The role of dendritic cell precursors in tumour vasculogenesis

In this review, we discuss the recent identification in vivo of a population of CD11c+ cells exhibiting simultaneous expression of both endothelial and dendritic cell markers, termed vascular leukocytes (VLCs). VLCs are highly represented in human ovarian carcinomas and, depending on the milieu, can assemble into functional blood vessels or act as antigen-presenting cells. The identification of dendritic cell precursors as bipotent cells has important implications for the physiopathology and therapy of tumours. VLCs emerge as a novel therapeutic target against tumour vascularisation

LOS PÉREZ. LUGAREJO, LAS HOYAS, SIBERIO, PARRALILLO [Material gráfico]

Copia digital. Madrid : Ministerio de Educación, Cultura y Deporte, 201

IRE1α–XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity

Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function 1?4 . However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer?an aggressive malignancy that is refractory to standard treatments and current immunotherapies 5?8 ?induces endoplasmic reticulum stress and activates the IRE1α?XBP1 arm of the unfolded protein response 9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1α?XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1α?XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α?XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts.Fil: Song, Minkyung. Weill Cornell Medicine; Estados UnidosFil: Sandoval, Tito A.. Weill Cornell Medicine; Estados UnidosFil: Chae, Chang-Suk. Weill Cornell Medicine; Estados UnidosFil: Chopra, Sahil. Weill Cornell Medicine; Estados UnidosFil: Tan, Chen. Weill Cornell Medicine; Estados UnidosFil: Rutkowski, Melanie R.. University of Virginia; Estados UnidosFil: Raundhal, Mahesh. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados UnidosFil: Chaurio, Ricardo A.. H. Lee Moffitt Cancer Center & Research Institute; Estados UnidosFil: Payne, Kyle K.. H. Lee Moffitt Cancer Center & Research Institute; Estados UnidosFil: Konrad, Csaba. Weill Cornell Medicine; Estados UnidosFil: Bettigole, Sarah E.. Quentis Therapeutics Inc.; Estados UnidosFil: Shin, Hee Rae. Quentis Therapeutics Inc.; Estados UnidosFil: Crowley, Michael J. P.. Weill Cornell Graduate School of Medical Sciences; Estados UnidosFil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Kossenkov, Andrew V.. The Wistar Institute; Estados UnidosFil: Motorykin, Ievgen. Weill Cornell Medicine,; Estados UnidosFil: Zhang, Sheng. Weill Cornell Medicine,; Estados UnidosFil: Manfredi, Giovanni. Weill Cornell Medicine,; Estados UnidosFil: Zamarin, Dmitriy. Memorial Sloan Kettering Cancer Center; Estados UnidosFil: Holcomb, Kevin. Weill Cornell Medicine,; Estados UnidosFil: Rodriguez, Paulo C.. H. Lee Moffitt Cancer Center & Research Institute; Estados UnidosFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Conejo Garcia, Jose R.. H. Lee Moffitt Cancer Center & Research Institute; Estados UnidosFil: Glimcher, Laurie H.. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados UnidosFil: Cubillos-Ruiz, Juan R.. Weill Graduate School Of Medical Sciences; Estados Unidos. Weill Graduate School Of Medical Sciences; Estados Unido

Characterization of TEM1/endosialin in human and murine brain tumors

<p>Abstract</p> <p>Background</p> <p><it>TEM1/endosialin </it>is an emerging microvascular marker of tumor angiogenesis. We characterized the expression pattern of <it>TEM1/endosialin </it>in astrocytic and metastatic brain tumors and investigated its role as a therapeutic target in human endothelial cells and mouse xenograft models.</p> <p>Methods</p> <p><it>In situ </it>hybridization (ISH), immunohistochemistry (IH) and immunofluorescence (IF) were used to localize <it>TEM1/endosialin </it>expression in grade II-IV astrocytomas and metastatic brain tumors on tissue microarrays. Changes in <it>TEM1/endosialin </it>expression in response to pro-angiogenic conditions were assessed in human endothelial cells grown <it>in vitro</it>. Intracranial U87MG glioblastoma (GBM) xenografts were analyzed in nude <it>TEM1/endosialin </it>knockout (KO) and wildtype (WT) mice.</p> <p>Results</p> <p><it>TEM1/endosialin </it>was upregulated in primary and metastatic human brain tumors, where it localized primarily to the tumor vasculature and a subset of tumor stromal cells. Analysis of 275 arrayed grade II-IV astrocytomas demonstrated <it>TEM1/endosialin </it>expression in 79% of tumors. Robust <it>TEM1/endosialin </it>expression occurred in 31% of glioblastomas (grade IV astroctyomas). <it>TEM1/endosialin </it>expression was inversely correlated with patient age. TEM1/endosialin showed limited co-localization with CD31, αSMA and fibronectin in clinical specimens. <it>In vitro</it>, <it>TEM1/endosialin </it>was upregulated in human endothelial cells cultured in matrigel. Vascular <it>Tem1/endosialin </it>was induced in intracranial U87MG GBM xenografts grown in mice. <it>Tem1/endosialin </it>KO vs WT mice demonstrated equivalent survival and tumor growth when implanted with intracranial GBM xenografts, although <it>Tem1/endosialin </it>KO tumors were significantly more vascular than the WT counterparts.</p> <p>Conclusion</p> <p><it>TEM1/endosialin </it>was induced in the vasculature of high-grade brain tumors where its expression was inversely correlated with patient age. Although lack of <it>TEM1/endosialin </it>did not suppress growth of intracranial GBM xenografts, it did increase tumor vascularity. The cellular localization of <it>TEM1/endosialin </it>and its expression profile in primary and metastatic brain tumors support efforts to therapeutically target this protein, potentially via antibody mediated drug delivery strategies.</p

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8+ T cells. However, their independent functional contributions in distinct CD8+ T cell subsets are not well understood. In this study, CD8+ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA+CD27+) and memory (CD45RA−CD27+) CD8+ T cells (CD28Hi), while its expression was significantly lower in effector (CD45RA+CD27−) CD8+ T cells (CD28Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8+ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8+ T cells. Co-stimulation of CD28Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8+ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8+ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8+ T cells. However, boosting a recall immune response via memory CD8+ T cells or vaccination to stimulate naïve CD8+ T cells would require CD28-mediated co-stimulation

Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

<p>Abstract</p> <p>Background</p> <p>Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs) with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application.</p> <p>Methods</p> <p>To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs) for the direct and rapid expansion of TILs isolated from primary cancer specimens.</p> <p>Results</p> <p>TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures.</p> <p>Conclusion</p> <p>Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.</p

Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection

<p>Abstract</p> <p>Background</p> <p>In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV).</p> <p>Methods</p> <p>Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII⁺-cell depletion) in nude mice.</p> <p>Results</p> <p>Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII⁺/CD31⁺vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII⁺-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis.</p> <p>Conclusions</p> <p>Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.</p

Comprehensive assessment of sequence variation within the copy number variable defensin cluster on 8p23 by target enriched in-depth 454 sequencing

<p>Abstract</p> <p>Background</p> <p>In highly copy number variable (CNV) regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS) approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach.</p> <p>Results</p> <p>As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations.</p> <p>Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs) including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods.</p> <p>Conclusion</p> <p>Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation.</p

Influence of bevacizumab, sunitinib and sorafenib as single agents or in combination on the inhibitory effects of VEGF on human dendritic cell differentiation from monocytes

Vascular endothelial growth factor (VEGF) inhibits differentiation and maturation of dendritic cells (DC), suggesting a potential immunosuppressive role for this proangiogenic factor. Bevacizumab, sorafenib and sunitinib target VEGF-mediated angiogenesis and are active against several types of cancer, but their effects on the immune system are poorly understood. In this study, VEGF and supernatants of renal carcinoma cell lines cultured under hypoxia were found to alter the differentiation of human monocytes to DC. Resulting DC showed impaired activity, as assessed by the alloreactive mixed T-lymphocyte reaction. Bevacizumab and sorafenib, but not sunitinib, reversed the inhibitory effects of VEGF, but not of those mediated by tumour supernatants. Dendritic cells matured under the influence of VEGF expressed less human leukocyte antigen-DR (HLA-DR) and CD86, and this effect was restored by bevacizumab and sorafenib. Finally, tumour-cell supernatants decreased interleukin-12 (IL-12) production by mature DC, and such inhibition was not restored by any of the tested drugs, delivered either as single agents or in combination. The deleterious effects of tumour-cell supernatants were mainly mediated by thermostable molecules distinct from VEGF. These results indicate that inhibition of the differentiation of monocytes to DC is a multifactorial effect, and that they support the development of combinations of angiogenesis inhibitors with immunological modulators

Anti-angiogenic effect of high doses of ascorbic acid

Pharmaceutical doses of ascorbic acid (AA, vitamin C, or its salts) have been reported to exert anticancer activity in vitro and in vivo. One proposed mechanism involves direct cytotoxicity mediated by accumulation of ascorbic acid radicals and hydrogen peroxide in the extracellular environment of tumor cells. However, therapeutic effects have been reported at concentrations insufficient to induce direct tumor cell death. We hypothesized that AA may exert anti-angiogenic effects. To test this, we expanded endothelial progenitor cells (EPCs) from peripheral blood and assessed, whether or not high dose AA would inhibit EPC ability to migrate, change energy metabolism, and tube formation ability. We also evaluated the effects of high dose AA on angiogenic activities of HUVECs (human umbilical vein endothelial cells) and HUAECs (human umbilical arterial endothelial cells). According to our data, concentrations of AA higher than 100 mg/dl suppressed capillary-like tube formation on Matrigel for all cells tested and the effect was more pronounced for progenitor cells in comparison with mature cells. Co-culture of differentiated endothelial cells with progenitor cells showed that there was incorporation of EPCs in vessels formed by HUVECs and HUAECs. Cell migration was assessed using an in vitro wound healing model. The results of these experiments showed an inverse correlation between AA concentrations relative to both cell migration and gap filling capacity. Suppression of NO (nitric oxide) generation appeared to be one of the mechanisms by which AA mediated angiostatic effects. This study supports further investigation into non-cytotoxic antitumor activities of AA