSIONS: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.

The Servier oncology pipeline in 2017.

Cancer is a complex, multifactorial disease that for years has been the focus of intensive research efforts to explore both the molecular and biological mechanisms involved and the development of novel agents to target these pathways. Servier is an independent French pharmaceutical company with a focus on oncology. Currently, Servier's commercial portfolio includes agents used to treat non-Hodgkin's lymphoma and metastatic colorectal cancer; Servier's oncology pipeline involves agents for the treatment of both solid and hematological tumors. The main areas of future research focus on the development of therapeutics targeting apoptosis or the active immune components involved in tumour development/maintenance. Servier intends to continue its focus on cutting-edge oncology innovation by collaborating with both industry and academia, and maintaining its strong patient-centered approach.

Neutrophils contribute to inflammatory lymphangiogenesis by increasing VEGF-A bioavailability and secreting VEGF-D.

Lymphangiogenesis is an important physiological response to inflammatory insult, acting to limit inflammation. Macrophages, dendritic cells, and lymphocytes are known to drive lymphangiogenesis. In this study, we show that neutrophils recruited to sites of inflammation can also coordinate lymphangiogenesis. In the absence of B cells, intranodal lymphangiogenesis induced during prolonged inflammation as a consequence of immunization is dependent on the accumulation of neutrophils. When neutrophils are depleted in wild-type mice developing skin inflammation in response to immunization or contact hypersensitization, lymphangiogenesis is decreased and local inflammation is increased. We demonstrate that neutrophils contribute to lymphangiogenesis primarily by modulating vascular endothelial growth factor (VEGF)-A bioavailability and bioactivity and, to a lesser extent, secreting VEGF-D. We further show that neutrophils increased VEGF-A bioavailability and bioactivity via the secretion of matrix metalloproteinases 9 and heparanase. Together, these findings uncover a novel function for neutrophils as organizers of lymphangiogenesis during inflammation.

Mesenchymal transition and dissemination of cancer cells is driven by myeloid-derived suppressor cells infiltrating the primary tumor.

In order to metastasize, cancer cells need to acquire a motile phenotype. Previously, development of this phenotype was thought to rely on the acquisition of selected, random mutations and thus would occur late in cancer progression. However, recent studies show that cancer cells disseminate early, implying the existence of a different, faster route to the metastatic motile phenotype. Using a spontaneous murine model of melanoma, we show that a subset of bone marrow-derived immune cells (myeloid-derived suppressor cells or MDSC) preferentially infiltrates the primary tumor and actively promotes cancer cell dissemination by inducing epithelial-mesenchymal transition (EMT). CXCL5 is the main chemokine attracting MDSC to the primary tumor. In vitro assay using purified MDSC showed that TGF-ß, EGF, and HGF signaling pathways are all used by MDSC to induce EMT in cancer cells. These findings explain how cancer cells acquire a motile phenotype so early and provide a mechanistic explanation for the long recognized link between inflammation and cancer progression.

Expansion of cortical and medullary sinuses restrains lymph node hypertrophy during prolonged inflammation.

During inflammation, accumulation of immune cells in activated lymph nodes (LNs), coupled with a transient shutdown in lymphocyte exit, results in dramatic cellular expansion. Counter-regulatory measures to restrain LN expansion must exist and may include re-establishment of lymphocyte egress to steady-state levels. Indeed, we show in a murine model that egress of lymphocytes from LNs was returned to steady-state levels during prolonged inflammation following initial retention. This restoration in lymphocyte egress was supported by a preferential expansion of cortical and medullary sinuses during late inflammation. Cortical and medullary sinus remodeling during late inflammation was dependent on temporal and spatial changes in vascular endothelial growth factor-A distribution. Specifically, its expression was restricted to the subcapsular space of the LN during early inflammation, whereas its expression was concentrated in the paracortical and medullary regions of the LN at later stages. We next showed that this process was mostly driven by the synergistic cross-talk between fibroblastic reticular cells and interstitial flow. Our data shed new light on the biological significance of LN lymphangiogenesis during prolonged inflammation and further underscore the collaborative roles of stromal cells, immune cells, and interstitial flow in modulating LN plasticity and function.

[The early steps of the metastatic process]. / Les étapes précoces du processus métastatique.

Metastasis is the main cause of cancer-related death. While the development of clinically detectable metastases occurs only at late time points, recent data obtained in mice and humans indicate that cancer cell dissemination is an early event in the progression of several types of cancer. However, disseminated cancer cells can remain dormant for prolonged periods of time. Then, how do cancer cells acquire the ability to disseminate so early? What are the selective pressures driving their dissemination? What are the signals controlling dormancy and why do some cancer cells eventually escape these controls? The present review presents our current understanding on these questions and how this novel paradigm could be translated to the clinic.

Chikungunya virus envelope-specific human monoclonal antibodies with broad neutralization potency.

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics in Africa and Asia. Infection by CHIKV is often characterized by long-lasting, incapacitating arthritis, and some fatal cases have been described among elderly and newborns. Currently, there is no available vaccine or specific treatment against CHIKV. Blood B cells from a donor with history of CHIKV infection were activated, immortalized, amplified, and cloned. Two human mAbs against CHIKV, 5F10 and 8B10, were identified, sequenced, and expressed in recombinant form for characterization. In a plaque reduction neutralization test, 5F10 and 8B10 show mean IC(50) of 72 and 46 ng/ml, respectively. Moreover, both mAbs lead to a strong decrease in extracellular spreading of infectious viral particles from infected to uninfected cells. Importantly, the mAbs neutralize different CHIKV isolates from Singapore, Africa, and Indonesia, as well as O'nyong-nyong virus, but do not recognize other alphaviruses tested. Both mAbs are specific for the CHIKV envelope: 5F10 binds to the E2 glycoprotein ectodomain and 8B10 to E1 and/or E2. In conclusion, these two unique human mAbs strongly, broadly, and specifically neutralize CHIKV infection in vitro and might become possible therapeutic tools against CHIKV infection, especially in individuals at risk for severe disease. Importantly, these mAbs will also represent precious tools for future studies on host-pathogen interactions and the rational design of vaccines against CHIKV.

Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated. METHODS: Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearest-template prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182. RESULTS: The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8(+) T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death. CONCLUSION: A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease.

Macrophage polarization to a unique phenotype driven by B cells.

Regulation of adaptive immunity by innate immune cells is widely accepted. Conversely, adaptive immune cells can also regulate cells of the innate immune system. Here, we report for the first time the essential role of B cells in regulating macrophage (Mφ) phenotype. In vitro B cell/Mφ co-culture experiments together with experiments in transgenic mice models for B-cell deficiency or overexpression showed B1 cells to polarize Mφ to a distinct phenotype. This was characterized by downregulated TNF-α, IL-1ß and CCL3, but upregulated IL-10 upon LPS stimulation; constitutive expression of M2 Mφ markers (e.g. Ym1, Fizz1) and overexpression of TRIF-dependent cytokines (IFN-ß, CCL5). Mechanistically, this phenotype was linked to a defective NF-κB activation, but a functional TRIF/STAT1 pathway. B1-cell-derived IL-10 was found to be instrumental in the polarization of these Mφ. Finally, in vivo relevance of B1-cell-induced Mφ polarization was confirmed using the B16 melanoma tumor model where adoptive transfer of B1 cells induced an M2 polarization of tumor-associated Mφ. Collectively, our results define a new mechanism of Mφ polarization wherein B1 cells play a key role in driving Mφ to a unique, but M2-biased phenotype. Future studies along these lines may lead to targeting of B1 cells to regulate Mφ response in inflammation and cancer.

Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is an aggressive malignancy with few treatment options. As the status of the tumour immune microenvironment can affect progression of established tumours, we evaluated potential immune mechanisms associated with survival in HCC. METHODS: Immune gene expression profiles were analyzed in tumour and non-tumour liver tissues from resected HCC patients using quantitative PCR and immunohistochemistry. Tumour-infiltrating leukocytes (TILs) were isolated to verify the expression of immune genes and to identify proliferating TILs. These parameters were analyzed statistically in relation with patient survival and tumour phenotype (apoptosis and proliferation). RESULTS: The immune microenvironment within tumours was found to be heterogeneous, although globally more inert compared to the adjacent non-tumour liver tissue. Univariate analysis in 61 patients identified a group of innate immune genes whose expression within tumours is positively associated with patient survival. TNF, IL6 and CCL2 are the most significant genes, with TNF being an independent predictor of survival in multivariate analysis. The gene set includes macrophage and NK-associated molecules such as TLR4, TLR3, CCR2, NCR3. Most of these molecules are expressed by TILs. Importantly, proliferating immune cells, predominantly NK and T cells, are present in tumours of patients with longer survival, and exclusively in areas devoid of proliferating tumour cells. NK and CD8(+) T cell densities are correlated positively with tumour apoptosis, and negatively with tumour proliferation. CONCLUSIONS: Hence, an inflammatory immune microenvironment within HCC tumours could be an important means to control tumour progression via TIL activation and proliferation.

Macrophages and cancer.

Macrophages are ubiquitous cells physiologically involved in a variety of processes including pathogen destruction, inflammation, tissue repair and remodeling. They have a highly plastic phenotype and their functional polarization is determined by cytokines and factors found within local microenvironments. The role of macrophages during tumor development is ambiguous. At late stages, tumor-associated macrophages are known to produce molecules directly promoting tumor growth, invasion, and metastasis; the so called "myeloid-derived suppressor cells" also suppress the adaptive anti-tumor immune response. However, if properly activated, macrophages may control initial tumor development, and pilot studies in cancer patients suggest that adoptive transfers could be beneficial as adjuvant treatment in patients with minimal residual disease. Indeed, a limited tumor mass will probably be insufficient to educate macrophages into a suppressive phenotype. Thus, the macrophage effect in vivo may be determined by a variety of factors including the tumor type and stage, the degree of macrophage infiltration and their functional polarization. Unfortunately, the in vivo mechanisms responsible for the anti-tumor activity of macrophages are still unclear. Current promising strategies to target tumor macrophages in vivo include pharmacological agents capable to re-polarize them towards a classically activated phenotype or to inhibit their suppressive properties.

Induction of multiple CD8+ T cell responses against the inducible Hsp70 employing an Hsp70 oligoepitope peptide.

The inducible heat shock protein Hsp70 has been described as a tumour antigen being frequently overexpressed in tumours of various histologic origins, with a role in tumourigenicity, as a critical event in tumour progression. A strategy to enhance the immune response to an antigen is the identification of multiple epitopes and the induction of a polyspecific response. Applied to tumour vaccination, such a polyspecific response should lead to a more robust antitumour efficacy. The long peptide Hsp70380-402 encompasses three nonamer peptides with a high affinity for HLA-A *0201. In a previous paper, we have shown that two of these nonamer peptides, p391 and p393, can raise CTL to recognize tumour cells overexpressing Hsp70. In the present paper, we demonstrate that the third nonamer peptide, p380, is a new epitope efficient in raising an antitumour immune response. The p380-402 polypeptide was able to induce an immune response against each of the three constituent epitopes both in vivo in HLA-A *0201 transgenic mice and in vitro with human PBMC. This polypeptide therefore constitutes an interesting candidate for the induction of multiple HLA-A *0201-restricted anti-Hsp70 antitumour CTL responses.

Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector.

We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.

Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells.

Dendritic cells (DCs) are the most potent antigen presenting cells. Major histocompatibility complex (MHC) class II molecule expression changes with maturation; immature DCs concentrate MHC class II molecules intracellularly, whereas maturation increases surface expression of MHC class II and costimulatory molecules to optimize antigen presentation. Signal transduction via MHC class II molecules localized in lipid microdomains has been described in B lymphocytes and in the THP-1 monocyte cell line. We have characterized MHC class II molecules throughout human DC maturation with particular attention to their localization in lipid-rich microdomains. Only immature DCs expressed empty MHC class II molecules, and maturation increased the level of peptide-bound heterodimers. Ligand binding to surface human leukocyte antigen (HLA)-DR induced rapid internalization in immature DCs. The proportion of cell-surface detergent-insoluble glycosphingolipid-enriched microdomain-clustered HLA-DR was higher in immature DCs despite the higher surface expression of HLA-DR in mature DCs. Constituents of HLA-DR containing microdomains included the src kinase Lyn and the cytoskeletal protein tubulin in immature DCs. Maturation modified the composition of the HLA-DR-containing microdomains to include protein kinase C (PKC)-delta, Lyn, and the cytoskeletal protein actin, accompanied by the loss of tubulin. Signaling via HLA-DR redistributed HLA-DR and -DM and PKC-delta as well as enriching the actin content of mature DC microdomains. The increased expression of HLA-DR as a result of DC maturation was therefore accompanied by modification of the spatial organization of HLA-DR. Such regulation could contribute to the distinct responses induced by ligand binding to MHC class II molecules in immature versus mature DCs.

Lymphadenectomy promotes tumor growth and cancer cell dissemination in the spontaneous RET mouse model of human uveal melanoma.

Resection of infiltrated tumor-draining lymph nodes (TDLNs) is a standard practice for the treatment of several cancers including breast cancer and melanoma. However, many randomized prospective trials have failed to show convincing clinical benefits associated with LN removal and the role of TDLNs in cancer dissemination is poorly understood. Here, we found in a well-characterized spontaneous mouse model of uveal melanoma that the growth of the primary tumor was accompanied by increased lymphangiogenesis and cancer cell colonization in the LNs draining the eyes. But, unexpectedly, early resection of the TDLNs increased the growth of the primary tumor and associated blood vessels as well as promoted cancer cell survival and dissemination. These effects were accompanied by increased tumor cell proliferation and expression of phosphorylated AKT. Topical application of a broad anti-inflammatory agent, Tobradex, or an oral treatment with cyclooxygenase-2 specific inhibitor, Celecoxib, reversed tumor progression observed after complete lymphadenectomy. Our study confirms the importance of tumor homeostasis in cancer progression by showing the enhancing effects of TDLN removal on tumor growth and cancer cell dissemination, and suggests that TDLN resection may only be beneficial if used in combination with anti-inflammatory drugs such as Tobradex and Celecoxib.

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.

Tumor stroma and chemokines control T-cell migration into melanoma following Temozolomide treatment.

The infiltration of T lymphocytes within tumors is associated with better outcomes in cancer patients, yet current understanding of factors that influence T-lymphocyte infiltration into tumors remains incomplete. In our study, Temozolomide (TMZ), a chemotherapeutic drug used to treat metastatic melanoma, induced T-cell infiltration into transplanted melanoma and into genitourinary (GU) tumors in mice developing spontaneous melanoma. In contrast, TMZ treatment did not increase T-cell infiltration into cutaneous tumors, despite similar increases in the expression of the (C-X-C) chemokines CXCL9 and CXCL10 in all sites after TMZ exposure. Our findings reveal that the matrix architecture of the GU tumor stroma, and its ability to present CXCL9 and CXCL10 after TMZ treatment played a key role in favouring T-cell infiltration. We subsequently demonstrate that modifications of these key elements by combined collagenase and TMZ treatment induced T-cell infiltration into skin tumors. T cells accumulating within GU tumors after TMZ treatment exhibited T helper type-1 effector and cytolytic functional phenotypes, which are important for control of tumor growth. Our findings highlight the importance of the interaction between tumor stroma and chemokines in influencing T-cell migration into tumors, thereby impacting immune control of tumor growth. This knowledge will aid the development of strategies to promote T-cell infiltration into cancerous lesions and has the potential to markedly improve treatment outcomes.

TLR3 agonist and Sorafenib combinatorial therapy promotes immune activation and controls hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is associated with high mortality and the current therapy for advanced HCC, Sorafenib, offers limited survival benefits. Here we assessed whether combining the TLR3 agonist: lysine-stabilized polyinosinic-polycytidylic-acid (poly-ICLC) with Sorafenib could enhance tumor control in HCC. Combinatorial therapy with poly-ICLC and Sorafenib increased apoptosis and reduced proliferation of HCC cell lines in vitro, in association with impaired phosphorylation of AKT, MEK and ERK. In vivo, the combinatorial treatment enhanced control of tumor growth in two mouse models: one transplanted with Hepa 1-6 cells, and the other with liver tumors induced using the Sleeping beauty transposon. Tumor cell apoptosis and host immune responses in the tumor microenvironment were enhanced. Particularly, the activation of local NK cells, T cells, macrophages and dendritic cells was enhanced. Decreased expression of the inhibitory signaling molecules PD-1 and PD-L1 was observed in tumor-infiltrating CD8+ T cells and tumor cells, respectively. Tumor infiltration by monocytic-myeloid derived suppressor cells (Mo-MDSC) was also reduced indicating the reversion of the immunosuppressive tumor microenvironment. Our data demonstrated that the combinatorial therapy with poly-ICLC and Sorafenib enhances tumor control and local immune response hence providing a rationale for future clinical studies.