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Simple Summary The introduction of immunotherapy within the usual strategies for cancer treatment has meant an enormous advance in the survival of patients with very restrictive prognoses. However, a high percentage of patients still cannot benefit from it. The lack of response to immunotherapy is mainly due to resistance from the tumor stroma itself. The lymphatic endothelium constitutes a permeable vascular system from the stroma specially dedicated to balancing tissue homeostasis and leukocyte emigration. Most solid carcinomas present lymphatic microvasculature that participates in tumor progression ambivalently. On one hand, it facilitates the transit of immune cells toward the lymph nodes and therefore promotes the antitumor response; on the other hand, they constitute an accessible route through which tumor cells metastasize to the lymph nodes. Due to this double function, it is not easy to establish strategies that educate the lymphatic endothelium only in its antitumor function without compromising its participation in the immune response. In this review, we study how combinations of radiotherapy and immunotherapy can modulate lymphatic function to use them in therapeutic approaches. The study of lymphatic tumor vasculature has been gaining interest in the context of cancer immunotherapy. These vessels constitute conduits for immune cells' transit toward the lymph nodes, and they endow tumors with routes to metastasize to the lymph nodes and, from them, toward distant sites. In addition, this vasculature participates in the modulation of the immune response directly through the interaction with tumor-infiltrating leukocytes and indirectly through the secretion of cytokines and chemokines that attract leukocytes and tumor cells. Radiotherapy constitutes the therapeutic option for more than 50% of solid tumors. Besides impacting transformed cells, RT affects stromal cells such as endothelial and immune cells. Mature lymphatic endothelial cells are resistant to RT, but we do not know to what extent RT may affect tumor-aberrant lymphatics. RT compromises lymphatic integrity and functionality, and it is a risk factor to the onset of lymphedema, a condition characterized by deficient lymphatic drainage and compromised tissue homeostasis. This review aims to provide evidence of RT's effects on tumor vessels, particularly on lymphatic endothelial cell physiology and immune properties. We will also explore the therapeutic options available so far to modulate signaling through lymphatic endothelial cell receptors and their repercussions on tumor immune cells in the context of cancer. There is a need for careful consideration of the RT dosage to come to terms with the participation of the lymphatic vasculature in anti-tumor response. Here, we provide new approaches to enhance the contribution of the lymphatic endothelium to radioimmuno-oncology.

Breast cancer is one of the most common and malignant cancers nowadays. Like many cancers, it is extensively regulated by pro-angiogenic and pro-lymphangiogenic factors, which induce rapid vasculature growth. This results in aberrant vessels which enhance a pro-tumor environment by preventing treatment against cancer to get to the tumor, inducing the malignancy of cancer cells, and inhibiting the immune response. In the last years, antiangiogenic treatment was suggested as a possible way to eliminate the tumor-supporting environment. This approach was soon replaced by the judicious use of these anti-angiogenic molecules in normalizing concentrations, to prevent pro-tumor effects, such as cell malignification. In a novel approach to prevent these processes to occur, we have designed a treatment based on the normalization of the tumoral vasculature of triple-negative breast carcinoma, so they return to their physiological state. For this aim, we have analyzed the effects of the blockage of VEGF-A and VEGF-C signaling pathways (angiogenesis and lymphangiogenesis), by targeting their receptors, VEGFR2 and VEGFR3, with inhibitor molecules (DC101 antibody and SAR131675 molecules).

The quantity of T-lymphocytes reaching the draining lymph nodes from tumors is likely important to mount effective distant responses and for the establishment of long term systemic memory. Looking into mechanisms behind lymphocyte egress, we directed our attention to leukocyte adhesion mechanisms inside tumors. Here we demonstrate that activated T-cells form intra-tumor aggregates in a LFA-1-ICAM-1-dependent fashion in mouse models of melanoma and breast cancer. We also provide evidence of the presence of T-cell clusters in primary human melanoma. Disruption of LFA-1-ICAM-1 interactions, and thereby T-cell clustering, enhances the arrival of activated CD8+ T-cells to tumor draining lymph nodes in both transplanted and spontaneous cancer models. Interestingly, upon ICAM-1 blockade, the expression of the chemotactic receptor CCR7 augments in tumor infiltrating lymphocytes and in in-vitro de-clustered T cells, as well as their ability to transmigrate across lymphatic endothelial cells. We propose that ICAM-1-mediated homotypic T-lymphocyte aggregation may serve as a tumor-mediated immune retention mechanism entrapping activated CD8+ T cells in the tumor microenvironment. Modulation of T-cell adhesion may be of use to improve the transit of activated lymphocytes toward the lymph nodes and their subsequent recirculation.

A catenary sequence of soils formed on reworked loess under semiarid climatic conditions was studied. Four soil profiles located on the piedmont of Sierra Chica de Córdoba, central Argentina,were described, classified and geochemically analyzed. All soils, developed on summit, shoulder, backslope, and toeslope positions, were classified as Mollisols. Decarbonatation-carbonatation, melanization, and argilluviation are the main pedogenic processes recognized in these soils, which appear to control the differentiation of genetic horizons along the catena. Different geochemical approaches indicate that there are not substantial variations in the chemical composition of the studied soils along the catena, with the exception of the soil in the toeslope position, which exhibits slightly differences. In general, all profiles show weak depletions of mobile elements (Ca, Na, Mg, Sr, U) in the upper continental crust UCC-normalized diagrams which are attributed to a slight chemical alteration. Other elements, such as Fe, Cr, Co and Ni, also exhibit depletions compared to UCC, which can be explained by the alteration of ferromagnesian silicates, but can also be an inherited feature from the parent material. The significant enrichment in As compared to UCC, evident in all profiles along the catena, is also a typical feature of the pampean plains´ loess of Argentina. So, the chemical differences in the profile located on the toeslope aremainly attributed to the supply of materials from local sources, i.e., crystalline basement and sedimentary rocks, due to its position in the catena. Statistical correlations and multivariate cluster analyses reinforce the assumption that the geochemistry of the studied soils is inherited from the parent material. In addition, chemical indices (CIA, ICV), elemental ratios (Ba/Sr, Rb/Sr) and the A-CN-K ternary diagram indicate an incipient degree of chemical alteration for thesesoils, compatible with the weathering regime prevailing in the region. Thus, the differentiation of genetic horizons along the catena is the result of weak weathering and pedogenic processes, which have not been strong enough to mask the chemical imprint of the parent material. Fil: Pasquini, Andrea Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Ciencias de la Tierra. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; Argentina Fil: Campodonico, Verena Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Ciencias de la Tierra. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; Argentina Fil: Rouzaut, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Ciencias de la Tierra. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; Argentina Fil: Giampaoli Viviana. Universidade de Sao Paulo; Brasil

Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions. Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix. In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs. We would like to acknowledge the support of the Spanish Ministry of Economy and Competitiveness, under grants number DPI2012-38090-C03-02 and DPI2015-64221-C2-2-R (COS), TEC2013-48552-C2-1-R, TEC2016-78052-R, TEC2015-73064-EXP (AMB) and the Torres Quevedo program PTQ-11-04778 (RP); the Spanish Ministry of Health (FIS PI13/02313) (AR); the Czech Science Foundation, under grant number 302/12/G157 (MK, MMaška); and the European Research Council (ERC) through project ERC-2012-StG 306751 (JMGA).

T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged T cell migration through afferent LVs in vivo. T cells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic T cell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. In vitro, blockade of endothelial cell-expressed ICAM-1 reduced T cell adhesion, crawling, and transmigration across lymphatic endothelium and decreased T cell advancement from capillaries into lymphatic collectors in skin explants. In vivo, T cell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that T cell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process., Cell Reports, 18 (4), ISSN:2666-3864, ISSN:2211-1247

Cancer related deaths are primarily due to tumor metastasis. To facilitate their dissemination to distant sites, cancer cells develop invadopodia, actin-rich protrusions capable of degrading the surrounding extracellular matrix (ECM). We aimed to determine whether β3 integrin participates in invadopodia formed by lung carcinoma cells, based on our previous findings of specific TGF-β induction of β3 integrin dependent metastasis in animal models of lung carcinoma. In this study, we demonstrate that lung carcinoma cells form invadopodia in response to TGF-β exposure. Invadopodia formation and degradation activity is dependent on β3 integrin expression since β3 integrin deficient cells are not able to degrade gelatin-coated surfaces. Even more, transient over-expression of SRC did not restore invadopodia formation in β3 integrin deficient cells. Finally, we observed that blockade of PLC-dependent signaling leads to more intense labeling for β3 integrin in invadopodia. Our results suggest that β3 integrin function, and location, in lung cancer cells are essential for invadopodia formation, and this integrin regulates the activation of different signal pathways necessary for the invasive structure. β3 integrin has been associated with poor prognosis and increased metastasis in several carcinoma types, including lung cancer. Our findings provide new evidence to support the use of targeted therapies against this integrin to combat the onset of metastases.

En el presente trabajo se relacionan parámetros magnéticos en perfiles de suelos con vegetación autóctona y suelos cultivados de la Serie Corralito en la provincia de Córdoba, Argentina. Ellos se encuentran en la misma región geomorfológica, bajo el mismo régimen climático, y en similar actitud topográfica y de drenaje. Se encontró una correlación entre la actividad agrícola y las propiedades magnéticas; que sugiere que la disparidad en la señal magnética de los suelos cultivados podría ser consecuencia del estado de crecimiento del cultivo, la siembra directa o el empleo de distintos abonos en las distintas parcelas.This paper deals with the results of magnetic parameters obtained from both native vegetation and cultivated soils of Corralito Series in the province of Córdoba, Argentina. The soils are in the same geomorphological region, under the same climatic regime, in similar topographic positions and similar drainage. We found a correlation between agriculture and magnetic properties, the disparity in the magnetic signal of cultivated soils may be a consequence of different growth stage of the crop, tillage or different use of fertilizers in the plots.

Summary: Cancer immunotherapy has great promise, but is limited by diverse mechanisms used by tumors to prevent sustained antitumor immune responses. Tumors disrupt antigen presentation, T/NK–cell activation, and T/NK–cell homing through soluble and cell-surface mediators, the vasculature, and immunosuppressive cells such as myeloid-derived suppressor cells and regulatory T cells. However, many molecular mechanisms preventing the efficacy of antitumor immunity have been identified and can be disrupted by combination immunotherapy. Here, we examine immunosuppressive mechanisms exploited by tumors and provide insights into the therapies under development to overcome them, focusing on lymphocyte traffic. Cancer Discov; 4(5); 522–6. ©2014 AACR.

BACKGROUND: Interleukin-8 (IL-8, CXCL8) is readily produced by human malignant cells. Dendritic cells (DC) both produce IL-8 and express the IL-8 functional receptors CXCR1 and CXCR2. Most human colon carcinomas produce IL-8. IL-8 importance in malignancies has been ascribed to angiogenesis promotion. PRINCIPAL FINDINGS: IL-8 effects on human monocyte-derived DC biology were explored upon DC exposure to recombinant IL-8 and with the help of an IL-8 neutralizing mAb. In vivo experiments were performed in immunodeficient mice xenografted with IL-8-producing human colon carcinomas and comparatively with cell lines that do not produce IL-8. Allogenic T lymphocyte stimulation by DC was explored under the influence of IL-8. DC and neutrophil chemotaxis were measured by transwell-migration assays. Sera from tumor-xenografted mice contained increasing concentrations of IL-8 as the tumors progress. IL-8 production by carcinoma cells can be modulated by low doses of cyclophosphamide at the transcription level. If human DC are injected into HT29 or CaCo2 xenografted tumors, DC are retained intratumorally in an IL-8-dependent fashion. However, IL-8 did not modify the ability of DC to stimulate T cells. Interestingly, pre-exposure of DC to IL-8 desensitizes such cells for IL-8-mediated in vitro or in vivo chemoattraction. Thereby DC become disoriented to subsequently follow IL-8 chemotactic gradients towards malignant or inflamed tissue. CONCLUSIONS: IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation.

Type I interferons (IFN-I) are well-known inducers of tumor cell apoptosis and antiangiogenesis via signaling through a common receptor interferon alpha receptor (IFNAR). IFNAR induces the Janus activated kinase-signal transducer and activation of transcription (JAK-STAT) pathway in most cells, along with other biochemical pathways that may differentially operate, depending on the responding cell subset, and jointly control a large collection of genes. IFNs-I were found to systemically activate natural killer (NK) cell activity. Recently, mouse experiments have shown that IFNs-I directly activate other cells of the immune system, such as antigen-presenting dendritic cells (DC) and CD4 and CD8 T cells. Signaling through the IFNAR in T cells is critical for the acquisition of effector functions. Cross-talk between IFNAR and the pathways turned on by other surface lymphocyte receptors has been described. Importantly, IFNs-I also increase antigen presentation of the tumor cells to be recognized by T lymphocytes. These IFN-driven immunostimulatory pathways offer opportunities to devise combinatorial immunotherapy strategies.

CD137 artificial costimulation results in complete tumor rejection in several mouse models. Type I interferons (IFN) exert antitumor effects through an array of molecular functions on malignant cells, tumor stroma and immune system cells. The fact that agonist anti-CD137 mAb induce tumor regressions in mice deficient in the unique receptor for Type I IFNs (IFNAR(-/-) ) indicated potential for treatment combinations. Indeed, combination of intratumor injections of mouse IFN-α and intraperitoneal injections of anti-CD137 mAb synergized as seen on subcutaneous lesions derived from the MC38 colon carcinoma, which is resistant to each treatment if given separately. Therapeutic activity was achieved both against lesions directly injected with IFN-α and against distant concomitant tumors. Experiments in bone marrow chimeras prepared with IFNAR(-/-) and WT mice concluded that expression of the receptor for Type I interferons is mainly required on cells of the hematopoietic compartment. Synergistic effects correlated with a remarkable cellular hyperplasia of the tumor draining lymph nodes (TDLNs). Enlarged TDLNs contained more plasmacytoid and conventional dendritic cells (DC) that more readily cross-presented. Importantly, numbers of both DC subtypes inversely correlated with the tumor size. Numbers of CD8 T cells specific for a dominant tumor antigen were increased at TDLNs by each separate treatment but only with slight augments due to the combination. Combined antitumor effects of the therapeutic strategy were also seen on subcutaneous TC-1 tumors established for 24 days before treatment onset. The described strategy is realistic because (i) agents of each kind are clinically available and (ii) equivalent procedures in humans are feasible.

Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag−/− mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti–ICAM-1 and anti–VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies. Cancer Res; 71(3); 801–11. ©2011 AACR.

Background Lung cancer is one of the most prevalent neoplasias in developed countries. Advances in patient survival have been limited and the identification of prognostic molecules is needed. Resistance to treatment is strongly related to tumor cell adhesion to the extracellular matrix and alterations in the quantity and nature of molecules constituting the tumor cell niche. Recently, transforming growth factor beta-induced protein (TGFBI), an extracellular matrix adaptor protein, has been reported to be differentially expressed in transformed tissues. Loss of TGFBI expression has been described in several cancers including lung carcinoma, and it has been suggested to act as a tumor suppressor gene. Results To address the importance of TGFBI expression in cancer progression, we determined its expression in NSCLC clinical samples using immunohistochemistry. We identified a strong association between elevated TGFBI expression and the response to chemotherapy. Furthermore, we transiently over-expressed and silenced TGFBI in human NSCLC cell lines. Cells over-expressing TGFBI displayed increased sensitivity to etoposide, paclitaxel, cisplatin and gemcitabine. We observed that TGFBI-mediated induction of apoptosis occurred through its binding to αvβ3 integrin. We also determined that full-length TGFBI did not induce caspase 3/7 activation but its proteolytic fragments that were < 3 kDa in size, were able to activate caspase 3, 7 and 8. This pro-apoptotic effect was blocked by anti-αvβ3 integrin antibodies. Conclusions The results shown here indicate that TGFBI is a predictive factor of the response to chemotherapy, and suggest the use of TGFBI-derived peptides as possible therapeutic adjuvants for the enhancement of responses to chemotherapy.

TGFbeta-induced protein (TGFBI) is an extracellular protein that mediates cell adhesion to collagen, laminin and fibronectin through its interaction with different beta integrins. We had previously reported that hypoxia-induced TGFBI mRNA expression in lymphatic endothelial cells (LEC). Here, we demonstrate that TGFBI can contribute to hypoxia-induced increases in LEC adhesion to the ECM. We show that while there are no changes in alpha1, alpha4, alphav, beta1, beta2, beta3, alpha5beta1, alphavbeta3, alphavbeta5 integrin expression on the LEC surface after hypoxia exposure, there exists an accumulation of TGFBI adaptor protein in LEC supernatants. We also demonstrate that hypoxia driven TGBFI expression is dependent on TGFbeta production by LEC. Furthermore, we show that TGFBI mediated LEC adhesion and migration through the ECM by its binding to the beta3 integrin. The identification of the specific mechanisms regulating LEC-ECM interactions may help us design new therapeutic applications for diseases in which lymphatic vessel function is compromised.

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PPi-dependent phosphorolysis catalysed by wild-type and AZT (3′-azido-3′-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template–primer (Kd=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template–primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.

Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.