Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM). Here, we show that angiogenesis also can be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and endothelial cells (ECs). As shown in vitro, this direct interaction is mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. It promotes both EC network formation and enhances directed migration toward basic fibroblast growth factor. Activation of αvβ3 and bone marrow tyrosine kinase on chromosome X (BMX) is required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells and CSCs in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting

WJMSC-Derived Small Extracellular Vesicle Enhance T Cell Suppression Through PD-L1

© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles Both mesenchymal stem cells (MSCs) and their corresponding small extracellular vesicles (sEVs, commonly referred to as exosomes) share similar immunomodulatory properties that are potentially beneficial for the treatment of acute graft versus host disease (aGvHD). We report that clinical grade Wharton\u27s Jelly-derived MSCs (WJMSCs) secrete sEVs enriched in programmed death-ligand 1 (PD-L1), an essential ligand for an inhibitory immune checkpoint. A rapid increase in circulating sEV-associated PD-L1 was observed in patients with aGvHD and was directly associated with the infusion time of clinical grade WJMSCs. In addition, in vitro inhibitory antibody mediated blocking of sEV-associated PD-L1 restored T cell activation (TCA), suggesting a functional inhibitory role of sEVs-PD-L1. PD-L1-deficient sEVs isolated from WJMSCs following CRISPR-Cas9 gene editing fail to inhibit TCA. Furthermore, we found that PD-L1 is essential for WJMSC-derived sEVs to modulate T cell receptors (TCRs). Our study reveals an important mechanism by which therapeutic WJMSCs modulate TCR-mediated TCA through sEVs or sEV-carried immune checkpoints. In addition, our clinical data suggest that sEV-associated PD-L1 may be not only useful in predicting the outcomes from WJMSC clinical administration, but also in developing cell-independent therapy for aGvHD patients

Direct In Vivo Evidence for Tumor Propagation by Glioblastoma Cancer Stem Cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC

All-Trans Retinoic Acid-Induced Deficiency of the Wnt/β-Catenin Pathway Enhances Hepatic Carcinoma Stem Cell Differentiation.

Retinoic acid (RA) is an important biological signal that directly differentiates cells during embryonic development and tumorigenesis. However, the molecular mechanism of RA-mediated differentiation in hepatic cancer stem cells (hCSCs) is not well understood. In this study, we found that mRNA expressions of RA-biosynthesis-related dehydrogenases were highly expressed in hepatocellular carcinoma. All-trans retinoic acid (ATRA) differentiated hCSCs through inhibiting the function of β-catenin in vitro. ATRA also inhibited the function of PI3K-AKT and enhanced GSK-3β-dependent degradation of phosphorylated β-catenin. Furthermore, ATRA and β-catenin silencing both increased hCSC sensitivity to docetaxel treatment. Our results suggest that targeting β-catenin will provide extra benefits for ATRA-mediated treatment of hepatic cancer patients

Context dependent role of the CD36--thrombospondin--histidine-rich glycoprotein axis in tumor angiogenesis and growth

<div><p>The angiogenic switch is a promising therapeutic target in cancer. Work by our laboratory and others has described an important endogenous anti-angiogenic pathway mediated by interactions of CD36, a receptor on microvascular endothelial cells, with proteins containing thrombospondin (TSP) type I repeat domains (TSR). Recent studies revealed that circulating Histidine Rich Glycoprotein (HRG) inhibits the anti-angiogenic potential of the CD36-TSR pathway by functioning as a decoy receptor that binds and sequesters TSR proteins. As tumors of different origin display variable expression profiles of numerous targets, we hypothesized that the TSP-CD36-HRG axis regulates vascularization and growth in the tumor microenvironment in a context, or tumor type, dependent manner. Growth of Lewis Lung Carcinoma (LL2) and B16F1 Melanoma tumor cell implants in syngeneic wild type (WT), <em>hrg</em>, or <em>cd36</em> null mice were used as a model to interrogate this signaling axis. LL2 tumor volumes were greater in <em>cd36</em> null mice and smaller in <em>hrg</em> null mice compared to WT. Immunofluorescent staining showed increased vascularity in <em>cd36</em> null vs. WT and WT vs. <em>hrg</em> null mice. No differences in tumor growth or vascularity were observed with B16F1 implants, consistent with lack of expression of TSP-1 in B16F1 cells. When TSR expression was induced in B16F1 cells by cDNA transfection, tumor growth and vascularity were similar to that seen with LL2 cells. These data show a role for CD36-mediated anti-angiogenic activity in the tumor microenvironment when TSR proteins are available and demonstrate that HRG modulates this activity. Further, they suggest a mechanism by which tumor microenvironments may regulate sensitivity to TSR containing proteins.</p> </div

ATRA enhanced differentiation of CD133⁺ hCSCs by down-regulating the Wnt/β-catenin signaling pathway.

<p>(<b>A</b>) ATRA treatment (concentrations from 10⁻⁹ M to 10⁻⁵ M) decreased the percentage of CD133-expressing HepG2 cells at day 5 <i>in vitro</i>. (<b>B</b>) Expression of the CD133 protein decreased in CD133⁺ hCSCs after treatment with 1 × 10⁻⁵ M, 5 × 10⁻⁵ M and 10 × 10⁻⁵ M ATRA for 12 or 48 hours. <i>Control</i>, normal cell culture. (<b>C</b>) ATRA treatment down-regulated the protein expression level of other stem cell markers SOX2, NANOG and OCT4 after 5 days ATRA exposure (concentrations from 10⁻⁹ M to 10⁻⁵ M). (<b>D</b>) ATRA treatment decreased the protein expression level of β-catenin and increased its phosphorylation after 5 days ATRA exposure (concentrations from 10⁻⁹ M to 10⁻⁵ M). (<b>E</b>) ATRA inactivated the PI3K-AKT signaling pathway in CD133⁺ hCSCs after 5 days ATRA exposure (concentrations from 10⁻⁹ M to 10⁻⁵ M). (<b>F-G</b>) Silencing β-catenin mRNA decreased both the mRNA (<b>F</b>) and protein (<b>G</b>) expression of stem cell markers SOX2, NANOG and OCT4. <i>Control</i>, normal cell culture; <i>PLKO</i>.<i>1</i>, cells treated with empty vector PLKO.1; <i>β-catenin shRNA</i>, cells treated with the PLKO.1-β-catenin-shRNA constructed vector. ***p < 0.001; **p < 0.01.</p

ATRA-induced differentiation of CD133⁺ hCSCs increased their sensitivity to docetaxel (DOC) treatment <i>in vitro</i>.

<p>(<b>A-B</b>) Survival and proliferation of CD133⁺ hCSCs after treatment with three concentrations (10⁻⁵ M, 10⁻⁶ M and 10⁻⁷ M) of ATRA. (<b>C-D</b>) Survival and proliferation of CD133⁺ hCSCs after treatment with three different concentrations (10⁻¹⁰ M, 10⁻⁹ M and 10⁻⁸ M) of DOC. (<b>E-F</b>) Combined treatment with ATRA (10⁻⁵ M, 10⁻⁶ M and 0⁻⁷ M) and DOC (10⁻⁸ M) decreased the growth of CD133⁺ hCSCs. (<b>A, B, C</b>) Growth curves of CD133⁺ hCSCs; (<b>D, E, F</b>) Quantitative analyses of CD133⁺ hCSC growth at day 5. ***p < 0.001; **p < 0.01. (<b>G-H</b>) ATRA treatment decreased the survival and proliferation of β-catenin mRNA knockdown-CD133⁺ hCSCs. (<b>I-J</b>) Knockdown of β-catenin mRNA decreased resistance of CD133⁺ hCSCs to DOC treatment (10⁻⁸ M). Concentration of ATRA in <b>G-J</b> is 10⁻⁶ M. (<b>G, I</b>) Growth curves of CD133⁺ hCSCs; (<b>H, J</b>) Quantitative analyses of CD133⁺ hCSC growth at day 3, day 4 and day 5. ***p < 0.001; **p < 0.01. (<b>K</b>) Molecular mechanism of ATRA-mediated differentiation of CD133⁺ hCSCs. Wnt/β-catenin was required to maintain the stemness of hCSCs. Binding of ATRA to RARs induced inactivation of the PI3K-AKT pathway, enhancing GSK-3β-dependent phosphorylation of β-catenin.</p