Abstract Tumors commonly exhibit a leaky vasculature and innate and/or adaptive immune cell infiltration. A recent study showed that a non-leaky and normalized mouse breast cancer vasculature after conditional deletion of the Shb gene in endothelial cells (EC) displayed a suppressive immune cell profile. To extend those findings further, regulation of immunomodulatory gene expression of normalized breast cancer EC was related to healthy human breast and human breast cancer EC gene expression. A considerable correlation was observed in the gene expression profiles of immunoregulatory genes between normal breast arterial EC and normalized ( Shb -deficient) EC, and these changes did not primarily seem to involve gene products forming conduits for tumor leukocyte transendothelial migration but other aspects of immune suppression as for example cytokine expression. In addition to breast cancer, healthy lung and colonic EC exhibited an immune suppressive phenotype compared to their lung cancer and colorectal cancer counterparts. It is concluded that normalized EC may confer a non-leaky and immune suppressed phenotype, an insight that may be exploited to enhance the efficacy of immunotherapy.

Tumor-associated macrophages are indispensable in the modulation of tumor angiogenesis, and their role in normalizing tumor vasculature is attracting current interest. We have recently discovered that macrophages position themselves perivascularly in injured, ischemic muscle, and that this is important for healing of the ischemic injury as they can take on mural cell functions including vessel maturation, as well as mural cell gene expression while losing their macrophage identity. The aim of the current study is to determine the importance of the mural cell-like macrophages for tumor vasculature. A murine model of breast cancer was used, where EO771 cells are injected into the mammary gland to induce tumor growth. To enable lineage tracing of macrophages in the tumor environment, Cx3cr1CreERT2xRosa26-TdTomato mice crossed with PdgfrβeGFP reporter mice was used, resulting in TdTomato expression in CX3CR1+ macrophages following tamoxifen administration. The presence of PDGFRβ-positive macrophages in the tumor microenvironment was confirmed using flow cytometry of tumor single-cell suspensions. Immunofluorescence of tumor sections demonstrated that these macrophages position themselves perivascularly. In conclusion, mural-cell-like macrophages are present in growing murine mammary tumors, and their role in the maturation of tumor vasculature is now being explored to understand the therapeutic potential of these cells. Research Council (K2012-99x, 65X-12219-15-6, 5570x-15043, O4X-08646, K2015-54X-12219-19-4), Ragnar Söderberg foundation, Knut and Alice Wallenberg foundation, Swedish Foundation for Strategic Research, Diabetes Wellness Sweden, The Swedish Cancer Society Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

The vasculature and the immune system both play roles in breast cancer progression and metastasis. In an experimental mouse model of Shb‐gene deficiency in endothelial cells, breast cancer lung metastasis correlated with immune suppression rather than with vascular leakage. The present study aimed to assess underlying gene expression changes in endothelial and immune cells responsible for this phenotype and to explore their relationship to human disease. Mouse endothelial cell Shb‐gene deficiency, leading to ‘vessel normalization’, resulted in altered expression of chemo/cytokine genes and upregulation of immune checkpoint genes in immune cells. Endothelial cells under these conditions exhibited gene expression patterns compatible with reduced angiogenesis and vascular leakage. Additionally, genes whose products relate to immune cell vascular transmigration and function were affected. In a human triple‐negative breast cancer cohort, tumors with reduced vascular leakage exhibited a higher relative proportion of regulatory T cells and larger tumor size. However, these changes were not associated with increased metastasis. In conclusion, a low leakage vascular phenotype reduces tumor cell intravasation/metastasis and modifies the immune response, which in the current context becomes pro‐tumoral.

Supplementary Table 1 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Methods, Tables and Figures

Supplementary Figure 4 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Figure Legends 1-7 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Figure 3 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

Supplementary Figures 5-7 from Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

an amazing journey under Arne's stewardshipThe day we have dreaded for several years has finally come.Our Editor-in-Chief (EIC), Professor Arne Andersson, has decided to retire after serving that role since 2006.During those years, Upsala Journal of Medical Sciences (UJMS) experienced a remarkable transformation from a local journal with an impact factor of 0.476 to a global open access player with a 2-year impact factor of 2.384 (5-year impact factor is 3.080).Ever since it was founded in 1865, UJMS has been our precious 'little gem' (1, 2).The journal is under the auspices of the Uppsala Medical Society (Upsala Läkareförening) and has served an important role as a venue for scientific exchange within the local medical community.The journal's recent development has allowed Uppsala medical science to thrive in an increasingly global environment, and the increased relevance of our Journal is the consequence of Arne Andersson's focused stewardship.Arne Andersson is a distinguished diabetes researcher who made important contributions with particular focus on pancreatic islet transplantation.He shared the role as EIC with Gunnar Ronquist from 2006 to 2012 after which he became a sole EIC.He grasped at an early stage the ongoing evolution of medical science with its diversification and specialization, and consequently, he implemented changes in journal policies to address this.This resulted in the establishment of an editorial team of associate editors and an editorial board including international members.He accepted 493 manuscripts as EIC after scrutinous review, always with the highest's scientific standards.His personal commitment to this process includes supervision of linguistic text revision in order for manuscripts to reach impeccable standards.The extreme diversity of highquality publications recently accepted can be illustrated by publications in the fields of antibiotic resistance (3), vascular biology (4), diabetes (5-7), microRNA in tumor biology (8, 9), fertility (10), point-of-care testing (11), and clinical aspects on phage therapy against bacterial infections (12).The journal has experienced additional changes during Arne Andersson's leadership.It has become an open access online journal, initially with both electronic and printed paper versions of all articles but since 2021 only with electronic publications.That transition was a necessity due to conditions imposed by the COVID-19 pandemic, and Arne Andersson had mixed feelings about this change, always preferring the printed version for reading.

The vasculature plays a major role in regulating the tumor immune cell response although the underlying mechanisms explaining such effects remain poorly understood. This review discusses current knowledge on known vascular functions with a viewpoint on how they may yield distinct immune responses. The vasculature might directly influence selective immune cell infiltration into tumors by its cell surface expression of cell adhesion molecules, expression of cytokines, cell junction properties, focal adhesions, cytoskeleton and functional capacity. This will alter the tumor microenvironment and unleash a plethora of responses that will influence the tumor's immune status. Despite our current knowledge of numerous mechanisms operating, the field is underexplored in that few functions providing a high degree of specificity have yet been provided in relation to the enormous divergence of responses apparent in human cancers. Further exploration of this field is much warranted.

Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src deficiency, VE-cadherin internalization is maintained and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.

Metastasis reflects both the inherent properties of tumor cells and the response of the stroma to the presence of the tumor. Vascular barrier properties, either due to endothelial cell (EC) or pericyte function, play an important role in metastasis in addition to the contribution of the immune system. The Shb gene encodes the Src homology-2 domain protein B that operates downstream of tyrosine kinases in both vascular and immune cells. We have investigated E0771.lmb breast carcinoma metastasis in mice with conditional deletion of the Shb gene using the Cdh5-CreERt2 transgene, resulting in inactivation of the Shb-gene in EC and some hematopoietic cell populations. Lung metastasis from orthotopic tumors, tumor vascular and immune cell characteristics, and immune cell gene expression profiles were determined. We found no increase in vascular leakage that could explain the observed increase in metastasis upon the loss of Shb expression. Instead, Shb deficiency in EC promoted the recruitment of monocytic/macrophagic myeloid-derived suppressor cells (mMDSC), an immune cell type that confers a suppressive immune response, thus enhancing lung metastasis. An MDSC-promoting cytokine/chemokine profile was simultaneously observed in tumors grown in mice with EC-specific Shb deficiency, providing an explanation for the expanded mMDSC population. The results demonstrate an intricate interplay between tumor EC and immune cells that pivots between pro-tumoral and anti-tumoral properties, depending on relevant genetic and/or environmental factors operating in the microenvironment.

The evolvement of the newly started subsection IJMS molecular oncology is discussed. The breadth and depth of the journal articles is alluded to. A bright future for this subsection is anticipated, developing into a top tier cancer journal.

Intravasation, vascular dissemination and metastasis of malignant tumor cells require their passage through the vascular wall which is commonly composed of pericytes and endothelial cells. We currently decided to investigate the relative contribution of these cell types to B16F10 melanoma metastasis in mice using an experimental model of host Shb gene (Src homology 2 domain-containing protein B) inactivation. Conditional inactivation of Shb in endothelial cells using Cdh5-CreERt2 resulted in decreased tumor growth, reduced vascular leakage, increased hypoxia and no effect on pericyte coverage and lung metastasis. RNAseq of tumor endothelial cells from these mice revealed changes in cellular components such as adherens junctions and focal adhesions by gene ontology analysis that were in line with the observed effects on leakage and junction morphology. Conditional inactivation of Shb in pericytes using Pdgfrb-CreERt2 resulted in decreased pericyte coverage of small tumor vessels with lumen, increased leakage, aberrant platelet-derived growth factor receptor B (PDGFRB) signaling and a higher frequency of lung metastasis without concomitant effects on tumor growth or oxygenation. Flow cytometry failed to reveal immune cell alterations that could explain the metastatic phenotype in this genetic model of Shb deficiency. It is concluded that proper pericyte function plays a significant role in suppressing B16F10 lung metastasis.

Mixed lineage leukemia (MLL) arises from several KMT2A-gene chromosomal translocations. Shb gene deficiency has been found to exhibit pleiotropic effects in different models of leukemia, and consequently, this study aimed to investigate MLL-AF9-induced leukemia in Shb deficiency. Bone marrow cells from wild type and Shb knockout (KO) mice were transduced with the MLL-AF9 gene. Shb KO MLL-AF9 cells proliferated at an increased rate, exhibited altered expression of certain cytokine genes (Kitl, Csf3, IL6, IL1b) and higher expression of cell cycle genes (Ccnd2, Ccne1). Mice receiving Shb KO MLL-AF9 cells showed longer latency without displaying any difference in rates of leukemic cell proliferation, indicating a dichotomy between the in vitro and in vivo phenotypes. The mice with Shb deficient MLL-AF9 cells had a lower content of leukemic bone marrow cells allowing elevated normal hematopoiesis, explaining the longer latency. Finally, Shb knockout GFP-positive bone marrow cells showed a higher percentage of cells expressing myeloid markers. The result suggests a role of Shb in the progression of leukemia and that the relevance of the Shb gene is context-dependent as inferred from the differences between the in vivo and in vitro responses. These findings help to obtain an increased understanding of human MLL-AF9 leukemia.

Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.

The tamoxifen-responsive conditional Cdh5-CreERT2 is commonly used for endothelial cell specific conditional deletion of loxP-flanked gene sequences. To address the role of endothelial cell Shb gene for B16F10 melanoma immune responses, tamoxifen-injected Cdh5-CreERT2 /WT and Cdh5-CreERT2 / Shb flox/flox mice received subcutaneous tumor cell injections. We observed a decrease of tumor myeloid cell Shb mRNA in the tamoxifen treated Cdh5-CreERT2 / Shb flox/flox mice, which was not present when the mice had undergone a preceding bone marrow transplantation using wild type bone marrow. Differences in CD4+/FoxP3+ Tregs were similarly abolished by a preceding bone marrow transplantation. In ROSA26-mTmG mice, Cdh5-CreERT2 caused detectable floxing in certain bone marrow populations and in spleen cells. Floxing in bone marrow could be detected two months after tamoxifen treatment. In the spleen, however, floxing was undetectable two months after tamoxifen treatment, suggesting that Cdh5-CreERT2 is operating in a non-renewable population of hematopoietic cells in this organ. These data suggest that conditional gene deletion in hematopoietic cells is a potential confounder in experiments attempting to assess the role of endothelial specific effects. A cautious approach to achieve an endothelial-specific phenotype would be to adopt a strategy that includes a preceding bone marrow transplantation.

To assess mechanisms responsible for breast carcinoma metastasis, 4T1 breast carcinomas were grown orthotopically in wild type or Shb knockout mice. Tumor growth, metastasis, vascular characteristics and immune cell properties were analyzed. Absence of Shb did not affect tumor growth although it increased lung metastasis. Shb knockout mouse tumors showed decreased redness and less developed vascular plexa located at the periphery of the tumors. No difference in overall tumor vascular density, leakage or pericyte coverage was noted between the genotypes although the average vessel size was smaller in the knockout. Tumors induced an increase of CD11b+ cells in spleen, lymph node, thymus, bone marrow and blood. Numbers of Shb knockout CD11b/CD8+ cells were decreased in lymph nodes and bone marrow of tumor bearing mice. Mice with tumors had reduced numbers of CD4+ lymphocytes in blood/lymphoid organs, whereas in most of these locations the proportion of CD4+ cells co-expressing FoxP3 was increased, suggesting a relative increase in Treg cells. This finding was reinforced by increased blood interleukin-35 (IL-35) in wild type tumor bearing mice. Shb knockout blood showed in addition an increased proportion of IL-35 expressing Treg cells, supporting the notion that absence of Shb further promotes tumor evasion from immune cell recognition. This could explain the increased number of lung metastases observed under these conditions. In conclusion, 4T1 tumors alter immune cell responses that promote tumor expansion, metastasis and escape from T cell recognition in an Shb dependent manner.

Abstract The plasma–protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and antitumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T-cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction–decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and colocalization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG–treated gliomas displayed decreased numbers of IL35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control antitumor immunity. Mol Cancer Ther; 17(9); 1961–72. ©2018 AACR.