Chondrogenic Regeneration Using Bone Marrow Clots and a Porous Polycaprolactone-Hydroxyapatite Scaffold by Three-Dimensional Printing

Scaffolds play an important role in directing three-dimensional (3D) cartilage regeneration. Our recent study reported the potential advantages of bone marrow clots (MC) in promoting extracellular matrix (ECM) scaffold chondrogenic regeneration. The aim of this study is to build a new scaffold for MC, with improved characteristics in mechanics, shaping, and biodegradability, compared to our previous study. To address this issue, this study prepared a 3D porous polycaprolactone (PCL)-hydroxyapatite (HA) scaffold combined with MC (Group A), while the control group (Group B) utilized a bone marrow stem cell seeded PCL-HA scaffold. The results of in vitro cultures and in vivo implantation demonstrated that although an initial obstruction of nutrient exchange caused by large amounts of fibrin and erythrocytes led to a decrease in the ratio of live cells in Group A, these scaffolds also showed significant improvements in cell adhesion, proliferation, and chondrogenic differentiation with porous recanalization in the later culture, compared to Group B. After 4 weeks of in vivo implantation, Group A scaffolds have a superior performance in DNA content, Sox9 and RunX2 expression, cartilage lacuna-like cell and ECM accumulation, when compared to Group B. Furthermore, Group A scaffold size and mechanics were stable during in vitro and in vivo experiments, unlike the scaffolds in our previous study. Our results suggest that the combination with MC proved to be a highly efficient, reliable, and simple new method that improves the biological performance of 3D PCL-HA scaffold. The MC-PCL-HA scaffold is a candidate for future cartilage regeneration studies.Cell & Tissue EngineeringBiotechnology & Applied MicrobiologyCell BiologySCI(E)[email protected]; [email protected]; [email protected]

Oncogenic state and cell identity combinatorially dictate the susceptibility of cells within glioma development hierarchy to IGF1R targeting

Glioblastoma is the most malignant cancer in the brain and currently incurable. It is urgent to identify effective targets for this lethal disease. Inhibition of such targets should suppress the growth of cancer cells and, ideally also precancerous cells for early prevention, but minimally affect their normal counterparts. Using genetic mouse models with neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) as the cells‐of‐origin/mutation, it is shown that the susceptibility of cells within the development hierarchy of glioma to the knockout of insulin‐like growth factor I receptor (IGF1R) is determined not only by their oncogenic states, but also by their cell identities/states. Knockout of IGF1R selectively disrupts the growth of mutant and transformed, but not normal OPCs, or NSCs. The desirable outcome of IGF1R knockout on cell growth requires the mutant cells to commit to the OPC identity regardless of its development hierarchical status. At the molecular level, oncogenic mutations reprogram the cellular network of OPCs and force them to depend more on IGF1R for their growth. A new‐generation brain‐penetrable, orally available IGF1R inhibitor harnessing tumor OPCs in the brain is also developed. The findings reveal the cellular window of IGF1R targeting and establish IGF1R as an effective target for the prevention and treatment of glioblastoma

Ascl2 Knockdown Results in Tumor Growth Arrest by miRNA-302b-Related Inhibition of Colon Cancer Progenitor Cells

Background: Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5–95 % of CD133 + population) and LS174T (0.45 % of CD133 + population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. Methodology/Principal Findings: Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133 + HT-29 cells was significantly higher than in CD133 2 HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2 % of cells staining CD133 + compared with 54.7 % in control shRNA-Ctr/HT-29 cells. The levels of ‘stemness ’ associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/ HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b

Expression of BNIP3 results in EndoG translocation.

<p>A, SH-SY5Y human neuroblastoma cells cells transfected with the full length BNIP3 or the mutant BNIP3ΔTM plasmids were immune-labeled with antibodies to BNIP3 and EndoG and counterstained with Hoechst 33342. Majority of BNIP3 expressing cells (82%) showed nuclear translocation of EndoG, while only 12% of cells expressing BNIP3ΔTM showed EndoG translocation. B and C, Western blot analysis of EndoG in mitochondrial and nuclear samples prepared from SH-SY5Y cells transfected with the indicated plasmids. Results shown represent the mean ±SD for combined data from 4 independent experiments. **, p<0.01.</p

Blocking VDAC abolishes BNIP3-induced mitochondrial release of EndoG.

<p>A, Mitochondria (1 mg/ml) were incubated with a recombinant GST-BNIP3 in the presence of 0.3 mg/ml of an anti-VDAC antibody or normal rabbit IgG in control. Incubation with the trunked BNIP3 (BNIP3ΔTM) was used as a control. The samples were separated into mitochondrial and supernatant fractions by centrifugation. a), Blocking VDAC prevented BNIP3 from integration into mitochondria. b) Analysis of EndoG from the supernatant showed that levels of EndoG released from mitochondria was reduced by the VDAC antibody blocking. c) Co-immunoprecipitation with a BNIP3 antibody to pull down BNIP3-interacting proteins showed that blocking VDAC abolished the integration of BNIP3 with mitochondria. B, Quantification of BNIP3 integration with mitochondria and mitochondrial release of EndoG affected by the VDAC antibody blocking. Results shown represent the mean ±SD for combined data from three independent experiments. **, p<0.01.</p

EndoG released from isolated mitochondria following BNIP3 induction is enzymatically active.

<p>Freshly isolated mitochondria were incubated with various recombinant proteins for 1 h at 37°C. The supernatants were collected and incubated with HEK293 nuclei for 2 h at 37°C. The DNA samples were loaded on a horizontal 1.8% agarose gel for DNA laddering assay. Shown is a representative image selected from three independent experiments.</p

BNIP3-induced mitochondrial release of EndoG.

<p>Freshly isolated mitochondria were incubated with the indicated recombinant proteins for 1 h and then separated into mitochondrial and supernatant fractions. In the group treated with both Bcl-2 and BNIP3, both were used at 0.5 µM. A and B, Western blotting analysis of BNIP3 and EndoG in the mitochondrial and supernatant fractions. Loading controls were performed with Cox IV and HSP60 antibodies. Results shown represent the mean ±SD for combined data from three independent experiments. ΔTM ^#, = BNIP3ΔTM; *, p<0.05; **, p<0.01. C, Amino acid sequence of EndoG. The tryptic peptide identified from the supernatant samples by mass fingerprinting is indicated in bold and underlined. D, Western blot analysis of cytochrome <i>c</i> and AIF. Both were not detectable in the supernatant samples.</p

BNIP3 induces MPT by interacting with VDAC.

<p>A, BNIP3-induced MPT. Calcein release was presented as the percentage of calcein escaped from mitochondria during the 30-minute incubation. **, p<0.01 when compared with the control. B, Inhibition of PT pores reduced BNIP3-induced EndoG release. Cyclosporine A (2 µM) was added to mitochondria in CFS buffer 10 min before the BNIP3-GST protein at different final concentrations as indicated. BNIP3, CoxIV and EndoG in mitochondrial and supernatant fractions were detected by Western blotting. C, Distribution of EndoG was determined by analyzing the total intensities of the EndoG bands. D, Identification of VDAC as an interacting partner of BNIP3. After mitochondria-BNIP3 incubation, interacting proteins of BNIP3 were precipitated with a BNIP3 antibody and analyzed by Mass spectrometry fingerprinting. The amino acid sequence of VDAC with the tryptic peptide identified from the immunoprecipitates is labelled in bold and underlined. E Western bloting analyses show interaction between BNIP3 and VDAC. a) Levels of GST-BNIP3 in the mitochondrial fraction after mitochondria-BNIP3 incubation as determined by Western blotting. b) After co-immunoprecipitation with a BNIP3 antibody, immunoblotting with a VDAC antibody reveals the presence of VDAC in the BNIP3 immunoprecipitates. c) BNIP3 was present in the precipitates when a VDAC antibody was used for co-immunoprecipitation. Shown are samples from three independent experiments. Cell lysates without BNIP3 treatment were used as controls.</p

Bottlenecks and opportunities in immunotherapy for glioma: a narrative review

Glioma is the most aggressive brain tumor having invasive ability and a highly heterogeneous phenotype. Many patients with glioma respond poorly to traditional surgery or temozolomide-based chemotherapy. Over the past few decades, developments in immunotherapeutic strategies have provided newer insights into the treatment of gliomas. Immunotherapy is based on the principle of normalization or recovery of T cell-mediated anti-tumor immunoreaction. Different innovative strategies have been used; these include enhancement of immunogenicity by administration of tumor antigens or dendritic cell vaccines, replenishment of cytotoxic T cells by adoptive T cell transfer, repair of exhausted T cells by immune checkpoint inhibitors, and the use of other immune activators such as oncolytic viruses. However, many immunotherapy-based clinical trials did not meet the expected therapeutic endpoints in patients with glioma. Gliomas use unique strategies to generate an immune-suppressive microenvironment; these include limiting immunogenicity and repressing T cell infiltration or activation. This may be addressed by the incorporation of immunotherapy with standard therapy or by use of certain innovative approaches such as tumor-treating fields. In this review, we summarize the updated immunotherapies in glioma and discuss current limitations and future prospects

Decreased expression of LATS1 is correlated with the progression and prognosis of glioma

<p>Abstract</p> <p>Background</p> <p>LATS1 is a tumor suppressor genes implicated in the pathogenesis of certain types of tumors, but its role is not known in human glioma.</p> <p>Methods</p> <p>Using real-time PCR and immunohistochemistry, we detected the mRNA and protein expression of LATS1 in glioma. The effect of LATS1 on cell growth and invasion were investigated.</p> <p>Results</p> <p>We found that mRNA and protein of LATS1 expression is significantly downregulated in glioma compared with normal control brain tissues. Furthermore, reduced LATS1 expression was markedly negatively correlated with WHO grade and KPS (p<0.001 and p<0.001) in glioma patients. Patients with lower LATS1 expression had a significantly shorter overall survival time than did patients with higher LATS1 expression. Multivariate analysis suggested that the level of LATS1 expression was an independent prognostic indicator (p<0.001) for the survival of patients with glioma. Forced expression of LATS1 in glioma U251 cells not only significantly suppressed cell growth, migration and invasion, but retarded cell cycle progression from G2/M to G1 in vitro. Finally, we found that overexpressed LATS1 markedly inhibited the expression level of cell cycle factor CCNA1.</p> <p>Conclusion</p> <p>These results indicate that LATS1 is an important candidate tumor suppressor and its downregulated expression may contribute to glioma progression.</p