Interaction of Glia Cells with Glioblastoma and Melanoma Cells under the Influence of Phytocannabinoids

Brain tumor heterogeneity and progression are subject to complex interactions between tumor cells and their microenvironment. Glioblastoma and brain metastasis can contain 30–40% of tumor-associated macrophages, microglia, and astrocytes, affecting migration, proliferation, and apoptosis. Here, we analyzed interactions between glial cells and LN229 glioblastoma or A375 melanoma cells in the context of motility and cell–cell interactions in a 3D model. Furthermore, the effects of phytocannabinoids, cannabidiol (CBD), tetrahydrocannabidiol (THC), or their co-application were analyzed. Co-culture of tumor cells with glial cells had little effect on 3D spheroid formation, while treatment with cannabinoids led to significantly larger spheroids. The addition of astrocytes blocked cannabinoid-induced effects. None of the interventions affected cell death. Furthermore, glial cell-conditioned media led to a significant slowdown in collective, but not single-cell migration speed. Taken together, glial cells in glioblastoma and brain metastasis micromilieu impact the tumor spheroid formation, cell spreading, and motility. Since the size of spheroid remained unaffected in glial cell tumor co-cultures, phytocannabinoids increased the size of spheroids without any effects on migration. This aspect might be of relevance since phytocannabinoids are frequently used in tumor therapy for side effects.

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.

Inhibitory Effects of LOXL2 Knockdown On Cellular Functions of Liver Cancer Stem Cells

Background and aim: Lysyl oxidase-like 2 (LOXL2) plays a role in tumor microenvironment formation and metastasis of hepatocellular carcinoma (HCC), which has a high mortality burden. Liver cancer stem cells (LCSCs) are related with the major malignant phenotypes of HCC. The function of LOXL2 in regulation of LCSCs remains unknown.Methods: CD133+HepG2 and CD133+Hep3B cells were sorted by fluorescence-activated cell sorting (FACS) from two human hepatoblastoma cell lines. Spheroid formation, apoptosis, cell cycle, as well as transwell assays were performed upon LOXL2 knock down in CD133+HepG2 and CD133+Hep3B cells. Protein and mRNA levels were quantified by Western blotting, Immunofluorescence and real-time PCR. Results: Knockdown of LOXL2 decreased spheroid formation, migration and invasion (p < 0.05), also induced apoptosis (p < 0.05) and cell cycle arrest (p < 0.05) in CD133+HepG2 and CD133+Hep3B cells. Knockdown of LOXL2 effectively inhibited expression of the anti-apoptosis proteins baculoviral IAP repeat-containing 3 (BIRC3) and murine double minute 2 (MDM2) (p < 0.01), as well as autophagy marker microtubule-associated protein 1 light chain 3 B (LC3) and autophagy gene ATG5 in CD133+HepG2 and CD133+Hep3B cells (p < 0.01). Conclusions: The results revealed that LOXL2 inhibition could reduce the proliferation and expansion of LCSCs, making LOXL2 inhibitors an attractive and novel therapeutic strategy of HCC.

pERK-mediated IL8 secretion can enhance the migration, invasion, and cisplatin resistance of CD10-positive oral cancer cells

Background Cancer stem cells (CSCs) drive tumor initiation and progression and participate in tumor chemoresistance. We recently discovered that oral squamous cell carcinoma (OSCC) cells that highly express CD10 (CD10H cells) present cancer stem cells (CSC)-associated characteristics, which, in turn, affect the tumor growth, epithelial-mesenchymal transition (EMT), and resistance to cisplatin. In this study, we further investigated this mechanism in vitro and in vivo. We hypothesized that IL8 might regulate migration, invasion, and cisplatin resistance of CD10-positive oral cancer cells through the ERK pathway. Methods CD10 MicroBead Kit was used to select HN6 cells with high and low expression of CD10. The target protein IL8 was screened via protein chip assay. Lentiviral transduction and specific inhibitor were applied to investigate the signaling pathway. Real-time PCR, Western blot, and immunohistochemistry were used to analyze the mRNA and protein expression; transwell assay, spheroid formation assay, and cell viability assay were used to study the cell biological behavior in vitro; xenograft animal model was used to evaluate the tumor formation rate in vivo. Results Overexpression of CD10 promoted CSC-related genes expression and enhanced migration, invasion, spheroid formation, and chemoresistance in HN6 cells. Moreover, the overexpression of IL8 was detected in OSCC tumor tissue and cell lines (HN6 and CAL27) overexpressing CD10. IL8 secreted by CD10H HN6 promoted migration and invasion and restored tumor chemosensitivity via the p-ERK signaling pathway, while the inhibition of IL8 increased the chemosensitivity to cisplatin. Conclusions IL8 secretion by CD10 positive cells promotes migration, invasion, and cisplatin resistance of OSCC via the p-ERK signaling pathway.

Complex Tumor Spheroid Formation and One-Step Cancer-Associated Fibroblasts Purification from Hepatocellular Carcinoma Tissue Promoted by Inorganic Surface Topography

In vitro cell models play important roles as testbeds for toxicity studies, drug development, or as replacements in animal experiments. In particular, complex tumor models such as hepatocellular carcinoma (HCC) are needed to predict drug efficacy and facilitate translation into clinical practice. In this work, topographical features of amorphous silicon dioxide (SiO2) are fabricated and tested for cell culture of primary HCC cells and cell lines. The topographies vary from pyramids to octahedrons to structures named fractals, with increased hierarchy and organized in periodic arrays (square or Hexagonal). The pyramids were found to promote complex 2D/3D tissue formation from primary HCC cells. It was found that the 2D layer was mainly composed of cancer-associated fibroblasts (CAFs), while the 3D spheroids were composed of tumor cells enwrapped by a CAF layer. Compared with conventional protocols for 3D cultures, this novel approach mimics the 2D/3D complexity of the original tumor by invading CAFs and a microtumor. Topographies such as octahedrons and fractals exclude tumor cells and allow one-step isolation of CAFs even directly from tumor tissue of patients as the CAFs migrate into the structured substrate. Cell lines form spheroids within a short time. The presented inorganic topographical surfaces stimulate complex spheroid formation while avoiding additional biological scaffolds and allowing direct visualization on the substrate.

Distinct and interchangeable growing patterns in colorectal cancer stem-like cells are regulated by Musashi-1

The dynamic and heterogeneous features of cancer stem-like cells (CSCs) have been widely recognized, but their nongenetic cellular plasticity mechanisms remain elusive. By using colorectal cancer organoids, we phenotypically tracked their spheroid formation and growth capacity to a single-cell resolution, and we discovered that the spheroid-forming cells exhibit a heterogeneous growth pattern, consisting of slow- and fast-growing spheroids. The isolated fast-growing spheroids seem to preserve a dual-growing pattern through multiple passages, whereas the isolated slow-growing spheroids are restricted to a slow-growing pattern. Notably, the spheroids of both patterns were tumorigenic. Moreover, the expression of CSC markers varied among the subpopulations with different growth patterns. The isolated slow-growing spheroids adopted the dual-growing pattern by various extrinsic triggers, in which Musashi-1 plays a key role. The slow-growing fraction was resistant to chemotherapy, and its successful isolation can provide an in vitro platform allowing us to elucidate their role in drug resistance.

Anti-metastatic effect of GV1001 on prostate cancer cells; roles of GnRHR-mediated Gαs-cAMP pathway and AR-YAP1 axis

Background Gonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi. However, the precise mechanism underlying the functions of Gαs signal in prostate cancer cells is still unclear. We have previously identified that GV1001, a fragment of the human telomerase reverse transcriptase, functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway. Here, we tried to reveal the potential mechanisms of which GV1001-stimulated Gαs-cAMP signaling pathway reduces the migration and metastasis of prostate cancer (PCa) cells. Methods The expression of epithelial-mesenchymal transition (EMT)-related genes was measured by western-blotting and spheroid formation on ultra-low attachment plate was detected after GV1001 treatment. In vivo Spleen-liver metastasis mouse model was used to explore the inhibitory effect of GV1001 on metastatic ability of PCa and the transwell migration assay was performed to identify whether GV1001 had a suppressive effect on cell migration in vitro. In order to demonstrate the interaction between androgen receptor (AR) and YAP1, co-immunoprecipitation (co-IP), immunofluorescence (IF) staining, chromatin immunoprecipitation (ChIP) were performed in LNCaP cells with and without GV1001 treatment. Results GV1001 inhibited expression of EMT-related genes and spheroid formation. GV1001 also suppressed in vivo spleen-liver metastasis of LNCaP cells as well as cell migration in vitro. GV1001 enhanced the phosphorylation of AR and transcription activity of androgen response element reporter gene through cAMP/protein kinase A pathway. Moreover, GV1001 increased Ser-127 phosphorylation of YAP1 and its ubiquitination, and subsequently decreased the levels of AR-YAP1 binding in the promoter region of the CTGF gene. In contrast, both protein and mRNA levels of NKX3.1 known for tumor suppressor gene and AR-coregulator were upregulated by GV1001 in LNCaP cells. YAP1 knockout using CRISPR/Cas9 significantly suppressed the migration ability of LNCaP cells, and GV1001 did not affect the cell migration of YAP1-deficient LNCaP cells. On the contrary, cell migration was more potentiated in LNCaP cells overexpressing YAP5SA, a constitutively active form of YAP1, which was not changed by GV1001 treatment. Conclusions Overall, this study reveals an essential role of AR-YAP1 in the regulation of PCa cell migration, and provides evidence that GV1001 could be a novel GnRHR ligand to inhibit metastasis of PCa via the Gαs/cAMP pathway.

The Effect of Fertilized Egg White on Growth, Migration, Differentiation and Genes Expression Profile in SW480 Colon Cancer Cell Line

Background: Embryonic microenvironments influence cancer stem cells properties, which leads to anti-cancer effects. Therefore, the current study investigates the effects of fertilized egg white, as an embryonic/fetal microenvironment, on survival, apoptosis, self-renewal characteristic, stemness properties, and migration capacity of SW480 colon cancer cells and 5-fluorouracil (5FU) resistant subgroup.Methods: MTT and Flow cytometry was used to study the cell viability and cell cycle analysis. Clonogenic, spheroid formation, and wound healing assays were used to evaluate cancer cells' self-renewal, stemness properties, and migration capacity. RT-PCR was performed to analyze NANOG, c-MYC, E-cadherin, and NDRG1 mRNA expression.Results: The SW480 colon carcinoma cell line and SW480-5FU chemo-resistant subpopulation cells were subjected to Fertilized Egg White (FEW). FEW decreased cell viability and increased the percentage of the sub-G1 stage in both cell lines. In addition, colony and spheroid formations were decreased in both cells, and the FEW inhibited the migration. Expression of NANOG and c-MYC were reduced in both cells. E-cadherin and NDRG1 expression increased in SW480 cells.Conclusion: FEW decreased the SW480 colon cancer cell line and the SW480-5FU chemo-resistant subpopulation growth and migration. Also, by the changes observed in gene expression and spheroid formation, we suggest the possibility of decreased stemness properties and induction of differentiation following fertilized egg white treatment.