CD11c-CD8 Spatial Cross Presentation: A Novel Approach to Link Immune Surveillance and Patient Survival in Soft Tissue Sarcoma.

Checkpoint inhibitors are slowly being introduced in the care of specific sarcoma subtypes such as undifferentiated pleomorphic sarcoma, alveolar soft part sarcoma, and angiosarcoma even though formal indication is lacking. Proper biomarkers to unravel potential immune reactivity in the tumor microenvironment are therefore expected to be highly warranted. In this study, intratumoral spatial cross presentation was investigated as a novel concept where immune cell composition in the tumor microenvironment was suggested to act as a proxy for immune surveillance. Double immunohistochemistry revealed a prognostic role of direct spatial interactions between CD11c+ antigen-presenting cells (APCs) and CD8+ cells in contrast to each marker alone in a soft tissue sarcoma (STS) cohort of 177 patients from the Karolinska University Hospital (MFS p = 0.048, OS p = 0.025). The survival benefit was verified in multivariable analysis (MFS p = 0.012, OS p = 0.004). Transcriptomics performed in the TCGA sarcoma cohort confirmed the prognostic value of combining CD11c with CD8 (259 patients, p = 0.005), irrespective of FOXP3 levels and in a CD274 (PD-LI)-rich tumor microenvironment. Altogether, this study presents a histopathological approach to link immune surveillance and patient survival in STS. Notably, spatial cross presentation as a prognostic marker is distinct from therapy response-predictive biomarkers such as immune checkpoint molecules of the PD-L1/PD1 pathway.

Pericytes regulate the blood-brain barrier.

The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.

The absence of pericytes does not increase the sensitivity of tumor vasculature to vascular endothelial growth factor-A blockade.

Recent progress with therapies targeting endothelial cells has drawn attention also to the pericytes as potential target cells for antiangiogenic therapy. Published data suggest that pericytes might confer resistance to vascular endothelial growth factor (VEGF) withdrawal in tumors. This hypothesis has been supported by experiments using tumors with reversible transgenic expression of VEGF-A as well as by individual pharmacologically targeting VEGF and platelet-derived growth factor receptor signaling in endothelial cells and pericytes using receptor tyrosine kinase (RTK) inhibitors with different specificities. However, the RTK inhibitors applied thus far are not entirely specific to the mentioned pathways, and therefore, the effects putatively attributed to pericyte targeting might reflect other antitumor effects. Here, we have reinvestigated the putative benefits of doubly targeting endothelial cells and pericytes in the treatment of experimental tumors. For this purpose, we used two highly specific tools, the pericyte-deficient pdgfb(ret/ret) mouse and the recently developed specific anti-VEGF-A antibody G6-31, which neutralizes both murine and human VEGF-A. We generated B16, Lewis lung carcinoma, and T241 subcutaneous tumors in both pdgfb(ret/ret) and control mice and treated these mice with G6-31. Our results fail to show any improved effect of VEGF inhibition, as measured by tumor growth or decrease in vascular density, in pericyte-deficient tumors compared with controls. Our observations suggest that additional targeting of pericytes does not increase the antitumor effect already generated by anti-VEGF drugs.

Identification of a core set of 58 gene transcripts with broad and specific expression in the microvasculature.

OBJECTIVE: Pathological angiogenesis is an integral component of many diseases. Antiangiogenesis and vascular targeting are therefore promising new therapeutic principles. However, few endothelial-specific putative drug targets have been identified, and information is still limited about endothelial-specific molecular processes. Here we aimed at determining the endothelial cell-specific core transcriptome in vivo. METHODS AND RESULTS: Analysis of publicly available microarray data identified a mixed vascular/lung cluster of 132 genes that correlated with known endothelial markers. Filtering against kidney glomerular/nonglomerular and brain vascular/nonvascular microarray profiles separated contaminating lung markers, leaving 58 genes with broad and specific microvascular expression. More than half of these have not previously been linked to endothelial functions or studied in detail before. The endothelial cell-specific expression of a selected subset of these, Eltd1, Gpr116, Ramp2, Slc9a3r2, Slc43a3, Rasip1, and NM_023516, was confirmed by real-time quantitative polymerase chain reaction and/or immunohistochemistry. CONCLUSIONS: We have used a combination of publicly available and own microarray data to identify 58 gene transcripts with broad yet specific expression in microvascular endothelium. Most of these have unknown functions, but many of them are predicted to be cell surface expressed or implicated in cell signaling processes and should therefore be explored as putative microvascular drug targets.

PDGF-B signaling is important for murine cardiac development: its role in developing atrioventricular valves, coronaries, and cardiac innervation.

We hypothesized that PDGF-B/PDGFR-beta-signaling is important in the cardiac contribution of epicardium-derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf-b-/- and Pdgfr-beta-/- mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for alphaSMA, periostin, ephrinB2, EphB4, VEGFR-2, Dll1, and NCAM was performed on wild-type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest-related innervation deficits. This implies a role for PDGF-B/PDGFR-beta-signaling specifically in the contribution of these cell lineages to cardiac development.

Generation and characterization of rgs5 mutant mice.

Regulators of G-protein signaling (RGS) are involved in a wide variety of functions, including olfaction, vision, and cell migration. RGS5 has a perivascular expression pattern and was recently identified as a marker for brain pericytes. This suggests a role for RGS5 in vascular development and pericyte biology. We have created a mouse line which lacks the rgs5 gene and replaced it with a green fluorescent protein (GFP) reporter (rgs5(GFP/GFP)). The mice are viable and fertile and display no obvious developmental defects, and the vasculature appears to develop normally with proper pericyte coverage. Also, no differences were observed in the vasculature under pathological conditions, such as tumor growth and oxygen-induced retinopathy. The GFP expression in pericytes of rgs5(GFP) mice allows detection and sorting of these cells, thereby providing a valuable novel tool for pericyte research.