Expression In Epithelial Cancer Research Articles

Generic semi-automated radiofluorination strategy for single domain antibodies: [18F]FB-labelled single domain antibodies for PET imaging of fibroblast activation protein-α or folate receptor-α overexpression in cancer

BackgroundRadiofluorination of single domain antibodies (sdAbs) via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) has shown to be a promising strategy in the development of sdAb-based PET tracers. While automation of the prosthetic group (PG) [18F]SFB production, has been successfully reported, no practical method for large scale sdAb labelling has been reported. Therefore, we optimized and automated the PG production, enabling a subsequently efficient manual conjugation reaction to an anti-fibroblast activation protein (FAP)-α sdAb (4AH29) and an anti-folate receptor (FR)-α sdAb (2BD42). Both the alpha isoform of FAP and the FR are established tumour markers. FAP-α is known to be overexpressed mainly by cancer-associated fibroblasts in breast, ovarian, and other cancers, while its expression in normal tissues is low or undetectable. FR-α has an elevated expression in epithelial cancers, such as ovarian, brain and lung cancers. Non-invasive imaging techniques, such as PET-imaging, using tracers targeting specific tumour markers can provide molecular information over both the tumour and its environment, which aides in the diagnosis, therapy selection and assessment of the cancer treatment.Results[18F]SFB was synthesized using a fully automated three-step, one-pot reaction. The total procedure time was 54 min and results in [18F]SFB with a RCP > 90% and a RCY d.c. of 44 ± 4% (n = 13). The manual conjugation reaction after purification produced [18F]FB-sdAbs with a RCP > 95%, an end of synthesis activity > 600 MBq and an apparent molar activity > 10 GBq/µmol. Overall RCY d.c., corrected to the trapping of [18F]F− on the QMA, were 9% (n = 1) and 5 ± 2% (n = 3) for [18F]FB-2BD42 and [18F]FB-4AH29, respectively.Conclusion[18F]SFB synthesis was successfully automated and upscaled on a Trasis AllInOne module. The anti-hFAP-α and anti-hFR-α sdAbs were radiofluorinated, yielding similar RCYs d.c. and RCPs, showing the potential of this method as a generic radiofluorination strategy for sdAbs. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging.

High TSPAN8 expression in epithelial cancer cell-derived small extracellular vesicles promote confined diffusion and pronounced uptake.

Small extracellular vesicles (sEVs) play a key role in intercellular communication. Cargo molecules carried by sEVs may affect the phenotype and function of recipient cells. Epithelial cancer cell‐derived sEVs, particularly those enriched in CD151 or tetraspanin8 (TSPAN8) and associated integrins, promote tumour progression. The mechanism of binding and modulation of sEVs to recipient cells remains elusive. Here, we used genetically engineered breast cancer cells to derive TSPAN8‐enriched sEVs and evaluated the impact of TSPAN8 on target cell membrane's diffusion and transport properties. The single‐particle tracking technique showed that TSPAN8 significantly promoted sEV binding via confined diffusion. Functional assays indicated that the transgenic TSPAN8‐sEV cargo increased cancer cell motility and epithelial‐mesenchymal transition (EMT). In vivo, transgenic TSPAN8‐sEV promoted uptake of sEVs in the liver, lung, and spleen. We concluded that TSPAN8 encourages the sEV‐target cell interaction via forced confined diffusion and significantly increases cell motility. Therefore, TSPAN8‐sEV may serve as an important direct or indirect therapeutic target.

Evolution of Thyroglobulin Loop Kinetics in EpCAM

Epithelial cell-activating molecule (EpCAM) is an important cancer biomarker and therapeutic target given its elevated expression in epithelial cancers. EpCAM is a type I transmembrane protein that forms cis-dimers along the thyroglobulin type-1A-like domain (TYD) in the extracellular region. The thyroglobulin loop (TY loop) within the TYD is structurally dynamic in the monomer state of human EpCAM, binding reversibly to a TYD site. However, it is not known if this flexibility is prevalent across different species. Here, we conduct over 17 μs of all-atom molecular dynamics simulations to study EpCAM TY loop kinetics of five different species, including human, mouse, chicken, frog, and fish. We find that the TY loop remains dynamic across evolution. In addition to the TYD binding site, we discover a second binding site for the TY loop in the C-terminal domain (CTD). Calculations of the dissociation rate constants from the simulation trajectories suggest a differential binding pattern of fish EpCAM and other organisms. Whereas fish TY loop has comparable binding for both TYD and CTD sites, the TY loops of other species preferably bind the TYD site. A hybrid construct of fish EpCAM with human TY loop restores the TYD binding preference, suggesting robust effects of the TY loop sequence on its dynamic behavior. Our findings provide insights into the structural dynamics of EpCAM and its implication in physiological functions.

Formulation of Medium Viscosity Chitosan-Pectin –MJ Protein Nanoparticles Conjugated with Anti-Ep-CAM and Its Cytotoxicity Against T47D Breast Cancer Cell Lines

Chitosan nanoparticle could become potential formula to protect protein degradation during therapy,since chitosan nanoparticles have “proton sponge hypothesis” mechanism on its protection. Chitosan and pectinis used as basic formula of drug delivery because of its biodegradable and biocompatible properties. Chitosanpectin nanoparticles can be formulated by polyelectrolit complex. EpCAM showed excessive expression inepithelial cancer cells thus can be used as a therapeutic biomarker. MJ protein, a Ribosome-Inactivating Proteins(RIPs) isolated from Mirabilis jalapa L had a higher cytotoxicity on malignant cells than normal cells. MJ proteinneed to be formulated to protect from proteosome degradation in endosome. The aim of this research was todevelop MJ protein-chitosan-pectin nanoparticles and conjugated with anti EpCAM for breast cancer therapy.Mj protein was extracted from M.jalapa leaves. RIPs activity was assayed by supercoiled DNA cleavage. MJprotein were loaded into chitosan nanoparticles using medium viscous chitosan and pectin as cross-linker withpolyelectrolit complex method. Anti EpCAM was conjugated to MJ protein-chitosan-pectin nanoparticles bycarbodiimide reaction and characterized for its entrapment efficiency, morphology by transmission electronmicroscope, particles size, and zeta potential. MJ protein nanoparticles conjugated anti EpCAM and withoutanti EpCAM were cytotoxicity assayed toward T47D and Vero cell lines. MJ protein was able to cleave thesupercoiled DNA into linear and nicked-circular ones. The nanoparticles optimal concentration of mediumviscous chitosan: MJ protein: pectin was 0.01%: 0.01%: 1% (m/v). A high entrapment efficiency of MJ proteinnanoparticles was 98.97 ± 0.07%. Morphology nanoparticles showed an amorphic structure with 200.00 nmparticles size. The nanoparticles conjugated anti EpCAM showed average particles size 367.67nm, polydispersityindex 0.332, and zeta potential +39.97mV. MJ protein-chitosan-pectin nanoparticles conjugated anti EpCAMand unconjugated both had higher cytotoxicity with the IC50 57.64 μg/mL and 46.84 μg/mL respectivelyagainst T47D and 99.38 μg/mL and 111.34 μg/mL against Vero cell lines compared to MJ protein with IC50 of3075.61 μg/mL against T47D and 3286.88 μg/mL against Vero cell lines. Both MJ protein-nanoparticles couldincrease the cytotoxicity effects about 50 times compared to the unformulated MJ protein activity, howeverhad less specificity toward T47D and Vero cell lines.

Prognostic Significance of FRA Expression in Epithelial Cancers using AQUA ® technology

Although agents that target FRA have advanced through clinical trials, comprehensive analyses of FRA expression in epithelial cancers compared with clinical variables and prognosis are limited. FRA expression was examined in non-small-cell lung cancer (NSCLC), ovarian cancer and endometrial cancer cohorts using AQUA(®) technology. For the NSCLC cohort, FRA expression was significantly higher in adenocarcinoma samples (p < 0.001) than other histologies, and in females (p = 0.003) versus males. High FRA expression was significantly associated with better survival in NSCLC cases (p = 0.01) while significantly and independently associated with worse prognosis in endometrial (p < 0.001) and ovarian cancers (p < 0.001). These studies confirm the prognostic value of FRA in multiple indications. The opposing prognostic effects observed may suggest differential biology.

Effect of short hairpin RNA targeting of fibroblast activation protein on tumor growth and angiogenesis in a 4T1 murine breast cancer model.

e13538 Background: Fibroblast activation protein(FAP) is an increased abnormal signal protein in the TME and has both dipeptidyl peptidase and endopeptidase activity. FAP has been recognized to influence cancer growth and invasion, through its non- enzyme function or proteolytic activities in concert with other moleculars such as CD26, uPAR, β1-integrin, and MMP-1. Since FAP is over expression in epithelial cancers and absent from normal adult tissues, it has long been suggested to be a target of antitumor therapy. Methods: 21 BALB/C female mice implanted with 4T1 breast cancer were enrolled and randomly divided into three groups (n=7 per group) .The experiment group(E-group) was administered with shRNA targeting of FAP every 48 hours in 2 weeks by local administration, and the two control groups with HK or 5%GS. Tumor volumes and weights were monitored. Expression of FAP was detected by RT-PCR and immunohistochemistry staining. Collagen fiber, microvessel density and cell apoptosis were investigated. Results: The expression of FAP was reduced about 50% by shRNA targeting of FAP; the tumor volume and weight of the E-group showed significant reduction of 40% compared with the controls (p=0.001). Knockdown of FAP increased accumulation of disorganized collagen fibers; the MVD in control groups was about 4 times at least compared to the E-group (p&lt;0.001) and apoptosis cells markedly increased 3 fold (p=0.0015). Conclusions: Knockdown of FAP by shRNA interference mediated inhibition of breast cancer growth, amelioration of the tumor microenvironment including increased accumulation of disorganized collagen, suppressed angiogenesis, and promotion of apoptosis. FAP contributes to the formation of TME and is a stimulative factor of breast cancer growth. Target of FAP may be a potential supplemental therapeutic method for breast cancer.

M30 Neoepitope Expression in Epithelial Cancer: Quantification of Apoptosis in Circulating Tumor Cells by CellSearch Analysis

This study aimed to detect the M30 neoepitope on circulating tumor cells (CTC) as a tool for quantifying apoptotic CTC throughout disease course and treatment. An automated sample preparation and analysis platform for computing CTC (CellSearch) was integrated with a monoclonal antibody (M30) targeting a neoepitope disclosed by caspase cleavage at cytokeratin 18 (CK18) in early apoptosis. The assay was validated using cell lines and blood samples from healthy volunteers and patients with epithelial cancer. M30-positive CTC could be detected in >70% of CTC-positive carcinoma patients, which were free for both chemotherapy and radiologic treatments. The fraction of M30-positive CTC varied from 50% to 80%, depending on the histotype. To investigate the potential application of the M30 CTC assay for the evaluation of response in early phase trials, CTC and M30-positive CTC were enumerated in a small case series of breast cancer patients during treatment. Results indicate that changes in the balance of M30-negative/positive CTC may be used as a dynamic parameter indicating an active disease, as documented by consistent radiologic findings. M30 expression on CTC is detectable by immunofluorescence. The M30-integrated test has potential for monitoring dynamic changes in the quote of apoptotic CTC (in addition to CTC count) to evaluate response in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research, in which there is a pressing need for informative circulating biomarkers.

A COX-2 inhibitor combined with chemoradiation of locally advanced rectal cancer

Dear Editor: We would like to comment on the paper by Jacobsen et al., which examined the possible effects of adding a COX-2 inhibitor to an established preoperative chemoradiotherapy regimen in locally advanced colorectal cancer. It is very unfortunate that the severe dermatological reaction reported by the authors occurred as this clearly limited the interpretability and usefulness of this study. Selective COX-2 inhibitors have also been criticised for severe cardiovascular side effects, but clearly these were not seen in this study, probably due to compliance issues and short treatment duration. The principle behind this investigation does merit further study. The role of aberrant COX-2 expression in epithelial cancers, for example, breast and colorectal cancer, is undisputed. More controversial is the proposition that measures to inhibit COX-2 expression might be beneficial in preventing malignancy or treating actual cancer patients. Results of epidemiological observation studies of NSAID or COX-2 inhibitor use generally support a preventitive role, but prospective randomised studies using these agents have not supported these findings. This could be for a number of reasons, for example, inappropriate dose regimens and use of patients at low risk of developing the disease in prospective trials. Expression of COX-2 can be interrupted by a multitude of agents, including non-selective COX-2 inhibitors such as aspirin, and also other selective COX-2 inhibitors. Many experimental studies have indicated COX-2 suppression with these agents as a means of cancer treatment or prevention to be theoretically possible, although there are issues with dosages required, treatment duration and whether tumours can eventually “escape” from COX-2 inhibition. A repeat of this study with a larger number of patients and a safer method of COX-2 inhibition (for example, lower dose of agent or use of a non-selective inhibitor, e.g. aspirin), together with a placebo arm, would give more useful information. Int J Colorectal Dis (2009) 24:863 DOI 10.1007/s00384-009-0685-3

Collagenase-3 (MMP-13) expression in epithelial cancers of the eyelids

To determine, by means of immunohistochemistry, tumoral expression of collagenase-3, a matrix metalloproteinase linked to cancer and to basal cell and squamous cell carcinomas of the eyelid. Retrospective review of 23 basal cell carcinomas and 25 squamous cell carcinomas of the eyelid treated at different hospitals during the last five years. We evaluated collagenase-3 expression and the possible relationship to patient and tumour characteristics which included age, sex, histological type, tumour location and surgical margins status. 65% of the basal cell carcinomas and 88% of the squamous cell carcinomas of the eyelids stained positively for collagenase-3. In both cases, the immunostaining was localized in the cytoplasm of the malignant cells and, occasionally in the surrounding stromal cells. Statistical analysis showed no significant association between collagenase-3 immunostaining and patients or tumour characteristics but the expression of this protein was more intense in the epithelial tumoral cells located at the invading front which could explain the aggressive behaviour of this kind of tumours.

Identification of epidermal growth factor receptor-derived peptides recognised by both cellular and humoral immune responses in HLA-A24 + non-small cell lung cancer patients

The epidermal growth factor receptor (EGFR) is one of the most appropriate target molecules for cancer therapy because of its high expression in epithelial cancers. A novel EGFR-tyrosine-kinase inhibitor, ZD1839, has been approved as a drug for non-small cell lung cancer (NSCLC), and many other agents are now being tested in clinical trials. Cytotoxic T lymphocyte (CTL)-directed epitope peptides could be another class of useful compounds in EGFR-targeted therapies. However, at present, there are no data on CTL-directed peptides of EGFR. Therefore, this study aimed to identify immunogenic EGFR-derived peptides in HLA-A24 + NSCLC patients. We report in this study three such EGFR-derived peptides at positions 54–62, 124–132 and 800–809. These peptides were recognised by both cellular and humoral immune responses in most of the peripheral blood mononuclear cells (PBMCs) and sera from NSCLC patients that we tested. These results may provide a scientific basis for the development of EGFR-based immunotherapy.

Identification of epidermal growth factor receptor-derived peptides recognised by both cellular and humoral immune responses in HLA-A24+ non-small cell lung cancer patients*1

The epidermal growth factor receptor (EGFR) is one of the most appropriate target molecules for cancer therapy because of its high expression in epithelial cancers. A novel EGFR-tyrosine-kinase inhibitor, ZD1839, has been approved as a drug for non-small cell lung cancer (NSCLC), and many other agents are now being tested in clinical trials. Cytotoxic T lymphocyte (CTL)-directed epitope peptides could be another class of useful compounds in EGFR-targeted therapies. However, at present, there are no data on CTL-directed peptides of EGFR. Therefore, this study aimed to identify immunogenic EGFR-derived peptides in HLA-A24+ NSCLC patients. We report in this study three such EGFR-derived peptides at positions 54–62, 124–132 and 800–809. These peptides were recognised by both cellular and humoral immune responses in most of the peripheral blood mononuclear cells (PBMCs) and sera from NSCLC patients that we tested. These results may provide a scientific basis for the development of EGFR-based immunotherapy.