IFN-γ-responsiveness of lymphatic endothelial cells inhibits melanoma lymphatic dissemination via AMPK-mediated metabolic control.

The integrity of the lymphatic system is critical for preventing the dissemination of tumor cells, such as melanoma, to distant parts of the body. IFN-γ is well studied as a negative regulator for lymphangiogenesis, which is strongly associated with cancer metastasis. However, the exact mechanisms underlying this process remain unclear. In the present study, we investigated whether IFN-γ signaling in lymphatic endothelial cells (LECs) affects tumor cell dissemination by regulating the barrier function of tumor-associated lymphatic vessels. Using LEC-specific IFN-γ receptor (IFN-γR) knockout mice, we found that the loss of IFN-γR in LECs increased the dissemination of melanoma cells into the draining lymph nodes. Notably, IFN-γ signaling in LECs inhibited trans-lymphatic endothelial cell migration of melanoma cells, indicating its regulation of lymphatic barrier function. Further investigations revealed that IFN-γ upregulated the expression of the tight junction protein Claudin-3 in LECs, while knockdown of Claudin-3 in LECs abolished IFN-γ-induced inhibition of trans-lymphatic endothelial migration activity. Mechanistically, IFN-γ inhibits AMPK signaling activation, which is involved in the regulation of fatty acid metabolism. Modulating fatty acid metabolism and AMPK activation in LECs also affected the lymphatic dissemination of melanoma cells, further confirming that this process is involved in IFN-γ-induced regulation of lymphatic barrier function. These results provide novel insights into how IFN-γ modulates tight junctions in LECs, inhibiting the dissemination of melanoma cells via the lymphatic vessels.

PARP inhibition synergizes with CD47 blockade to promote phagocytosis by tumor-associated macrophages in homologous recombination-proficient tumors.

AIMS: PARP inhibitors (PARPi) are known to exert anti-tumor effects in patients with BRCA-mutated (BRCAmut) or homologous recombination (HR)-deficient cancer, but recent clinical investigations have suggested that this treatment may also be beneficial in patients with HR-proficient tumors. In this study, we aimed to investigate how PARPi exerts anti-tumor effects in non-BRCAmut tumors. MAIN METHODS: BRCA wild-type, HR-deficient-negative ID8 and E0771 murine tumor cells were treated in vitro and in vivo with olaparib, a clinically approved PARPi. The effects on tumor growth in vivo were determined in immune-proficient and -deficient mice and alterations of immune cell infiltrations were analyzed with flow cytometry. Tumor-associated macrophages (TAMs) were further investigated with RNA-seq and flow cytometry. In addition, we confirmed olaparib's effect on human TAMs. KEY FINDINGS: Olaparib did not affect HR-proficient tumor cell proliferation and survival in vitro. However, olaparib significantly decreased tumor growth in C57BL/6 and SCID-beige mice (defective in lymphoid development and NK cell activity). Olaparib increased macrophage numbers in the tumor microenvironment, and their depletion diminished the anti-tumor effects of olaparib in vivo. Further analysis revealed that olaparib improved TAM-associated phagocytosis of cancer cells. Notably, this enhancement was not solely reliant on the "Don't Eat Me" CD47/SIRPα signal. In addition, compared to monotherapy, the concomitant administration of αCD47 antibodies with olaparib improved tumor control. SIGNIFICANCE: Our work provides evidence for broadening the application of PARPi in HR-proficient cancer patients and paves the way for developing novel combined immunotherapy to upgrade the anti-tumor effects of macrophages.

Spleen-selective co-delivery of mRNA and TLR4 agonist-loaded LNPs for synergistic immunostimulation and Th1 immune responses.

Spleen is an ideal site for initiating and amplifying antigen-specific immune response. However, spleen-selective antigen delivery has limited tumor therapeutic efficacy owing to an inadequate cytotoxic T-cell immune response. In this study, we designed a spleen-selective mRNA vaccine that delivered unmodified mRNA and Toll-like Receptor (TLR) agonists to the spleen after systemic administration, resulting in a sufficient and persistent antitumor cellular immune response with potent tumor immunotherapeutic efficacy. To establish potent tumor vaccines (sLNPs-OVA/MPLA), we co-loaded stearic acid doped lipid nanoparticles with ovalbumin (OVA)-coding mRNA and TLR4 agonists (MPLA). We found that sLNPs-OVA/MPLA facilitated tissue-specific mRNA expression in the spleen after intravenous injection and elicited enhanced adjuvant activity with Th1 immune responses by activating multiple TLRs. In a prophylactic mouse model, sLNPs-OVA/MPLA induced a potent antigen-specific cytotoxic T cell immune response and ultimately prevented the growth of EG.7-OVA tumors with persistent immune memory protection. In addition, sLNPs-OVA/MPLA effectively delayed the tumor growth of EG.7-OVA subcutaneously transplanted lymphoma and lung metastasis formation of B16F10-OVA intravenously injected melanoma. This study showed that the co-delivery of mRNA antigens and appropriate TLR agonists could significantly improve the antitumor immunotherapeutic efficacy of spleen-targeted mRNA vaccines via synergistic immunostimulation and Th1 immune responses.

Targeting CD276 by CAR-T cells induces regression of esophagus squamous cell carcinoma in xenograft mouse models.

Esophageal cancer, including esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), has a poor prognosis and limited therapeutic options. Chimeric antigen receptor (CAR)-T cells represent a potential ESCC treatment. In this study, we examined CD276 expression in healthy and esophageal tumor tissues and explored the tumoricidal potential of CD276-targeting CAR-T cells in ESCC. CD276 was strongly and homogenously expressed in ESCC and EAC tumor lesions but mildly in healthy tissues, representing a good target for CAR-T cell therapy. We generated CD276-directed CAR-T cells with a humanized antigen-recognizing domain and CD28 or 4-1BB co-stimulation. CD276-specific CAR-T cells efficiently killed ESCC tumor cells in an antigen-dependent manner both in vitro and in vivo. In patient-derived xenograft models, CAR-T cells induced tumor regression and extended mouse survival. In addition, CAR-T cells generated from patient T cells demonstrated potent cytotoxicity against autologous tumor cells. Our study indicates that CD276 is an attractive target for ESCC therapy, and CD276-targeting CAR-T cells are worth testing in ESCC clinical trials.

Tie2-expressing monocytes as a novel angiogenesis-related cellular biomarker for non-small cell lung cancer.

To investigate the clinical value of Tie2-expressing monocytes (TEMs) in the early diagnosis of lung cancer and assess its correlation with angiogenesis, a total of 184 patients with non-small cell lung cancer (NSCLC), 101 patients with benign pulmonary disease (BPD), and 77 healthy controls were enrolled in our study. The distribution of TEMs in lung tissue was determined by immunofluorescence staining. Lung microvascular density was assessed by immunohistochemical staining. Receiver-operating characteristic (ROC) curve analysis was performed to assess the diagnostic value of TEM frequency. Patients with NSCLC were followed up for 26 months. We found that the TEM frequency in peripheral blood monocytes of patients with NSCLC was significantly greater than that in patients with BPD and healthy controls. TEM frequency showed a correlation with NSCLC recurrence. The majority of TEMs in tumor tissues were localized around blood vessels; tumoral TEM frequency showed a positive correlation with microvascular density. High percentage of TEMs in the peripheral blood was associated with poor overall survival. ROC curve analysis revealed the potential diagnostic value of circulating TEM frequency in NSCLC. Thus, we believe that TEM frequency is related to angiogenesis in tumor tissues and may serve as a diagnostic marker for NSCLC.

CCL5-armed oncolytic virus augments CCR5-engineered NK cell infiltration and antitumor efficiency.

BACKGROUND: Natural killer (NK) cells have potent antitumor activities. Nevertheless, adoptive transfer therapy of NK cells has gained very limited success in patients with solid tumors as most infused NK cells remain circulating in the peripheral blood instead of entering tumor sites. Chemokines and their receptors play important roles in NK cell distribution. Enhancing chemokine receptors on immune cells to match and be driven to tumor-specific chemokines may improve the therapeutic efficacy of NK cells. METHODS: The CCR5-CCL5 axis is critical in NK cell homing to tumor sites. Thus, we analyzed CCR5 expression on NK cells from patients with cancer and healthy donors. We then upregulated CCR5 and CCL5 with lentiviruses and oncolytic viruses in NK and tumor cells, respectively. Animal experiments were also carried out to test the efficacy of the combination of oncolytic virus with NK cells. RESULTS: In NK cells from patients with various solid tumors or healthy subjects, CCR5 was expressed at low levels before and after expansion in vitro. CCR5-engineered NK cells showed enhanced tumor infiltration and antitumor effects, but no complete regressions were noted in the in vivo tumor models. To further improve therapeutic efficacy, we constructed CCL5-expressing oncolytic vaccinia virus. In vitro data demonstrated that vaccinia virus can produce CCL5 in tumor cells while infectivity remained unaffected. Supernatants from tumor cells infected by CCL5-modified vaccinia virus enhanced the directional movement of CCR5-overexpressed NK cells but not green fluorescent protein (GFP)-expressing cells. More importantly, NK cells were resistant to the vaccinia virus and their functions were not affected after being in contact. In vivo assays demonstrated that CCL5-expressing vaccinia virus induced a greater accumulation of NK cells within tumor lesions compared with that of the prototype virus. CONCLUSION: Enhancement of matched chemokines and chemokine receptors is a promising method of increasing NK cell homing and therapeutic effects. Oncolytic vaccinia viruses that express specific chemokines can synergistically augment the efficacies of NK cell-based therapy.

TNF Receptor 2 Makes Tumor Necrosis Factor a Friend of Tumors.

Tumor necrosis factor (TNF) is widely accepted as a tumor-suppressive cytokine via its ubiquitous receptor TNF receptor 1 (TNFR1). The other receptor, TNFR2, is not only expressed on some tumor cells but also on suppressive immune cells, including regulatory T cells and myeloid-derived suppressor cells. In contrast to TNFR1, TNFR2 diverts the tumor-inhibiting TNF into a tumor-advocating factor. TNFR2 directly promotes the proliferation of some kinds of tumor cells. Also activating immunosuppressive cells, it supports immune escape and tumor development. Hence, TNFR2 may represent a potential target of cancer therapy. Here, we focus on expression and role of TNFR2 in the tumor microenvironment. We summarize the recent progress in understanding how TNFR2-dependent mechanisms promote carcinogenesis and tumor growth and discuss the potential value of TNFR2 in cancer treatment.

PPMP, a novel tubulin-depolymerizing agent against esophageal cancer in patient-derived tumor xenografts.

Esophageal cancer is one of the least studied and deadliest cancers worldwide with a poor prognosis due to limited options for treatment. Chemotherapy agents such as the microtubule-targeting compounds are the mainstay of palliation for advanced esophageal cancer treatment. However, the toxicity and side effects of tubulin-binding agents (TBAs) have promoted the development of novel, more potent but less toxic TBAs. Herein, we identified 2-[4-(3,4-dimethoxyphenyl)-3-methyl-1H-pyrazol-5-yl]-5-[(2-methylprop-2-en-1-yl)oxy] phenol (PPMP) as a novel TBA for esophageal cancer treatment. PPMP markedly inhibited tubulin polymerization, and decreased viability and anchorage-independent growth of esophageal cancer cell lines, effects that were accompanied by G2/M arrest and apoptosis. Importantly, we produced patient-derived esophageal cancer xenografts to evaluate the therapeutic effect of PPMP in a setting that best mimics the clinical context in patients with esophageal cancer. Overall, we identified PPMP as a novel microtubule-destabilizing compound and as a new therapeutic agent against esophageal carcinoma.

Circulating Prostaglandin Biosynthesis in Colorectal Cancer and Potential Clinical Significance.

BACKGROUND: Colorectal cancer (CRC) represents the third leading cause of cancer-related death in the United States. Lack of reliable biomarkers remains a critical issue for early detection of CRC. In this study, we investigated the potential predictive values of circulating prostaglandin (PG) biosynthesis in CRC risk. METHODS: Profiles of circulating PG biosynthesis and platelet counts were determined in healthy subjects (n = 16), familial adenomatous polyposis (FAP) patients who were classified as regular aspirin users (n = 14) or nonusers (n = 24), and CRC patients with (n = 18) or without FAP history (n = 20). Immunohistochemistry staining was performed on biopsy samples. RESULTS: Analysis of circulating PG biosynthesis unexpectedly revealed that CRC progression is accompanied by a pronounced elevation of circulating thromboxane A2 (TXA2) levels. When a circulating TXA2 level of 1000 pg/mL was selected as a practical cutoff point, 95% of CRC patients were successfully identified. Further study suggested that the TXA2 pathway is constitutively activated during colorectal tumorigenesis and required for anchorage-independent growth of colon cancer cells. CONCLUSIONS: This study established the importance of the TXA2 pathway in CRC pathophysiology, and laid the groundwork for introducing a TXA2-targeting strategy to CRC prevention, early detection and management.

Caffeic acid directly targets ERK1/2 to attenuate solar UV-induced skin carcinogenesis.

Caffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical present in coffee and reportedly has anticancer activities. However, the underlying molecular mechanisms and targeted proteins involved in the suppression of carcinogenesis by caffeic acid are not fully understood. In this study, we report that caffeic acid significantly inhibits colony formation of human skin cancer cells and EGF-induced neoplastic transformation of HaCaT cells dose-dependently. Caffeic acid topically applied to dorsal mouse skin significantly suppressed tumor incidence and volume in a solar UV (SUV)-induced skin carcinogenesis mouse model. A substantial reduction of phosphorylation in mitogen-activated protein kinase signaling was observed in mice treated with caffeic acid either before or after SUV exposure. Caffeic acid directly interacted with ERK1/2 and inhibited ERK1/2 activities in vitro. Importantly, we resolved the cocrystal structure of ERK2 complexed with caffeic acid. Caffeic acid interacted directly with ERK2 at amino acid residues Q105, D106, and M108. Moreover, A431 cells expressing knockdown of ERK2 lost sensitivity to caffeic acid in a skin cancer xenograft mouse model. Taken together, our results suggest that caffeic acid exerts chemopreventive activity against SUV-induced skin carcinogenesis by targeting ERK1 and 2.

3,6,2',4',5'-Pentahydroxyflavone, an orally bioavailable multiple protein kinase inhibitor, overcomes gefitinib resistance in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most lethal cancer, causing more than 150,000 deaths in the United States in 2013. The receptor tyrosine kinase inhibitors such as gefitinib are not perfect clinical therapeutic agents for NSCLC treatment due to primary or acquired tyrosine kinase inhibitor resistance. Herein, 3,6,2',4',5'-pentahydroxyflavone (36245-PHF) was identified as a multiple kinase inhibitor for NSCLC treatment based on the computational screening of a natural products database. 36245-PHF was shown to inhibit PI3K and Aurora A and B kinases and overcome gefitinib-resistant NSCLC growth. Our data clearly showed that 36245-PHF markedly inhibited anchorage-independent growth of gefitinib-resistant NSCLC cell lines and exerted a substantial chemotherapeutic effect following oral administration in a gefitinib-resistant NSCLC xenograft model. The evidence from three different subsequent methodological approaches, in vitro, ex vivo, and in vivo, all confirmed that 36245-PHF as a multiple protein kinase inhibitor. Overall, we identified 36245-PHF as a multiple protein kinase inhibitor and as a novel therapeutic agent to overcome gefitinib-resistant NSCLC growth, which could provide a new option for clinical NSCLC oral treatment.

TRAF4 is a critical molecule for Akt activation in lung cancer.

TRAF4 is an adapter protein overexpressed in certain cancers, but its contributions to tumorigenesis are unclear. In lung cancer cells and primary lung tumors, we found that TRAF4 is overexpressed. RNA interference-mediated attenuation of TRAF4 expression blunted the malignant phenotype in this setting, exerting inhibitory effects on cell proliferation, anchorage-independent growth, and tumor development in a xenograft mouse model. Unexpectedly, we discovered that TRAF4, but not Skp2, was required for activation of the pivotal cell survival kinase Akt through ubiquitination. Furthermore, TRAF4 attenuation impaired glucose metabolism by inhibiting expression of Glut1 and HK2 mediated by the Akt pathway. Overall, our work suggests that TRAF4 offers a candidate molecular target for lung cancer prevention and therapy.

Construction of a novel oncolytic adenoviral vector and its biological characteristics.

In this study, we aimed to construct an effective and safe oncolytic adenoviral vector for cancer treatment with gene therapy. First, the promoter of the catalytic subunit of human telomerase (hTERTp), adenovirus early region 1a gene (E1A) and thymidine kinase gene of human herpes virus type 1 (HSV-1-TK) were amplified by using PCR from genomic DNA of 293A cells and wild-type HSV-1 (wHSV-1). These specially-prepared elements were inserted into an adenoviral shuttle vector in the opposite and the same directions of left inverted terminal repeat (L-ITR), respectively, to construct pENTR-E1A-IRES-TK-hTERTp (pEITH) and pENTR-hTERTp-E1A-IRES-TK (pHEIT). LR reaction between adenoviral shuttle vectors (pEITH and pHEIT) and the backbone vector DEST was carried out to establish adenoviral expression vectors pAd-E1A-IRES-TK-hTERTp (pAd-EITH) and pAd-hTERTp-E1A-IRES-TK (pAd-HEIT). Recombinant adenovirus Ad-EITH and Ad-HEIT were produced by transfecting 293A cells and purified for the subsequent studies of titer measurement, replication capability with and without acyclovir (ACV) and antitumor ability with and without ganciclovir (GCV) to evaluate the biological characteristics. Adenoviral shuttle vectors pEITH and pHEIT and expression vectors pAd-EITH and pAd-HEIT were successfully constructed, and recombinant adenoviruses Ad-EITH and Ad-HEIT with high titer were produced. The results of replication and cytotoxicity assays showed that Ad-EITH and Ad-HEIT replicated in the hTERTp (+) human nasopharyngeal carcinoma cell line CNE and expressed the TK gene effectively leading to the death of tumor cells. In addition, there were still some Ad-HEIT particles replicating in the hTERTp (-) human osteosarcoma U-2OS cells and human lung HFL-1 fibroblasts compared to Ad-EITH which was hardly able to replicate in U-2OS and HFL-1 cells. In addition, we also observed an interesting phenomenon, that the replication of Ad-EITH could be inhibited by antiviral drug ACV on account of the expression of HSV-1-TK gene making Ad-EITH sensitive to ACV. In conclusion, a novel oncolytic adenoviral vector Ad-EITH was produced which can be used for cancer-specific and efficient viral replication, and its safety is potentially improved as replication can be inhibited by ACV in vitro.

Increased Nanog expression promotes tumor development and Cisplatin resistance in human esophageal cancer cells.

BACKGROUND/AIMS: Nanog plays a key role in stem cell self-renewal and pluripotency differentiation in embryonic stem cells ( ESCs). Recently, some studies reported that abnormal expression of Nanog could be detected in several tumors, indicating that Nanog might be related to tumor development. However, studies on the correlation between Nanog expression and esophageal cancer are sparse. METHODS: In this study, we established two esophageal cancer cell lines 9706-Nanog and 9706-shNanog which stably expressed Nanog and Nanog-short-hairpin RNA (shRNA) genes. RESULTS: We found that Nanog expression could promote the proliferation and invasiveness of the cancer cells, and inhibit the apoptosis. We also treated 9706-Nanog, EC9706 and 9706-shNanog cell lines with cisplatin and evaluated the drug sensitivity of the three cell lines. We found that the sensitivity of cisplatin was decreased with increased expression of Nanog. The expression of MDR-1 was also increased in 9706Nanog cells. CONCLUSIONS: Nanog may play an important role in human esophageal cancer development, and could be used as a therapeutic target in esophageal cancer treatment.

Pilot and pivotal study to evaluate the bioequivalence of two paroxetine 40 mg tablet formulations in healthy Chinese subjects.

The purpose of this study was to conduct a pilot study in order to obtain reliable results for further planning of a well-designed pivotal trial comparing the bioequivalence (BE) of two paroxetine tablet formulations in healthy Chinese subjects. Before conducting the pivotal trial, the pilot trial enrolled 14 subjects to help in study design, establishing the recruitment period, determining pharmacokinetics (PK) time points and sample size, and assessing BE of the two formulations. The single-center, randomized, open-label, single-dose, two period crossover study with a 7-day washout interval was conducted after obtaining information from the fasted pilot trial in 72 healthy volunteers for a pivotal study under fed and fasted conditions, respectively. There were 19 PK sample collection time points employed in both the pilot and pivotal trials. A sensitive and specific liquid chromatography- tandem mass spectrometry (LC-MS/ MS) method was developed and validated for determining paroxetine in human plasma. BE between two articles was determined by calculating 90% confidence intervals (CIs) for the ratio of Cmax 91.38 - 110.39% for the pilot trial, 99.81 - 114.08% for pivotal trial under fasted condition, and 94.06 - 110.41% for pivotal trial under fed condition, AUC(0-t) 96.06 - 110.52% for pilot study, 100.88 - 113.05% for the pivotal trial under fasted condition, and 97.08 - 106.06% for pivotal study under fed condition, and AUC(0-∞) 96.17 - 110.42% for the pilot study, 100.85 - 112.81% for the pivotal trial under fasted condition and 97.22 - 106.14% for the pivotal study under fed condition, respectively. These values for the test and reference products are within the 80 - 125% interval proposed by FDA and EMEA. It was concluded that the proposed method was successfully applied to a PK study in healthy Chinese volunteers, and results showed from both the pilot and pivotal studies that the two paroxetine formulations are bioequivalent in their rates and extent of absorption.