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The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor cytotoxic T lymphocytes. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin-mediated cDC1 depletion prior to immunotherapy treatment with anti-PD-1 and/or anti-CD137 immunostimulatory monoclonal antibodies (mAbs) completely ablated anti-tumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti-PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors. This work was supported by Spanish Ministry of Economy and Competitiveness and Spanish Ministry of Research (MINECO SAF2014-52361-R and SAF 2017-83267-C2-1R and PID2020-112892RB-100, PID2020-113174-RA-100 [AEI/FEDER,UE], financed by MCIN/AEI/10.13039/501100011033), Cancer Research Institute under the CRI-CLIP, Asociación Española Contra el Cancer (AECC) Foundation under Grant GCB15152947MELE, Joint Translational Call for Proposals 2015 (JTC 2015) TRANSCAN-2 (code: TRS-2016-00000371), projects PI14/01686, PI13/00207, PI16/00668, PI19/01128, funded by Instituto de Salud Carlos III and co-funded by European Union (ERDF, “A way to make Europe”), European Commission within the Horizon 2020 Programme (PROCROP - 635122), Gobierno de Navarra Proyecto LINTERNA Ref: 0011–1411, Mark Foundation, Fundación BBVA and Fundación Olga Torres. AT is supported by the Ramon y Cajal program from the Spanish Ministry of Science (RYC2019-026406-I financiada por MCIN/AEI /10.13039/501100011033 y por El FSE invierte en tu futuro). Sí

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798–811. ©2018 AACR.

Figure S1 shows that CD8 T cells with high mitochondrial transmembrane potential after CD137 stimulation have enlarged mitochondria. Fig S2 shows that CD137 induced mitochondrial changes are not due to enrichment of specific CD8 T cells subsets. Figure S3 shows that mitochondrial enhanced functions induced by CD137 activation are not a consequence of autocrine secretion of cytokines. Fig S4 shows that spare respiratory capacity induced by CD137 activation is dependent on fatty acid oxidation. Figure S5 explores regulation of mitochondrial biogenesis and fusion related genes upon CD137 activation. Fig S6 shows functional effects of OPA knockdown in CD8 T cells. Figure S7 shows the implication of Akt pathway on enhanced mitochondrial function by CD137 activation. Figure S8 shows that CD137KO CD8 T cells have intrinsic mitochondrial defects upon CD28 activation. Figure S9 shows that CD137 activation enhances mitochondrial parameters in tumor infiltrating lymphocytes. Fig S10 shows engrafment abilities and memory markers of OTI cells in B16OVA tumor bearing mice upon OPA silencing and CD137 mAb treatment.

PDF file - 946K, Anti-CTLA-4 mAb treatment fails to extend survival of c-myc OVA tg+

PDF file - 953K, TILs recognize unknown antigens in JMJ cell lines.

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Purpose: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen.Experimental Design: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays.Results: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8+ and CD4+ T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy.Conclusion: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151–62. ©2013 AACR.

single dose radiotherapy and hypofractionated doses increase the level of CD137 and PD-1 expression on the surface of tumor-infiltrating T cells.

Supplementary figures 1-5 Supplementary methods

PDF file - 2976K, HCC cell lines derived from c-myc OVA tg+ mice express OVA and functional antigen presenting molecules.

Radiotherapy reduced the density and absolute numbers of CD4+, CD8+ and CD25+FOXP3+ T cells and Myeloid-derived suppressor cells both in the irradiated and non-irradiated lesions.

The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor CTLs. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin–mediated cDC1 depletion prior to immunotherapy treatment with anti–PD-1 and/or anti-CD137 immunostimulatory mAbs completely ablated antitumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti–PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors.Significance:These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.

PDF file - 1620K, Combo3 treatment induces infiltrates of blastic T lymphocytes and increases the CD8/Treg ratio.

Video S3

Statistical comparisons for figures 1, 2, 4 and supplementary figure 1.

4T1-derived spontaneous lung metastases

PDF file - 2166K, Granzyme B, perforin and FasL are upregulated on CD8+ T cells after Combo3 treatment.

Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in transplanted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3–dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8+ T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracellular IFNγ in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8+ TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from surgically excised human carcinomas that were irradiated ex vivo. These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strategies, even if the disease is left outside the field of irradiation. Cancer Res; 76(20); 5994–6005. ©2016 AACR.

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supplementary figure legends and supplementary material and methods section.

Mouse external beam irradiation setting and combinations of radiotherapy and anti-CD137 and/or anti-PD1 immunostimulatory monoclonal antibodies are safe and effective to treat MC38-derived bilateral tumors of which only one of the tumor sites is irradiated.

Ex-vivo irradiation of human carcinoma surgical expecimens gives rise to more intense expression of CD137 and PD-1 on infiltrating lymphocytes.

A course of radiotherapy plus immunostimulatory monoclonal antibodies anti-CD137 and anti-PD-1 mAb increases CD4 and CD8 T cells in the tumor infiltrate as well as the number of tumor antigen-specific CD8 T cells.

PDF file - 1818K, Activated T cells and tumor infiltrating lymphocytes express PD-1, B7-H1 (PD-L1), CD137 and OX40.

video S1

PDF file - 4662K, Three week old mice present established multifocal hepatocellular carcinomas and neonatal mice express OVA mRNA in their livers.

Video S2

Supplementary Figures 1-5 from Agonist Anti-CD137 mAb Act on Tumor Endothelial Cells to Enhance Recruitment of Activated T Lymphocytes

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.

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.

Collapsin response mediator protein 2 (CRMP2) is an adaptor protein that adds tubulin dimers to the growing tip of a microtubule. First described in neurons, it is now considered a ubiquitous protein that intervenes in processes such as cytoskeletal remodeling, synaptic connection and trafficking of voltage channels. Mounting evidence supports that CRMP2 plays an essential role in neuropathology and, more recently, in cancer. We have previously described a positive correlation between nuclear phosphorylation of CRMP2 and poor prognosis in lung adenocarcinoma patients. In this work, we studied whether this cytoskeleton molding protein is involved in cancer cell migration. To this aim, we evaluated CRMP2 phosphorylation and localization in the extending lamella of lung adenocarcinoma migrating cells using in vitro assays and in vivo confocal microscopy. We demonstrated that constitutive phosphorylation of CRMP2 impaired lamella formation, cell adhesion and oriented migration. In search of a mechanistic explanation of this phenomenon, we discovered that CRMP2 Ser522 phospho-mimetic mutants display unstable tubulin polymers, unable to bind EB1 plus-Tip protein and the cortical actin adaptor IQGAP1. In addition, integrin recycling is defective and invasive structures are less evident in these mutants. Significantly, mouse xenograft tumors of NSCLC expressing CRMP2 phosphorylation mimetic mutants grew significantly less than wild-type tumors. Given the recent development of small molecule inhibitors of CRMP2 phosphorylation to treat neurodegenerative diseases, our results open the door for their use in cancer treatment.

Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in transplanted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3–dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8+ T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracellular IFNγ in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8+ TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from surgically excised human carcinomas that were irradiated ex vivo. These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strategies, even if the disease is left outside the field of irradiation. Cancer Res; 76(20); 5994–6005. ©2016 AACR.

T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR. This project was supported by imCORE Network (Roche Genentech), MINECO (SAF2014-52361-R, 2017-83267-C2-1-R; I. Melero), European Commission VII Framework and Horizon 2020 programs (IACT and PROCROP), Fundacion de la Asociaci on Espa nola Contra el C ~ ancer (AECC), and Fundacion BBVA. A. Teijeira receives support from a Juan de la Cierva contract, MINECO. Electron and light microscopy CIMA services as well as Flow Cytometry CIMA facilities (Diego Alignani) and personnel at blood bank of Navarra are acknowledged for their technical support. We are also grateful to Drs. Lasarte and HervasStubbs for scientific discussion and to Dr. Paul Miller for editing the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sí

Background It is now obvious that the majority of cellular transcripts do not code for proteins, and a significant subset of them are long non-coding RNAs (lncRNAs). Many lncRNAs show aberrant expression in cancer, and some of them have been linked to cell transformation. However, the underlying mechanisms remain poorly understood and it is unknown how the sequences of lncRNA dictate their function. Results Here we characterize the function of the p53-regulated human lncRNA LINC-PINT in cancer. We find that LINC-PINT is downregulated in multiple types of cancer and acts as a tumor suppressor lncRNA by reducing the invasive phenotype of cancer cells. A cross-species analysis identifies a highly conserved sequence element in LINC-PINT that is essential for its function. This sequence mediates a specific interaction with PRC2, necessary for the LINC-PINT-dependent repression of a pro-invasion signature of genes regulated by the transcription factor EGR1. Conclusions Our findings support a conserved functional co-dependence between LINC-PINT and PRC2 and lead us to propose a new mechanism where the lncRNA regulates the availability of free PRC2 at the proximity of co-regulated genomic loci. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1331-y) contains supplementary material, which is available to authorized users.

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.

Purpose/Objectives The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Materials/Methods Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGFβ in this process anti-TGFβ blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGFβ and /or anti-ICAM1 blocking mAb. Results We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGFβ. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Conclusion Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy.

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.