Mechanically defined microenvironment promotes stabilization of microvasculature, which correlates with the enrichment of a novel Piezo-1+ population of circulating CD11b+/CD115+ monocytes

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long-term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long-term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b+ myeloid and CD11b+ /CD115+ monocytes that express the stretch activated cation channel Piezo-1, which is enriched prominently in the clot-like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment

EphrinB2/EphB4 signaling regulates non-sprouting angiogenesis by VEGF

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best-understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine-tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF-R2 activation by VEGF nor its internalization, but it modulates VEGF-R2 downstream signaling through phospho-ERK1/2.; In vivo; inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF-induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential

TIA-1 Cytotoxic Granule-Associated RNA Binding Protein Improves the Prognostic Performance of CD8 in Mismatch Repair-Proficient Colorectal Cancer

Evidence suggests a confounding effect of mismatch repair (MMR) status on immune response in colorectal cancer. The identification of innate and adaptive immune cells, that can complement the established prognostic effect of CD8 in MMR-proficient colorectal cancers patients, representing 85% of all cases, has not been performed

Functional validation of cancer stem cell markers in primary human colorectal cancer and established cell lines

In an increasing number of cancers, CSCs have been defined on the basis of the self-renewal and tumor initiation capacity by functional assays. It has been also suggested that CSC populations might be responsible for chemo- and radio-therapy resistance within tumors and ultimately for the post-therapeutic tumor recurrence. The identification of markers identifying CSC is fundamental for the validation of the CSC paradigm and for the development of new CSC-specific drugs and novel therapeutic approach. CD133, CD44 and CD166 have been proposed as putative CSC markers in CRC. These findings have opened the field for an extensive validation of the markers and their use for the development of specific anti-CSC therapy. In this work, I addressed a) the prognostic relevance of the expression of CSC surface markers in CRC clinical specimens, b) the “in vivo” tumorigenicity of primary CRC derived cells, as related to their expression of putative CSC surface markers, c) the possibility of using cells derived from established CRC cell lines expressing CSC surface markers as CSC cellular model, and finally d) the development of innovative culture models of potential relevance for the screening of anti CRC compounds. I have analyzed the surface markers expression in correlation with stem cell-like functional features, but no consistent results was found confirming stemness property associated with expression of those markers. These results obviously question the validity of putative surface CRC-SC markers. Taken together these data might suggest that their expression and CSC functional features might be associated with some degree of plasticity, potentially related to tumor microenvironmental characteristics being lost in conventionally cultured tumor cell lines and in primary tumor derived cell suspensions. Based on this background I have investigated the possibility to perform 3D culture of CRC cell lines to assess whether these systems might provide useful insights for the interpretation of our data. My findings clearly document the plasticity of gene expression profiles of cultured CRC cells depending on their three-dimensional architectures. Most importantly, I demonstrate that major gene expression modulation events only occur when culture in 3D spheroids is associated with ischemia and necrosis. I also showed that the gene expression of putative CSC markers increases with the occurrence of ischemia and necrosis in the core of a 3D spheroid

Engineering of an angiogenic niche by perfusion culture of adipose-derived stromal vascular fraction cells

Abstract In vitro recapitulation of an organotypic stromal environment, enabling efficient angiogenesis, is crucial to investigate and possibly improve vascularization in regenerative medicine. Our study aims at engineering the complexity of a vascular milieu including multiple cell-types, a stromal extracellular matrix (ECM), and molecular signals. For this purpose, the human adipose stromal vascular fraction (SVF), composed of a heterogeneous mix of pericytes, endothelial/stromal progenitor cells, was cultured under direct perfusion flow on three-dimensional (3D) collagen scaffolds. Perfusion culture of SVF-cells reproducibly promoted in vitro the early formation of a capillary-like network, embedded within an ECM backbone, and the release of numerous pro-angiogenic factors. Compared to static cultures, perfusion-based engineered constructs were more rapidly vascularized and supported a superior survival of delivered cells upon in vivo ectopic implantation. This was likely mediated by pericytes, whose number was significantly higher (4.5-fold) under perfusion and whose targeted depletion resulted in lower efficiency of vascularization, with an increased host foreign body reaction. 3D-perfusion culture of SVF-cells leads to the engineering of a specialized milieu, here defined as an angiogenic niche. This system could serve as a model to investigate multi-cellular interactions in angiogenesis, and as a module supporting increased grafted cell survival in regenerative medicine

CD133+, CD166+CD44+, and CD24+CD44+ phenotypes fail to reliably identify cell populations with cancer stem cell functional features in established human colorectal cancer cell lines

Increasing evidence that cancers originate from small populations of so-called cancer stem cells (CSCs), capable of surviving conventional chemotherapies and regenerating the original tumor, urges the development of novel CSC-targeted treatments. Screening of new anticancer compounds is conventionally conducted on established tumor cell lines, providing sufficient material for high-throughput studies. Whether tumor cell lines might comprise CSC populations resembling those of primary tumors, however, remains highly debated. We have analyzed the expression of defined phenotypic profiles, including CD133+, CD166+CD44+, and CD24+CD44+, reported as CSC-specific in human primary colorectal cancer (CRC), on a panel of 10 established CRC cell lines and evaluated their correlation with CSC properties. None of the putative CSC phenotypes consistently correlated with stem cell-like features, including spheroid formation ability, clonogenicity, aldehyde dehydrogenase-1 activity, and side population phenotype. Importantly, CRC cells expressing putative CSC markers did not exhibit increased survival when treated with chemotherapeutic drugs in vitro or display higher tumorigenicity in vivo. Thus, the expression of CD133 or the coexpression of CD166/CD44 or CD24/CD44 did not appear to reliably identify CSC populations in established CRC cell lines. Our findings question the suitability of cell lines for the screening of CSC-specific therapies and underline the urgency of developing novel platforms for anticancer drug discovery

Anticancer activity of paclitaxel-loaded keratin nanoparticles in two-dimensional and perfused three-dimensional breast cancer models

Purpose: Taxanes are highly effective cytotoxic drugs for progressing breast cancer treatment. However, their poor solubility and high toxicity urge the development of innovative formulations of potential clinical relevance. Materials and methods: By using a simple and straightforward aggregation method, we have generated paclitaxel (PTX) loaded in keratin nanoparticles (KER-NPs-PTX). Their activities were tested against human breast cancer MCF-7 and MDA MB 231 cell lines in conventional two-dimensional (2D) cultures and in a dynamic three-dimensional (3D) model with perfused bioreactor (p3D). Moreover, KER-NPs-PTX activity was compared to free PTX and to PTX loaded in albumin nanoparticles (HSA-NPs-PTX). Cell viability, induction of apoptosis, and gene expression analysis were used as readouts. Results: In 2D cultures, KER-NPs-PTX was able to inhibit tumor cell viability and to induce apoptosis similarly to PTX and HSA-NPs-PTX. In the p3D model, a lower sensitivity of tumor cells to treatments was observed. Importantly, only KER-NPs-PTX was able to induce a statistically significant increase in apoptotic cell percentages following 24 h treatment for MCF-7 (16.7±4.0 early and 11.3±4.9 late apoptotic cells) and 48 h treatment for MDA MB 231 (21.3±11.2 early and 10.5±1.8 late apoptotic cells) cells. These effects were supported, at least for MCF-7 cells, by significant increases in the expression of proapoptotic BAX gene (5.8±0.5) 24 h after treatment and of cleaved caspase 3 (CC3) protein. Conclusion: KER-NPs-PTX, generated by a simple procedure, is characterized by high water solubility and enhanced PTX-loading ability, as compared to HSA-NPs-PTX. Most importantly, it appears to be able to exert effective anticancer activities on breast cancer cells cultured in 2D or in p3D models

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features

Abstract Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system

The hyaluronan-mediated motility receptor RHAMM promotes growth, invasiveness and dissemination of colorectal cancer.

In colorectal cancer (CRC), RHAMM is an independent adverse prognostic factor. The aim of the study was therefore to investigate on the role of RHAMM as a potential direct driver of cell proliferation and migration in CRC cell lines and to identify pathways dependent on RHAMM in human CRC. Proliferation, cell cycle alterations and invasive capacity were tested in two RHAMM- and control- knockdown CRC cell lines by flow cytometry and in vitro assays. Tumorigenicity and metastasis formation was assessed in immunodeficient mice. RNA-Seq and immunohistochemistry was performed on six RHAMM+/- primary CRC tumors. In vitro, silencing of RHAMM inhibited CRC cell migration and invasion by 50% (p<0.01). In vivo, RHAMM knockdown resulted in slower growth, lower tumor size (p<0.001) and inhibition of metastasis (p<0.001). Patients with RHAMM-high CRC had a worse prognosis (p=0.040) and upregulated pathways for cell cycle progression and adhesion turnover. RHAMM overexpression is correlated with increased migration and invasion of CRC cells, leads to larger, fast growing tumors, and its downregulation essentially abolishes metastasis in mouse models. RHAMM is therefore a promising therapeutic target in all CRC stages as its inhibition affects growth and dissemination of the primary CRC as well as the metastases

TIA-1 expression on TILs.

<p>Single cell suspensions obtained from freshly excised tumor specimens (n = 10) were surface stained with FITC- or APC-labeled antibodies specific for the indicated surface markers and intracellularly stained with PE-labeled anti-TIA-1 antibodies. A) Percentages of total TIA-1+ cells B) Percentages of CD8+ cells, TCRγδ cells, neutrophils (identified as CD66b+ cells), CD4+ cells, macrophages (identified as CD16+ CD56- cells), NK cells (CD16+ CD56+) and NKT cells (Vα24-Jα18 TCR+) observed within gated TIA-1+ cells. C) Representative dot-plot illustrating co-expression of TIA-1 and CD8 molecules within gated lymphocytes on total cells obtained from a clinical specimen. D) Percentages of TIA-1+ and TIA-1- cells within gated CD8+ cells.</p