Turning anecdotal irradiation-induced anticancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE); even combining IR with immune checkpoint inhibitors has shown only anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug, disulfiram (DSF), complexed with copper (DSF/Cu) to induce tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anticancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs relative to spontaneous lung metastasis. In addition, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anticancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral (i.t.) injection of DSF/Cu and IR(12Gy) demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8+ and CD4+ cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, dendritic cells (DC), and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8+ and CD4+ cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anticancer immune response that results in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

Turning anecdotal irradiation-induced anti-cancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE). However, combining IR with immune checkpoint inhibitors has shown anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug disulfiram (DSF) and copper complex (DSF/Cu) via induction of tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anti-cancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs, i.e., spontaneous lung metastasis. Additionally, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anti-cancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral injection of DSF/Cu and IR demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8 + and CD4 + cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, DC, and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8 + and CD4 + cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anti-cancer immune response, resulting in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., the absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

USP15 Enhances the Proliferation, Migration, and Collagen Deposition of Hypertrophic Scar-Derived Fibroblasts by Deubiquitinating TGF-ßR1 In Vitro.

BACKGROUND: Hypertrophic scar is a fibroproliferative disorder caused by skin injury. The incidence of hypertrophic scar following trauma or burns is 40 to 70 percent or 70 percent, respectively. It has been shown that transforming growth factor (TGF) ß1/Smad signaling plays a crucial role in hypertrophic scar, and that USP15 can regulate the activity of TGFß1/Smad signaling to affect the progression of the disease. However, the underlying mechanism of USP15 in hypertrophic scar remains unclear. The authors hypothesized that USP15 was up-regulated and enhanced the proliferation, migration, invasion, and collagen deposition of hypertrophic scar-derived fibroblasts by deubiquitinating TGF-ß receptor I (TßRI) in vitro. METHODS: Fibroblasts were isolated from human hypertrophic scars in vitro. The knockdown and overexpression of USP15 in hypertrophic scar-derived fibroblasts were performed using lentivirus infection. The effect of USP15 on hypertrophic scar-derived fibroblast proliferation, migration, and invasion, and the expression of TßRI, Smad2, Smad3, α-SMA, COL1, and COL3, were detected by Cell Counting Kit-8, scratch, invasion, quantitative real-time polymerase chain reaction, and Western blot assays. The interaction between USP15 and TßRI was detected by co-immunoprecipitation and ubiquitination assays. RESULTS: The authors demonstrated that USP15 knockdown significantly inhibited the proliferation, migration, and invasion of hypertrophic scar-derived fibroblasts in vitro and down-regulated the expression of TßRI, Smad2, Smad3, α-SMA, COL1, and COL3; in addition, USP15 overexpression showed the opposite trends (p < 0.05). Co-immunoprecipitation and ubiquitination assays revealed that USP15 interacted with TßRI and deubiquitinated TßRI. CONCLUSION: USP15 enhances the proliferation, migration, invasion, and collagen deposition of hypertrophic scar-derived fibroblasts by deubiquitinating TßRI in vitro.

Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament.

BACKGROUND: The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remain to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy-associated gene expression was correlated with the expression of osteogenic differentiation genes. METHODS: Expression of autophagy-associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy-associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson's correlation. The expression of autophagy-associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. RESULTS: In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001), and collagen type 1 (COL 1) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy-associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP, and COL 1 by 63.2% (P < 0.01), 52% (P < 0.01), and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively. CONCLUSIONS: Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.

The roles of autophagy in osteogenic differentiation in rat ligamentum fibroblasts: Evidence and possible implications.

Autophagy, a macromolecular degradation process, plays a pivotal role in cell differentiation and survival. This study was designed to investigate the role of autophagy in the osteogenic differentiation in ligamentum fibroblasts. Rat ligamentum fibroblasts were isolated from the posterior longitudinal ligament and cultured in osteogenic induction medium. Ultrastructural analysis, immunofluorescence assay, western blot, flow cytometry, and lysosomal activity assessment were performed to determine the presence and activity of autophagy in the cells. The mineralization deposit and osteogenic gene expressions were evaluated to classify the association between autophagy activity and the bone formation ability of the spinal ligament cells. The influence of leptin and endothelin-1 on the autophagy activity was also evaluated. Our study demonstrated that autophagy was present and increased in the ligament cells under osteogenic induction. Inhibition of autophagy with either pharmacologic inhibitors (Bafilomycin A and 3-methyladenine) or Belcin1 (BECN1) knocking down weakened the mineralization capacity, decreased the gene expressions of COL1A1, osteocalcin (Ocn), and runt-related transcription factor 2 (Runx2) in the ligamentum fibroblasts and increased cell apoptosis. The Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-BECN1 autophagic pathway was activated in the osteogenic differentiating ligamentum fibroblasts. Leptin significantly increased the autophagy activity in the ligament cells under osteogenic induction. These discoveries might improve our understanding for the mechanism of ossification of the posterior longitudinal ligament (OPLL) and provide new approaches on the prevention and treatment of this not uncommon disease.

Correction to: Induction of immunogenic cell death in radiation-resistant breast cancer stem cells by repurposing anti-alcoholism drug disulfiram.

An amendment to this paper has been published and can be accessed via the original article.

Induction of immunogenic cell death in radiation-resistant breast cancer stem cells by repurposing anti-alcoholism drug disulfiram.

BACKGROUND: The current successful clinical use of agents promoting robust anti-tumor immunity in cancer patients warrants noting that radiation therapy (RT) induces immunogenic cell death (ICD) of tumor cells, which can generate anti-tumor immune responses. However, breast cancer stem cells (BCSCs) are resistant to RT and RT alone usually failed to mount an anti-tumor immune response. METHODS: High aldehyde dehydrogenase activity (ALDH)bright and CD44+/CD24-/ESA+ cancer cells, previously shown to have BCSC properties, were isolated from human MDA-MB-231 and UACC-812 breast cancer cell lines by flow cytometer. Flow sorted BCSCs and non-BCSCs were further tested for their characteristic of stemness by mammosphere formation assay. Induction of ICD in BCSCs vs. non-BCSCs in response to different in vitro treatments was determined by assessing cell apoptosis and a panel of damage-associated molecular pattern molecules (DAMPs) by flow and enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that ionizing radiation (IR) triggered a lower level of ICD in BCSCs than non-BCSCs. We then investigated the ability of disulfiram/cooper (DSF/Cu) which is known to preferentially induce cancer stem cells (CSCs) apoptosis to enhance IR-induced ICD of BCSCs. The results indicate that DSF/Cu induced a similar extent of IDC in both BCSCs and non-BCSCs and rendered IR-resistant BCSCs as sensitive as non-BCSCs to IR-induced ICD. IR and DSF/Cu induced ICD of BCSCs could be partly reversed by pre-treatment of BCSCs with a reactive oxygen species (ROS) scavenger and XBP1s inhibitors. CONCLUSION: DSF/Cu rendered IR-resistant BCSCs as sensitive as non-BCSCs to IR-induced ICD. Our data demonstrate the potential of IR and DSF/Cu to induce ICD in BCSCs and non-BCSCs leading to robust immune responses against not only differentiated/differentiating breast cancer cells but also BCSCs, the root cause of cancer formation, progression and metastasis.

MicroRNA-495 downregulates AQP1 and facilitates proliferation and differentiation of osteoblasts in mice with tibial fracture through activation of p38 MAPK signaling pathway.

Osteoblasts are implicated in the building of the vertebrate skeleton. The current study aimed to investigate the role of microRNA-495 (miR-495) in the osteoblasts of mice with tibial fractures and the underlying mechanism involving in aquaporin-1 (AQP1) and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Initially, a microarray-based analysis was performed to screen the differentially expressed genes and miRNAs associated with tibial fracture. Following the establishment of a tibial fracture mouse model, the positive rate of the AQP1 protein in the fracture tissue was detected by immunohistochemistry (IHC). Next, to verify the binding site between miR-495 on AQP1, bioinformatics data were employed in addition to the application of a dual-luciferase reporter gene assay. The osteoblast cell line MC3T3-E1 was treated with miR-495 mimic, miR-495 inhibitor and Anisomycin to explore the potent effects of miR-495 on proliferation and differentiation of osteoblasts in mice with tibial fracture. The expression of miR-495, AQP1, p38 MAPK, PCNA, Cyclin D1, OCN, and OPN was subsequently evaluated by RT-qPCR and Western blot analysis. Cell viability, the number of calcium nodules and alkaline phosphatase (ALP) activity were detected by MTT assay, alizarin red staining, and ALP activity assay, respectively. Our results revealed that miR-495 was down-regulated while AQP1 was up-regulated in the mice with tibial fractures. AQP1 was verified as a target gene of miR-495. When the cells were treated with overexpressed miR-495 or activated p38 MAPK signaling pathway, elevated expression of PCNA, Cyclin, D1, OCN, and OPN along with an increased amount of calcium nodules, higher cell viability, and enhanced ALP activity was detected, while the expression of AQP1 was reduced. Collectively, the key findings of the present study support the notion that overexpressed miR-495 may activate the p38 MAPK signaling pathway to inhibit AQP1 and to promote the proliferation and differentiation of osteoblasts in mice with tibial fracture.

Tension induces intervertebral disc degeneration via endoplasmic reticulum stress-mediated autophagy.

BACKGROUND: Intervertebral disc degeneration is a common degenerative disease. The present study aimed to explore the role and mechanism of tension-induced endoplasmic reticulum stress in intervertebral disc degeneration. METHODS: Intervertebral disc degeneration models of SD rat were analyzed for apoptosis, the expression of Poly(ADP-ribose) polymerase (PARP), Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP using immunohistochemistry, qPCR and Western blot analysis. Annulus fibrosus cells of intervertebral disc were isolated, subjected to cyclic deformation stress and analyzed for ROS and apoptosis, lysosome activity and expression of genes. The cells were knockdown with siRNA or treated with endoplasmic reticulum stress inhibitor 4-PBA and assayed for ROS, apoptosis, lysosome activity and gene expression. RESULTS: Compared with the controls, intervertebral disc degeneration was observed through X-rays examinations and HS staining. Apoptosis and expression of PARP, Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP were significantly increased in the intervertebral disc tissue of the models. In mechanic mimic experiments, the primary annulus fibrosus cells were subjected to 18% cyclic deformation, ROS and apoptosis as well as the activity of lysosome were increased. Similarly, the expression of PARP, Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP was also increased significantly after deformation treatment. On other hand, when the cells were treated with 9 mM 4-PBA and/or CHOP-siRNA4, the apoptosis rate, ROS level, lysosome activity and expression of PARP, Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP were significantly reduced. CONCLUSIONS: Autophagy reaction mediated by endoplasmic reticulum stress plays important rale in tension-induced intervertebral disc degeneration. Intervertebral disc degeneration likely results from interactions between autophagy, apoptosis and reticulum stress, and is ROS-dependent.

The influence of the stiffness of GelMA substrate on the outgrowth of PC12 cells.

Recent studies have shown the importance of cell-substrate interaction on neurone outgrowth, where the Young's modulus of the matrix plays a crucial role on the neurite length, migration, proliferation, and morphology of neurones. In the present study, PC12 cells were selected as the representative neurone to be cultured on hydrogel substrates with different stiffness to explore the effect of substrate stiffness on the neurone outgrowth. By adjusting the concentration of gelatin methacryloyl (GelMA), the hydrogel substrates with the variation of stiffnesses (indicated by Young's modulus) from approximately 3-180 KPa were prepared. It is found that the stiffness of GelMA substrates influences neuronal outgrowth, including cell viability, adhesion, spreading, and average neurite length. Our results show a critical range of substrate's Young's modulus that support PC12 outgrowth, and modulate the cell characteristics and morphology. The present study provides an insight into the relationship between the stiffness of GelMA hydrogel substrates and PC12 cell outgrowth, and helps the design and optimization of tissue engineering scaffolds for nerve regeneration.

Retracted: Inhibition of microRNA-940 suppresses the migration and invasion of human osteosarcoma cells through the secreted frizzled-related protein 1-mediated Wnt/ß-catenin signaling pathway.

Osteosarcoma (OS) is the most common malignant tumor of bone with a high potential for metastasis. This study intends to explore whether microRNA-940 (miR-940) affects the development of OS cells and the underlying mechanism. OS and adjacent normal tissues were collected from OS patients; the OS cell line with the highest expression of miR-940 was selected, which was then subjected to transfection of miR-940 mimic, miR-940 inhibitor, siRNA-secreted frizzled-related protein 1 (SFRP1) or LiCl (agonists of Wnt/ß-catenin pathway) to identify regulation of miR-940 to OS cells through SFRP1. The targeting relationship between miR-940 and SFRP1 was verified using dual-luciferase reporter gene assay. Reverse-transcription quantitative polymerase chain reaction and Western blot assay were performed to determine miR-940, SFRP1, ß-catenin, and cyclinD1 and apoptosis-related genes Fas, Bax, and Bcl-2. MTT (3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide) assay, scratch test, transwell assay, and flow cytometry were carried out to detect proliferation, migration, invasion, and apoptosis, respectively. Nude mice models were established to observe the tumor formation. Higher expression of miR-940, ß-catenin, and cyclinD1 and lower SFRP1 expression were identified in OS tissues. miR-940 targeted and negatively regulated SFRP1 expression. Furthermore, upregulated miR-940 expression activated the Wnt/ß-catenin signaling pathway in OS. With the treatment of miR-940 mimic, LiCL, or siRNA-SFRP1, OS cells showed promoted proliferation, migration, invasion, tumor formation, and impeded apoptosis (further reflected by elevated Bcl-2 expression and reduced Fas and Bax expression). The study demonstrates that miR-940 can promote the proliferation, migration, and invasion but suppress the apoptosis of human OS cells by downregulating SFRP1 through activating Wnt/ß-catenin signaling pathway.

[Differential expression profile of microRNA between hyperplastic scar and normal skin].

OBJECTIVE: To explore the miRNA differential expression profiles of hyperplastic scar and normal skin so as to further elucidate the pathogenesis of hyperplastic scar and search for new therapeutic targets. METHODS: The total RNA was extracted from 5 human hyperplastic scar and normal skin tissues by Trizol. The specimens were collected from the First Affiliated Hospital of Nanchang University from November 2010 to May 2011, and purified by mirVana(TM) miRNA Isolation Kit and then labeled and hybridized by miRNA Complete Labeling and Hyb Kit. The images of hybridization were analyzed by the Feature Extraction (v10.7) software and the microarray results confirmed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In hyperplastic scar, 92 miRNA genes were up-regulated and 13 down-regulated. The most significantly up-regulated miRNAs were hsa-miR-564 and hsa-miR-936, etc. while hsa-miR-451, hsa-miR-223, hsa-miR-363 and hsa-miR-29b-1* became significantly down-regulated. The findings of RT-PCR on hsa-miR-21 and hsa-miR-451 of regulation were in a high concordance with the microarray results. CONCLUSION: Distinct differences of miRNA expression between human hyperplastic scar and normal skin, it may be closely correlated with the formation, development and evolution of hyperplastic scar.