A Comparative Study of Cell Culture Conditions during Conversion from Primed to Naive Human Pluripotent Stem Cells.

The successful reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) represented a turning point in the stem cell research field, owing to their ability to differentiate into any cell type with fewer ethical issues than human embryonic stem cells (hESCs). In mice, PSCs are thought to exist in a naive state, the cell culture equivalent of the immature pre-implantation embryo, whereas in humans, PSCs are in a primed state, which is a more committed pluripotent state than a naive state. Recent studies have focused on capturing a similar cell stage in human cells. Given their earlier developmental stage and therefore lack of cell-of-origin epigenetic memory, these cells would be better candidates for further re-differentiation, use in disease modeling, regenerative medicine and drug discovery. In this study, we used primed hiPSCs and hESCs to evaluate the successful establishment and maintenance of a naive cell stage using three different naive-conversion media, both in the feeder and feeder-free cells conditions. In addition, we compared the directed differentiation capacity of primed and naive cells into the three germ layers and characterized these different cell stages with commonly used pluripotent and lineage-specific markers. Our results show that, in general, naive culture NHSM medium (in both feeder and feeder-free systems) confers greater hiPSCs and hESCs viability and the highest naive pluripotency markers expression. This medium also allows better cell differentiation cells toward endoderm and mesoderm.

Discoidin Domain Receptor 2 Expression as Worse Prognostic Marker in Invasive Breast Cancer.

Discoidin domain receptor 2 (DDR2) is arising as a promising therapeutic target in breast carcinoma (BC). The ability of DDR2 to bind to collagen promotes protumoral responses in cancer cells that influence the tumor microenvironment (TME). Nonetheless, the interrelation between DDR2 expression and TME modulation during BC progression remains poorly known. For this reason, we aim to evaluate the correlation between intratumoral expression of DDR2 and the infiltration of the main TME cell populations, cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs). First, collagen and DDR2 expression levels were analyzed in human invasive BC samples. Then, DDR2 status correlation with tumor aggressiveness and patient survival were retrieved from different databases. Subsequently, the main pathways, cell types, and tissues correlated with DDR2 expression in BC were obtained through bioinformatics approach. Finally, we studied the association of DDR2 expression with the recruitment of CAFs and TAMs. Our findings showed that, together with the expected overexpression of TME markers, DDR2 was upregulated in tumor samples. Besides, we uncovered that altered TME markers were linked to DDR2 expression in invasive BC patients. Consequently, DDR2 modulates the stromal reaction through CAFs and TAMs infiltration and could be used as a potential worse prognostic factor in the treatment response of invasive BC.

Tumor DDR1 deficiency reduces liver metastasis by colon carcinoma and impairs stromal reaction.

Tumor DDR1 acts as a key factor during the desmoplastic response surrounding hepatic colorectal metastasis. Hepatic sinusoidal cell-derived soluble factors stimulate tumor DDR1 activation. DDR1 modulates matrix remodeling to promote metastasis in the liver through the interaction with hepatic stromal cells, specifically liver sinusoidal endothelial cells and hepatic stellate cells.

Inhibition of COX-2 Impairs Colon Cancer Liver Metastasis through Reduced Stromal Cell Reaction.

Liver colonization is initiated through the interplay between tumor cells and adhesion molecules present in liver sinusoidal endothelial cells (LSECs). This crosstalk stimulates tumor COX-2 upregulation and PGE2 secretion. To elucidate the role of the LSEC intercellular adhesion molecule-1 (ICAM-1) in the prometastatic response exerted by tumor and stromal COX-2, we utilized celecoxib (CLX) as a COX-2 inhibitory agent. We analyzed the in vitro proliferative and secretory responses of murine C26 colorectal cancer (CRC) cells to soluble ICAM-1 (sICAM-1), cultured alone or with LSECs, and their effect on LSEC and hepatic stellate cell (HSC) migration and in vivo liver metastasis. CLX reduced sICAM-1-stimulated COX-2 activation and PGE2 secretion in C26 cells cultured alone or cocultured with LSECs. Moreover, CLX abrogated sICAM-1-induced C26 cell proliferation and C26 secretion of promigratory factors for LSECs and HSCs. Interestingly, CLX reduced the protumoral response of HSC, reducing their migratory potential when stimulated with C26 secretomes and impairing their secretion of chemotactic factors for LSECs and C26 cells and proliferative factors for C26 cells. In vivo, CLX abrogated the prometastatic ability of sICAM-1-activated C26 cells while reducing liver metastasis. COX-2 inhibition blocked the creation of a favorable tumor microenvironment (TME) by hindering the intratumoral recruitment of activated HSCs and macrophages in addition to the accumulation of fibrillar collagen. These results point to COX-2 being a key modulator of processes initiated by host ICAM-1 during tumor cell/LSEC/HSC crosstalk, leading to the creation of a prometastatic TME in the liver.

Silencing of sinusoidal DDR1 reduces murine liver metastasis by colon carcinoma.

Liver metastasis depends on the collagenous microenvironment generated by hepatic sinusoidal cells (SCs). DDR1 is an atypical collagen receptor linked to tumor progression, but whether SCs express DDR1 and its implication in liver metastasis remain unknown. Freshly isolated hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs), that conform the SCs, expressed functional DDR1. HSCs expressed the largest amounts. C26 colon carcinoma secretomes increased DDR1 phosphorylation in HSCs and KCs by collagen I. Inhibition of kinase activity by DDR1-IN-1 or mRNA silencing of DDR1 reduced HSCs secretion of MMP2/9 and chemoattractant and proliferative factors for LSECs and C26 cells. DDR1-IN-1 did not modify MMP2/9 in KCs or LSECs secretomes, but decreased the enhancement of C26 migration and proliferation induced by their secretomes. Gene array showed that DDR1 silencing downregulated HSCs genes for collagens, MMPs, interleukins and chemokines. Silencing of DDR1 before tumor inoculation reduced hepatic C26 metastasis in mice. Silenced livers bore less tumor foci than controls. Metastatic foci in DDR1 silenced mice were smaller and contained an altered stroma with fewer SCs, proliferating cells, collagen and MMPs than foci in control mice. In conclusion, hepatic DDR1 promotes C26 liver metastasis and favors the pro-metastatic response of SCs to the tumor.

Inhibition of DDR1 reduces invasive features of human A375 melanoma, HT29 colon carcinoma and SK-HEP hepatoma cells.

DDR1 is a receptor tyrosine kinases for collagen and an adverse prognostic factor in primary and metastatic tumors.Despite this, DDR1 signaling and its functional consequences in tumor development remain unclear. RT-PCR and Western blot show that A375, colon carcinoma HT29 and liver carcinoma SK-HEP human cell lines express functional DDR1 that phosphorylates in response to collagen type I. Chemical inhibition of DDR1 phosphorylation or DDR1 mRNA silencing reduced AKT and ERK phosphorylation, expression of ICAM1 and VCAM1, Ki67 and secretion of MMP9. DDR1 silenced cells showed reduced adhesion to collagen type I, MMP-dependent invasion, and chemotactic and proliferative responses to collagen type I. Our work indicates an essential role for DDR1 signaling in key prometastatic features of collagen type I in human carcinoma cells.

Liver sinusoidal endothelial cell ICAM-1 mediated tumor/endothelial crosstalk drives the development of liver metastasis by initiating inflammatory and angiogenic responses.

The prometastatic stroma generated through tumor cells/host cells interaction is critical for metastatic growth. To elucidate the role of ICAM-1 on the crosstalk between tumor and primary liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), implicated in tumor adhesion and angiogenesis, we performed in vitro cocultures and an in vivo model of liver metastasis of colorectal cancer (CRC). ICAM-1 blockade in the LSECs decreased the adhesion and transmigration of tumor cells through an LSEC in vitro and vivo. Cocultures of C26 cells and LSECs contained higher amounts of IL-1ß, IL-6, PGE-2, TNF-α and ICAM-1 than monocultures. C26 cells incubated with sICAM-1 secreted higher amounts of PGE-2, IL-6, VEGF, and MMPs, while enhanced the migration of LSECs and HSCs. HSCs cultures activated by media from C26 cells pretreated with sICAM-1 contained the largest amounts of VEGF and MMPs. C26 cell activation with sICAM-1 enhanced their metastasizing potential in vivo, while tumor LFA-1 blockade reduced tumor burden and LSECs and HSC-derived myofibroblasts recruitment. In vivo ICAM-1 silencing produced similar results. These findings uncover LSEC ICAM-1 as a mediator of the CRC metastatic cascade in the liver and identifies it as target for the inhibition of liver colonization and metastatic progression.

Ocoxin Oral Solution Exerts an Antitumoral Effect in Pancreatic Cancer and Reduces the Stromal-Mediated Chemoresistance.

OBJECTIVES: Pancreatic carcinoma is one of the most aggressive cancers overcoming chemoresistance. Thus, novel compounds to complement the current antitumor agents are in need. Ocoxin oral solution (OOS) has proven antioxidant, anti-inflammatory, and antistromagenic properties. The aim of this study was to analyze the effect of OOS in an experimental pancreatic cancer model and its implication in stroma-related chemoresistance to paclitaxel and gemcitabine. METHODS: Murine pancreatic carcinoma 266-6 cells were treated with OOS to analyze cell cycle and to perform a mRNA comparative microarray study. Then the viability was assessed in combination with paclitaxel and/or gemcitabine. Chemoresistance induced by the medium taken from fibroblast cultures was also investigated on 6 human pancreatic carcinoma cell lines. Furthermore, an experimental model of pancreatic cancer was carried out to study the effect of OOS in vivo. RESULTS: Ocoxin oral solution enhances the cytotoxic effect of paclitaxel and gemcitabine, while it ameliorates the chemoresistance induced by fibroblast-derived soluble factors in human pancreatic cancer cells. The OOS also promotes the regulation of the expression of genes that are altered in pancreatic carcinoma and slows down 266-6 cell pancreatic tumor development in vivo. CONCLUSIONS: Ocoxin oral solution could be a potential complement to the chemotherapeutic drugs for pancreatic adenocarcinoma.

CXCR4 receptor blockage reduces the contribution of tumor and stromal cells to the metastatic growth in the liver.

The liver is a common site for the metastatic spread of primary malignancies including colorectal cancer, and liver metastasis is a main cause of death in cancer patients. This is due to the complexity of the interactions taking place in the liver between tumor and stromal cells. In fact, cancer­associated fibroblasts (CAFs) have been shown to support tumor growth through the CXCL12/CXCR4 axis. However, along with cancer cells, myeloid­derived suppressor cells (MDSCs), immature dendritic cells with immunosuppressive potential, also express CXCR4. It has recently been demonstrated that reducing CXCL12 availability in the tumor microenvironment decreases liver metastasis. Therefore, blocking CXCL12 chemokine receptor CXCR4 may be a successful approach to diminish the metastatic spread of colorectal cancer to the liver. However, the subjacent mechanisms by which this chemokine influences the tumor are not fully understood. Thus, in order to uncover the role of CXCR4 during tumor cell/liver fibroblast crosstalk driving liver metastasis, the CXCR4 antagonist AMD3100 was used for in vitro studies and in an in vivo approach using an orthotopic model of liver metastasis in immune competent mice through intrasplenic injection of grafted C26 cells. In vitro blockage of CXCR4 led to an impaired migratory potential of tumor and hepatic stellate cells (HSCs) and a reduced tumor response to CXCL12. In vivo administration of AMD3100 to tumor­bearing mice resulted in attenuated metastatic development in the liver, which was accompanied by an impaired infiltration of αSMA­expressing cells within the tumors. In addition, a reduced CD11+Ly6G+ cell count in the liver was directly correlated with a reduction in MDSC numbers in the blood of AMD3100­treated mice compared to the vehicle­treated mice. Therefore, disruption of the CXCR4/CXCL12 axis by CXCR4 antagonist AMD3100 blocked the contribution of both cancer and stromal cells to the metastatic cascade in the liver.

Role of liver ICAM-1 in metastasis.

Intercellular adhesion molecule (ICAM)-1, is a transmembrane glycoprotein of the immunoglobulin (Ig)-like superfamily, consisting of five extracellular Ig-like domains, a transmembrane domain and a short cytoplasmic tail. ICAM-1 is expressed in various cell types, including endothelial cells and leukocytes, and is involved in several physiological processes. Furthermore, it has additionally been reported to be expressed in various cancer cells, including melanoma, colorectal cancer and lymphoma. The majority of studies to date have focused on the expression of the ICAM-1 on the surface of tumor cells, without research into ICAM-1 expression at sites of metastasis. Cancer cells frequently metastasize to the liver, due to its unique physiology and specialized liver sinusoid capillary network. Liver sinusoidal endothelial cells constitutively express ICAM-1, which is upregulated under inflammatory conditions. Furthermore, liver ICAM-1 may be important during the development of liver metastasis. Therefore, it is necessary to improve the understanding of the mechanisms mediated by this adhesion molecule in order to develop host-directed anticancer therapies.