Author Correction: A whole-cell tumor vaccine modified to express fibroblast activation protein induces antitumor immunity against both tumor cells and cancer-associated fibroblasts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Corrigendum: A whole-cell tumor vaccine modified to express fibroblast activation protein induces antitumor immunity against both tumor cells and cancer-associated fibroblasts.

This corrects the article DOI: 10.1038/srep14421.

The Promotion of Erythropoiesis via the Regulation of Reactive Oxygen Species by Lactic Acid.

The simultaneous increases in blood lactic acid and erythrocytes after intense exercise could suggest a link between lactate and the erythropoiesis. However, the effects of lactic acid on erythropoiesis remain to be elucidated. Here, we utilized a mouse model to determine the role of lactic acid in this process in parallel with studies using leukaemic K562 cells. Treatment of K562 cells in vitro with lactic acid increased the mRNA and protein expression of haemoglobin genes and the frequency of GPA+ cells. Also, increases in haematocrit and CD71-/Ter119+ erythroid cells were observed in lactic acid-treated mice, which showed a physiological increase in blood lactate. Mouse bone marrow CD34+/CD117- cells showed an increase in erythroid burst-forming units after stimulation with lactic acid in vitro. Furthermore, lactic acid increased the intracellular reactive oxygen species (ROS) content in bone marrow and in K562 cells. Erythroid differentiation induced in Haematopoietic Stem Cells (HSCs) and K562 cells by lactic acid was abolished by reducing ROS levels with SOD or 2-mercaptoethanol, which suggests that ROS is a critical regulator of this process. These findings provide a better understanding of the role of lactic acid in cellular metabolism and physiological functions.

Inhibition of A20 expression in tumor microenvironment exerts anti-tumor effect through inducing myeloid-derived suppressor cells apoptosis.

Myeloid-derived suppressor cells (MDSCs) are known to play important roles in the development of immunosuppressive tumor microenvironment. A20 is a zinc-finger protein which could negatively regulate apoptosis in several cell types. However, the role of A20 in tumor microenvironment remains largely unknown. In this study, we found that A20 was over-expressed in MDSCs. The treatment of tumor-bearing mice with small interfering RNA targeting A20 (si-A20) inhibited the growth of tumors. The infiltration of MDSCs was dramatically reduced after si-A20 treatment, as compared to control groups, whereas the numbers of dendritic cells and macrophages were not affected. Also, injection of si-A20 improved T cell mediated tumor-specific immune response. Depletion of MDSCs with anti-Gr1 antibody showed similar antitumor effect and improved T cell response. TNF-α was highly expressed after si-A20 injection. Furthermore, si-A20 induced apoptosis of MDSCs in the presence of TNF-α both in vivo and in vitro. Cleaved Caspase-3 and Caspase-8 were elevated with the activation of JNK pathway after the induction of MDSC apoptosis by si-A20. Thus, our findings suggested that knockdown of A20 in tumor site inhibited tumor growth at least through inducing the apoptosis of MDSCs. A20 might be a potential target in anticancer therapy.

Folate-Modified Lipoplexes Delivering the Interleukin-12 Gene for Targeting Colon Cancer Immunogene Therapy.

The incidence and mortality rate of colorectal cancer increase every year, making it a serious threat to human health. Targeted immunogene therapy is a novel method of treating this type of cancer. Colon cancer overexpresses folate receptor α (FRα) and folate-modified liposomes for colon cancer immunogene therapy may suppress tumor growth effectively. In this study, F-PLP/pIL12, an FRα-targeted lipoplex loading plasmid interleukin-12 (pIL12) was prepared and its physicochemical properties were characterized. Then the antitumor effect of F-PLP/pIL12 was studied in an in vivo model of CT-26 colon cancer. F-PLP/pIL12 was associated with about 56.6% tumor growth inhibition compared with the saline control. The production of malignant ascites was significantly less pronounced than in controls, and there were fewer tumor nodules and less overall tumor mass (P < 0.01). There was more IL12 expression and IFN-γ secretion in F-PLP/pIL12-treated tumor tissues, but there was less FRα expression. The antitumor mechanisms involved inducing tumor cell apoptosis, reducing microvessel density, and stimulating TNF-α secretion. In addition, there were fewer M2 macrophages in the tumor microenvironment of tissues stimulated with F-PLP/pIL12, which also activated the natural killer cells. H&E staining of vital organs suggested that F-PLP/pIL12 is safe for use in intraperitoneally administered cancer therapy. It was here concluded that F-PLP/plL12 may be a suitable targeting formulation for colon cancer immunogene therapy.

A whole-cell tumor vaccine modified to express fibroblast activation protein induces antitumor immunity against both tumor cells and cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are common components of the tumor-suppressive microenvironment, and are a major determinant of the poor outcome of therapeutic vaccination. In this study, we modified tumor cells to express the fibroblast activation protein (FAP), which is highly expressed by CAFs, to potentially improve whole-cell tumor vaccines by targeting both tumor cells and CAFs. Tumor cells were transfected with murine FAP plasmids bearing the cationic lipid DOTAP. Its antitumor effects were investigated in three established tumor models. Vaccination with tumor cells expressing FAP eliminated solid tumors and tumors resulting from hematogenous dissemination. This antitumor immune response was mediated by CD8+ T cells. Additionally, we found that CAFs were significantly reduced within the tumors. Furthermore, this vaccine enhanced the infiltration of CD8+ T lymphocytes, and suppressed the accumulation of immunosuppressive cells in the tumor microenvironment. Our results indicated that the FAP-modified whole-cell tumor vaccine induced strong antitumor immunity against both tumor cells and CAFs and reversed the immunosuppressive effects of tumors by decreasing the recruitment of immunosuppressive cells and enhancing the recruitment of effector T cells. This conclusion may have important implications for the clinical use of genetically modified tumor cells as cancer vaccines.

Cationic nanocarriers induce cell necrosis through impairment of Na(+)/K(+)-ATPase and cause subsequent inflammatory response.

Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na(+) overload was found to accompany the cell death. Depletion of Na(+) in culture medium or pretreatment of cells with the Na(+)/K(+)-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na(+)/K(+)-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na(+)/K(+)-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na(+)/K(+)-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.

Suppression of peritoneal tumorigenesis by placenta-derived mesenchymal stem cells expressing endostatin on colorectal cancer.

MSCs-based therapy for cancer is a relatively new but rapidly growing area of research. Human term placenta, an attractive source of MSCs (PMSCs), appears to have great advantage due to its easy access without invasive procedures, its lack of ethical issues and its high-throughput and young age. In the present study, we isolated MSCs from placenta and characterized their morphology and differentiation capacities. We next investigated the underlying antitumor effects and the potential mechanism of PMSCs to express endostatin using adenoviral transduction (Ad-Endo) in a colorectal peritoneal carcinomatosis (CRPC) mouse model. For in vitro experiments, the migratory potential of Ad-Endo-PMSCs towards tumor cells was demonstrated using a double-chamber assay, and the anti-angiogenesis ability of endostatin from engineered PMSCs was evaluated using the tube formation assay. For the in vivo study, mice harboring CT26 colorectal cancer indicated a significant reduction in tumor nodules and a prolongation of survival following Ad-Endo-PMSCs therapy. These observations were associated with significantly decreased tumor cell proliferation and blood vessel counts as well as increased tumor cell apoptosis. These data suggested the potential of PMSCs-based gene therapy for the targeted delivery of therapeutic proteins in cancer.

Synergistic antitumor effect of recombinant adeno-associated virus-mediated pigment epithelium-derived factor with hyperthermia on solid tumor.

Adeno-associated virus (AAV) is an ideal choice for gene delivery; however, its further development has been limited owing to its low transduction efficiency. DNA-damaging agents can improve AAV-mediated transgene expression. Hyperthermia, as one of the oldest documented tumor treatment modalities, can cause DNA damage as well. However, combined treatment consisting of hyperthermia and AAV-mediated gene therapy has not been reported yet. In this work we investigated whether therapy consisting of AAV-mediated pigment epithelium-derived factor (PEDF) delivery combined with hyperthermia has synergistic antitumor effect on established solid tumors. We produced the recombinant AAV encoding PEDF (rAAV-PEDF). The therapeutic effect of rAAV-PEDF plus hyperthermia was evaluated in a subcutaneous fibrosarcoma mouse model, and the possible mechanism of antitumor effect was investigated. We found that rAAV-PEDF could infect a murine fibrosarcoma cell line (Meth-A) and express PEDF protein with bioactivity in vitro. In addition, in vivo experiments suggested that the combination of rAAV-PEDF with hyperthermia could significantly suppress tumor growth and prolong survival time of treated mice. Immunofluorescence studies indicated that the combination therapy could inhibit angiogenesis and induce apoptosis in tumor tissues. An immunohistochemistry assay of tumor tissue showed that PEDF expression in the combined treatment group was significantly higher than in the rAAV-PEDF group, which implied that hyperthermia could improve the expression of PEDF protein in vivo. No significant differences were observed in each group by hematoxylin-eosin staining of major organs, serum chemistry test, and complete blood assay. These results indicate that the combination of rAAV-PEDF with hyperthermia has synergistic therapeutic effects on established solid tumors, with no side effects. In addition, hyperthermia could improve AAV-mediated transgene expression, which suggests that hyperthermia could serve as a promising adjunctive therapy for AAV-mediated gene therapy.

[REGgamma promotes malignant behaviors of lung cancer cells].

OBJECTIVE: To determine the expression of proteasome aotivator gamma (REGgamma) in human lung cancer tissues and cell lines and its association with malignant biological behaviors. METHODS: Immunohistochemistry (IHC) was used to detect the expression of REGgamma in lung cancer and normal lung tissues. The expressions of REGgamma in lung cancer cells and normal epithelial cells were determined by Western blot. The H1975 lung cancer stable cell lines with different levels of REGgamma expression were constructed and their proliferations were evaluated by MTT assay. PI staining was used to assess the influence of REGgamma on cell growth cycle. The effect of REGgamma on the migration of lung cancer cells were observed with the cell scratch experiment. RESULTS: Lung cancer tissues had significantly higher levels of REGgamma expression than normal tissues. Similarly, lung cancer cell lines showed higher levels of REGgamma expression than the normal epithelial cell line. The overexpression of REGgamma enhanced cancer cell proliferations (P < 0.05), promoted more cells into the S+G2/M phase (P < 0.05) and promoted the migration of cancer cells (P < 0.05). All of these effects were reversed after suppression of REGgamma. CONCLUSION: REGgamma facilitates malignant biological behaviors of lung cancers.

Enhanced efficacy of combination therapy with adeno­associated virus-delivered pigment epithelium-derived factor and cisplatin in a mouse model of Lewis lung carcinoma.

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, and the antitumor effect of adeno-associated virus (AAV)-mediated PEDF expression has been demonstrated in a range of animal models. The combined treatment of low-dose chemotherapy and gene therapy inhibits the growth of solid tumors more effectively than current traditional therapies or gene therapy alone. In the present study, the effect of treatment with an AAV2 vector harboring the human PEDF (hPEDF) gene in combination with low-dose cisplatin on the growth of Lewis lung carcinoma (LLC) in mice was assessed. LLC cells were infected with AAV-enhanced green fluorescent protein (EGFP) in the presence or absence of cisplatin, and then the effect of cisplatin on AAV-mediated gene expression was evaluated by image and flow cytometric analysis. Tumor growth, survival time, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD) and apoptotic index were analyzed in C57BL/6 mice treated with AAV-hPEDF, cisplatin or cisplatin plus AAV-hPEDF. The results of the present study provide evidence that cisplatin treatment is able to enhance AAV-mediated gene expression in LLC cells. In addition, the combined treatment of cisplatin plus AAV­hPEDF markedly prolonged the survival time of the mice and inhibited tumor growth, resulting in significant suppression of tumor angiogenesis and induction of tumor apoptosis in vivo, and also protected against cisplatin-related toxicity. These findings suggest that combination of AAV-hPEDF and cisplatin has potential as a novel therapeutic strategy for lung cancer.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts.

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.

Systemically administered liposome-encapsulated Ad-PEDF potentiates the anti-cancer effects in mouse lung metastasis melanoma.

BACKGROUND: The use of adenoviral vector for gene therapy is still an important strategy for advanced cancers, however, the lack of the requisite coxsackie-adenovirus receptor in cancer cells and host immune response to adenovirus limit the application of adenoviral vector in vivo. METHOD: We designed the antiangiogenic gene therapy with recombinant PEDF adenovirus (Ad-PEDF) encapsulated in cationic liposome (Ad-PEDF/Liposome), and investigated the anti-tumor efficacy of Ad-PEDF/Liposome complex on inhibition of tumor metastasis. RESULTS: We found that systemic administration of Ad-PEDF/liposome was well tolerated and resulted in marked suppression of tumor growth, and was more potent than uncoated Ad-PEDF to induce apoptosis in B16-F10 melanoma cells and inhibit murine pulmonary metastases in vivo. After Ad-luciferase was encapsulated with liposome, its distribution decreased in liver and increased in lung. The anti-Ad IgG level of Ad-PEDF/Liposome was significantly lower than Ad-PEDF used alone. CONCLUSION: The present findings provide evidences of systematic administration of cationic liposome-encapsulated Ad-PEDF in pulmonary metastatic melanoma mice model, and show an encouraging therapeutic effect for further exploration and application of more complexes based on liposome-encapsulated adenovirus for more cancers.

AAV2-mediated gene transfer of VEGF-Trap with potent suppression of primary breast tumor growth and spontaneous pulmonary metastases by long-term expression.

Vascular endothelial growth factor (VEGF) is an important signaling protein and a predominant mediator of angiogenesis in tumor growth and metastasis. Therefore, antagonism of the VEGF pathway results in inhibition of abnormal angiogenesis, then suppression of tumor growth and metastasis. VEGF-Trap, a high-affinity soluble decoy receptor, is currently in phase II clinical trails, and has demonstrated more efficacy in different types of solid tumors by intravenous injection every two weeks. In our study, we used recombinant AAV2 as a delivery vehicle to achieve long-lasting expression of VEGF Trap protein in a mouse model for the first time. We report that AAV2-VEGF-Trap can be safely administered and sustained expression in vivo via a single intravenously administration, simultaneously suppressing primary tumor growth and preventing the pulmonary metastases of 4T1 tumors. Decreased microvessel density and increased tumor cell apoptosis were observed in the treatment group. AAV2-VEGF-Trap can obviously decrease not only the concentration of VEGF in sera, but also the concentration of other angiogenic factors, such as aFGF, bFGF, angiopoietin-1 and others. These studies suggest that AAV-mediated long-term expression of VEGF-Trap is a useful and safe tool to block tumor progression and inhibit spontaneous pulmonary metastases.

Interleukin-13 peptide kinoid vaccination attenuates allergic inflammation in a mouse model of asthma.

Asthma is an atopic disorder with increasing frequency and severity in developed nations. Interleukin-13 (IL-13) is one of the most critical mediators of asthma pathology. In the present study, we developed a vaccine comprised of a keyhole limpet hemocyanin-mIL-13 heterocomplex immunogen to persistently neutralize excessive endogenous IL-13. Our results showed that the IL-13 peptide kinoid vaccine could induce sustained and high titer of IL-13-specific IgG when using aluminum hydroxide as an adjuvant, and could suppress the accumulation of eosinophils as well as IL-13 levels in bronchoalveolar lavage fluid (BALF). In addition, total IgE and ovalbumin (OVA)-specific IgE in serum were significantly inhibited. This study also showed that vaccination could prevent airway inflammation and epithelial cell proliferation with goblet cell hyperplasia in a mouse model of acute asthma. In summary, our findings suggest that the IL-13 peptide kinoid can serve as an innovative and effective vaccine against asthma.

AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice.

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, and mechanisms through which PEDF exerts its antitumour activity have recently been defined. The aim of our research was to evaluate the ability of adeno-associated virus (AAV) vector-mediated transfer of human PEDF to inhibit lewis lung carcinoma (LCC) cell growth. Intratumoural injection of AAV-PEDF caused significant reduction of the tumour volume and prolonged the survival time of mice bearing LLC cells, which were associated with decreased microvessel density and increased apoptosis in the tumours. AAV vectors represent a very promising tool for cancer gene therapy. No noticeable toxicity concerning AAV was detected as inferred from monitoring changes in animal body weight as well as basic organ structure and histological morphology, and by analyzing mouse liver and kidney function. Our findings indicate that AAV-mediated PEDF gene expression may offer an active approach to inhibit LLC growth and that treatment with AAV-PEDF may provide a promising therapeutic strategy in lung cancer treatment.

bFGF peptide combined with the pVAX-8CpG plasmid as adjuvant is a novel anticancer vaccine inducing effective immune responses against Lewis lung carcinoma.

Due to the poor immunogenicity of subunit protein antigens, there is a need to use adjuvants in order to generate effective immune responses. Basic fibroblast growth factor (bFGF) is one of the best characterized pro-angiogenic cytokine and is a candidate target for anticancer therapy. We used truncated bFGF (tbFGF) combined with engineered pVAX-nCpG as novel adjuvant to immunize mice in order to inhibit tumor angiogenesis and suppress tumor growth. In our study, the results demonstrated that the mice immunized with tbFGF-alum-pVAX-8CpG produced a better tumor-suppression effect compared with the other groups, apart from the group treated with tbFGF-alum-CpG. In addition, the function of immune modulation of pVAX-8CpG was similar to CpG ODNs. The vaccine composed of tbFGF, alum and pVAX-8CpG effectively inhibited tumor angiogenesis and induced strong antitumor immune responses. The antitumor activity induced by the vaccine tbFGF-alum-pVAX-8CpG was not only associated with the antigen-specific antibody, but also with the killing activity of cytotoxic cells. This indicates that alum-pVAX-8CpG may be an innovative adjuvant for cancer vaccines.

A novel combined conjugate vaccine: enhanced immunogenicity of bFGF with CRM197 as a carrier protein.

Tumor growth is partly dependent on tumor-associated angiogenesis, which is regulated by angiogenic growth factors. As the first angiogenic growth factor to be identified, basic fibroblast growth factor (bFGF) plays a major role in angiogensis and tumor growth and has been an effective target for anti-tumor therapy. However, due to its low immunogenicity, injection with bFGF alone cannot stimulate the body to produce a strong immune response. In this study, we investigated the role of CF (containing bFGF and CRM197) assisted by CpG and alum in enhancing antigen-specific immune response and suppressing the growth of murine colon carcinoma. The results revealed that compared to bFGF, CF could not stimulate NIH-3T3 fibroblast proliferation even at a concentration of 10 µg/ml in vitro. In vivo, the CF-CpG-alum produced a stronger antigen-specific immune response and inhibited tumor growth. The anti-tumor activity was associated with generating antigen-specific antibody, suppressing angiogenesis, promoting the apoptosis of tumor cells and inducing the mixed Th1 and Th2 responses. This indicates that CRM197 may be an innovative intramolecular adjuvant and provides a rational preservation for mouse CT26 colon carcinoma.

Adenoviral vectors modified by heparin-polyethyleneimine nanogels enhance targeting to the lung and show therapeutic potential for pulmonary metastasis in vivo.

Polyethyleneimine (PEI) is a well-known cationic polymer that has previously been shown to have significant potential to deliver genes in vitro and in vivo. However, PEI is non-degradable and exhibits a high cytotoxicity as its molecular weight increases. The clinical application for systemic administration of adenoviral (Ad) vectors is limited, as these vectors do not efficiently penetrate solid tumor masses due to a common deficiency of Coxsackie Adenovirus Receptor (CAR) on the tumor surface. In this study, we conjugated low molecular weight PEI (Mn = 1,800) to heparin (Mn = 4,000-6,000) to create a new type of cationic degradable nanogel (HPEI) that was then used to modify Ad vectors. The resulting HPEI-Ad complexes were used to infect CT26 and HeLa cells in vitro. Additionally, the HPEI-Ad complexes were administrated in vivo via intravenous injection, and tissue distribution was assessed using luciferase assays; the therapeutic potential of HPEI-Ad complexes for pulmonary metastasis mediated by CT26 cells was also investigated. In vitro, HPEI-Ad complexes enhanced the transfection efficiency in CT26 cells, reaching 36.3% compared with 0.1% of the native adenovirus. In vivo, HPEI-Ad complexes exhibited greater affinity for lung tissue than the native adenovirus and effectively inhibited the growth of pulmonary metastases mediated by CT26 cells. Our results indicate that Ad vectors modified by HPEI nanogels to form HPEI-Ad complexes enhanced transfection efficiency in CT26 cells that lacked CAR, targeted to the lung and demostrated a potential therapy for pulmonary metastasis.