Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: Molecular identification and expression analysis

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNF alpha, LTA, TNFSF15 and fish TNFa proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LIPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamity molecules are present in marine invertebrates. (C) 2008 Elsevier Ltd. All rights reserved.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Evaluation of tumor response, disease control, progression-free survival, and time to progression as potential surrogate end points in metastatic breast cancer.

PURPOSE: Overall survival (OS) can be observed only after prolonged follow-up, and any potential effect of first-line therapies on OS may be confounded by the effects of subsequent therapy. We investigated whether tumor response, disease control, progression-free survival (PFS), or time to progression (TTP) could be considered a valid surrogate for OS to assess the benefits of first-line therapies for patients with metastatic breast cancer. PATIENTS AND METHODS: Individual patient data were collected on 3,953 patients in 11 randomized trials that compared an anthracycline (alone or in combination) with a taxane (alone or in combination with an anthracycline). Surrogacy was assessed through the correlation between the end points as well as through the correlation between the treatment effects on the end points. RESULTS: Tumor response (survival odds ratio [OR], 6.2; 95% CI, 5.3 to 7.0) and disease control (survival OR, 5.5; 95% CI, 4.8 to 6.3) were strongly associated with OS. PFS (rank correlation coefficient, 0.688; 95% CI, 0.686 to 0.690) and TTP (rank correlation coefficient, 0.682; 95% CI, 0.680 to 0.684) were moderately associated with OS. Response log ORs were strongly correlated with PFS log hazard ratios (linear coefficient [rho], 0.96; 95% CI, 0.73 to 1.19). Response and disease control log ORs and PFS and TTP log hazard ratios were poorly correlated with log hazard ratios for OS, but the confidence limits of rho were too wide to be informative. CONCLUSION: No end point could be demonstrated as a good surrogate for OS in these trials. Tumor response may be an acceptable surrogate for PFS.

Attenuation of inflammatory events in human intervertebral disc cells with a tumor necrosis factor antagonist.

STUDY DESIGN: The inflammatory responses of primary human intervertebral disc (IVD) cells to tumor necrosis factor α (TNF-α) and an antagonist were evaluated in vitro. OBJECTIVE: To investigate an ability for soluble TNF receptor type II (sTNFRII) to antagonize TNF-α-induced inflammatory events in primary human IVD cells in vitro. SUMMARY OF BACKGROUND DATA: TNF-α is a known mediator of inflammation and pain associated with radiculopathy and IVD degeneration. sTNFRs and their analogues are of interest for the clinical treatment of these IVD pathologies, although information on the effects of sTNFR on human IVD cells remains unknown. METHODS: IVD cells were isolated from surgical tissues procured from 15 patients and cultured with or without 1.4 nmol/L TNF-α (25 ng/mL). Treatment groups were coincubated with varying doses of sTNFRII (12.5-100 nmol/L). Nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-6 (IL6) levels in media were quantified to characterize the inflammatory phenotype of the IVD cells. RESULTS: Across all patients, TNF-α induced large, statistically significant increases in NO, PGE₂, and IL6 secretion from IVD cells compared with controls (60-, 112-, and 4-fold increases, respectively; P < 0.0001). Coincubation of TNF-α with nanomolar doses of sTNFRII significantly attenuated the secretion of NO and PGE₂ in a dose-dependent manner, whereas IL6 levels were unchanged. Mean IC₅₀ values for NO and PGE₂ were found to be 35.1 and 20.5 nmol/L, respectively. CONCLUSION: Nanomolar concentrations of sTNFRII were able to significantly attenuate the effects of TNF-α on primary human IVD cells in vitro. These results suggest this sTNFR to be a potent TNF antagonist with potential to attenuate inflammation in IVD pathology.

Hand-held spectroscopic device for in vivo and intraoperative tumor detection: contrast enhancement, detection sensitivity, and tissue penetration.

Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a hand-held spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2-5 × 10(-11) M for the indocyanine dye and 0.5-1 × 10(-13) M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5-10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.

Stereocomplexed poly(lactic acid)-poly(ethylene glycol) nanoparticles with dual-emissive boron dyes for tumor accumulation.

Responsive biomaterials play important roles in imaging, diagnostics, and therapeutics. Polymeric nanoparticles (NPs) containing hydrophobic and hydrophilic segments are one class of biomaterial utilized for these purposes. The incorporation of luminescent molecules into NPs adds optical imaging and sensing capability to these vectors. Here we report on the synthesis of dual-emissive, pegylated NPs with "stealth"-like properties, delivered intravenously (IV), for the study of tumor accumulation. The NPs were created by means of stereocomplexation using a methoxy-terminated polyethylene glycol and poly(D-lactide) (mPEG-PDLA) block copolymer combined with iodide-substituted difluoroboron dibenzoylmethane-poly(L-lactide) (BF2dbm(I)PLLA). Boron nanoparticles (BNPs) were fabricated in two different solvent compositions to study the effects on BNP size distribution. The physical and photoluminescent properties of the BNPs were studied in vitro over time to determine stability. Finally, preliminary in vivo results show that stereocomplexed BNPs injected IV are taken up by tumors, an important prerequisite to their use as hypoxia imaging agents in preclinical studies.

Performance metrics of an optical spectral imaging system for intra-operative assessment of breast tumor margins.

As many as 20-70% of patients undergoing breast conserving surgery require repeat surgeries due to a close or positive surgical margin diagnosed post-operatively [1]. Currently there are no widely accepted tools for intra-operative margin assessment which is a significant unmet clinical need. Our group has developed a first-generation optical visible spectral imaging platform to image the molecular composition of breast tumor margins and has tested it clinically in 48 patients in a previously published study [2]. The goal of this paper is to report on the performance metrics of the system and compare it to clinical criteria for intra-operative tumor margin assessment. The system was found to have an average signal to noise ratio (SNR) >100 and <15% error in the extraction of optical properties indicating that there is sufficient SNR to leverage the differences in optical properties between negative and close/positive margins. The probe had a sensing depth of 0.5-2.2 mm over the wavelength range of 450-600 nm which is consistent with the pathologic criterion for clear margins of 0-2 mm. There was <1% cross-talk between adjacent channels of the multi-channel probe which shows that multiple sites can be measured simultaneously with negligible cross-talk between adjacent sites. Lastly, the system and measurement procedure were found to be reproducible when evaluated with repeated measures, with a low coefficient of variation (<0.11). The only aspect of the system not optimized for intra-operative use was the imaging time. The manuscript includes a discussion of how the speed of the system can be improved to work within the time constraints of an intra-operative setting.

Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.

BACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.

Targeting A20 decreases glioma stem cell survival and tumor growth.

Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.

A Plasmonic Gold Nanostar Theranostic Probe for In Vivo Tumor Imaging and Photothermal Therapy.

Nanomedicine has attracted increasing attention in recent years, because it offers great promise to provide personalized diagnostics and therapy with improved treatment efficacy and specificity. In this study, we developed a gold nanostar (GNS) probe for multi-modality theranostics including surface-enhanced Raman scattering (SERS) detection, x-ray computed tomography (CT), two-photon luminescence (TPL) imaging, and photothermal therapy (PTT). We performed radiolabeling, as well as CT and optical imaging, to investigate the GNS probe's biodistribution and intratumoral uptake at both macroscopic and microscopic scales. We also characterized the performance of the GNS nanoprobe for in vitro photothermal heating and in vivo photothermal ablation of primary sarcomas in mice. The results showed that 30-nm GNS have higher tumor uptake, as well as deeper penetration into tumor interstitial space compared to 60-nm GNS. In addition, we found that a higher injection dose of GNS can increase the percentage of tumor uptake. We also demonstrated the GNS probe's superior photothermal conversion efficiency with a highly concentrated heating effect due to a tip-enhanced plasmonic effect. In vivo photothermal therapy with a near-infrared (NIR) laser under the maximum permissible exposure (MPE) led to ablation of aggressive tumors containing GNS, but had no effect in the absence of GNS. This multifunctional GNS probe has the potential to be used for in vivo biosensing, preoperative CT imaging, intraoperative detection with optical methods (SERS and TPL), as well as image-guided photothermal therapy.

HPV16 antibodies as risk factors for oropharyngeal cancer and their association with tumor HPV and smoking status.

BACKGROUND: Antibodies (Abs) to the HPV16 proteome increase risk for HPV-associated OPC (HPVOPC). The goal of this study was to investigate the association of a panel of HPV16 Abs with risk for OPC as well as the association of these Abs with tumor HPV and smoking status among patients with OPC. METHODS: IgG Abs to the HPV16 antigens E1, E2, E4, E5, E6, E7, L1, L2 were quantified using a programmable ELISA assay. Sera were obtained from 258 OPC patients at diagnosis and 250 healthy controls. HPV16 tumor status was measured by PCR for 137 cases. Multivariable logistic regression was used to calculate odds ratios for the association of HPV16 Abs with risk for OPC. RESULTS: HPV16 E1, E2, E4, E5, E6, E7 and L1-specific IgG levels were elevated in OPC patients compared to healthy controls (p<0.05). After multivariable adjustment, Ab positivity for NE2, CE2, E6, and/or E7 was associated with OPC risk (OR [95% CI], 249.1 [99.3-624.9]). Among patients with OPC, Ab positivity for these antigens was associated with tumor HPV status, especially among never or light smokers (OR [95% CI], 6.5 [2.1-20.1] and OR [95% CI], 17.5 [4.0-77.2], respectively). CONCLUSIONS: Antibodies to HPV16 proteins are associated with increased risk for HPVOPC. Among patients with OPC, HPV16 Abs are associated with tumor HPV status, in particular among HPV positive patients with no or little smoking history.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.

Impact of tumor board recommendations on treatment outcome for locally advanced head and neck cancer

Background/Aims: To identify physician selection factors in the treatment of locally advanced head and neck cancer and how treatment outcome is affected by Tumor Board recommendations. Methods: A retrospective analysis of 213 patients treated for locally advanced head and neck cancer in a single institution was performed. All treatments followed Tumor Board recommendations: 115 patients had chemotherapy and radiation, and 98 patients received postoperative radiation. Patient characteristics, treatment toxicity, locoregional control and survival between these two treat- ment groups were compared. Patient survival was compared with survival data reported in randomized studies of locally advanced head and neck cancer. Results: There were no differences in comorbidity factors, and T or N stages between the two groups. A statistically significant number of patients with oropharyngeal and oral cavity tumors had chemoradiation and postoperative radiation, respectively (p < 0.0001). Grade 3-4 toxicities during treatment were 48 and 87% for the postoperative radiation and chemoradiation groups, respectively (p = 0.0001). There were no differences in survival, locoregional recurrences and distant metastases between the two groups. Patient survival was comparable to survival rates reported by randomized studies of locally advanced head and neck cancer. Conclusion: Disease sites remained the key determining factor for treatment selection. Multidisciplinary approaches provided optimal treatment outcome for locally advanced head and neck cancer, with overall survival in these patients being comparable to that reported in randomized clinical trials. Copyright © 2008 S. Karger AG.