Aberrant activation of the Hedgehog signalling pathway in squamous cell carcinoma of the vulva as a potential target for cancer therapy

In a previous study, we showed that the Hedgehog (Hh) signalling pathway is aberrantly activated in vulval squamous cell carcinoma (VSCC). In this study, we further validated our findings on a prospective cohort of primary VSCC cases, where immunohistochemical staining confirmed that key Hh pathway components were overexpressed in VSCC compared to normal vulval epithelium. We also undertook a series of in vitro studies to determine the extent of Hh pathway activation in VSCC-derived cell lines, and examine the consequences of pathway inhibition on the growth of these cells. We found that of six cell lines tested, four displayed elevated baseline Hh pathway activity that was dependent on SHH ligand, or in one case, a PTCH1 gene mutation. Hh signalling appeared necessary to sustain cell growth, as SHH ligand depletion with Robotikinin or SMO inhibition, either with chemical inhibitors (Itraconazole or LDE-225) or SMO-specific siRNA, attenuated GLI1 activity and cell proliferation in both monolayer and organotypic raft culture. Furthermore, treatment of Hh-dependent cell lines with SMO inhibitors sensitised cells to Cisplatin. Findings from our study offer us the opportunity to explore further the development of targeted chemotherapy for women with VSCC driven by aberrant Hh activation.

The Major Constituent of Green Tea, Epigallocatechin-3-Gallate (EGCG), Inhibits the Growth of HPV18-Infected Keratinocytes by Stimulating Proteasomal Turnover of the E6 and E7 Oncoproteins

Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.

A Novel In Vitro Culture Model System to Study Merkel Cell Polyomavirus–Associated MCC Using Three-Dimensional Organotypic Raft Equivalents of Human Skin

Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro using an organotypic (raft) culture system that is traditionally used to recapitulate the dermal and epidermal equivalents of skin in three dimensions (3D). In the optimal culture condition, MCPyV+ MCC cells were embedded in collagen between the epidermal equivalent comprising human keratinocytes and a dermal equivalent containing fibroblasts, resulting in MCC-like lesions arising within the dermal equivalent. The presence and organization of MCC cells within these dermal lesions were characterized through biomarker analyses. Interestingly, co-culture of MCPyV+ MCC together with keratinocytes specifically within the epidermal equivalent of the raft did not reproduce human MCC morphology, nor were any keratinocytes necessary for MCC-like lesions to develop in the dermal equivalent. This 3D tissue culture system provides a novel in vitro platform for studying the role of MCPyV T antigens in MCC oncogenesis, identifying additional factors involved in this process, and for screening potential MCPyV+ MCC therapeutic strategies.

Combining Segmentation Network and Nonsubsampled Contourlet Transform for Automatic Marine Raft Aquaculture Area Extraction from Sentinel-1 Images

Marine raft aquaculture (MFA) plays an important role in the marine economy and ecosystem. With the characteristics of covering a large area and being sparsely distributed in sea area, MFA monitoring suffers from the low efficiency of field survey and poor data of optical satellite imagery. Synthetic aperture radar (SAR) satellite imagery is currently considered to be an effective data source, while the state-of-the-art methods require manual parameter tuning under the guidance of professional experience. To preclude the limitation, this paper proposes a segmentation network combined with nonsubsampled contourlet transform (NSCT) to extract MFA areas using Sentinel-1 images. The proposed method is highlighted by several improvements based on the feature analysis of MFA. First, the NSCT was applied to enhance the contour and orientation features. Second, multiscale and asymmetric convolutions were introduced to fit the multisize and strip-like features more effectively. Third, both channel and spatial attention modules were adopted in the network architecture to overcome the problems of boundary fuzziness and area incompleteness. Experiments showed that the method can effectively extract marine raft culture areas. Although further research is needed to overcome the problem of interference caused by excessive waves, this paper provides a promising approach for periodical monitoring MFA in a large area with high efficiency and acceptable accuracy.

Integrating Greenhouse Cherry Tomato Production with Biofloc Tilapia Production

Integration of intensive aquaculture systems with greenhouse plant production has been shown to improve aquaculture water quality conditions and improve plant nutrient use efficiency. The majority of the focus of integrated systems has involved raft culture or true hydroponics. Little work has been done on soilless culture utilizing drip irrigation. This study investigates the feasibility of integrating biofloc Nile tilapia (Oreochromis niloticus) production with greenhouse cherry tomato production (Solanum lycopersicum var. cerasiforme). Nile tilapia (157 g/fish) were stocked at 40 fish/m3 and grown for 149 days. The cherry tomato cvs. “Favorita” and “Goldita” were grown with aquaculture effluent (AE) waste and compared to plants grown with conventional fertilizer (CF) in soilless culture. Plants were grown for 157 days. Few differences in yield occurred between treatments until fish harvest (117 DAT). Post fish harvest, there was an 18.4% difference in total yield between CF and AE at crop termination for “Favorita”. Differences in yield between AE and CF were observed for “Goldita” at fish harvest (117 DAT) and crop termination (157 DAT). Results from this study suggest the potential for successful integration of cherry tomato grown in a substrate-based system with AE from a tilapia biofloc production system.

In Vitro Organotypic Systems to Model Tumor Microenvironment in Human Papillomavirus (HPV)-Related Cancers

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

Targeting DNA Damage Response as a Strategy to Treat HPV Infections

Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90–99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.

Risk of Human Pathogen Internalization in Leafy Vegetables During Lab-Scale Hydroponic Cultivation

Controlled environment agriculture (CEA) is a growing industry for the production of leafy vegetables and fresh produce in general. Moreover, CEA is a potentially desirable alternative production system, as well as a risk management solution for the food safety challenges within the fresh produce industry. Here, we will focus on hydroponic leafy vegetable production (including lettuce, spinach, microgreens, and herbs), which can be categorized into six types: (1) nutrient film technique (NFT), (2) deep water raft culture (DWC), (3) flood and drain, (4) continuous drip systems, (5) the wick method, and (6) aeroponics. The first five are the most commonly used in the production of leafy vegetables. Each of these systems may confer different risks and advantages in the production of leafy vegetables. This review aims to (i) address the differences in current hydroponic system designs with respect to human pathogen internalization risk, and (ii) identify the preventive control points for reducing risks related to pathogen contamination in leafy greens and related fresh produce products.

Coastal Aquaculture Mapping from Very High Spatial Resolution Imagery by Combining Object-Based Neighbor Features

Coastal aquaculture plays an important role in the provision of seafood, the sustainable development of regional and global economy, and the protection of coastal ecosystems. Inappropriate planning of disordered and intensive coastal aquaculture may cause serious environmental problems and socioeconomic losses. Precise delineation and classification of different kinds of aquaculture areas are vital for coastal management. It is difficult to extract coastal aquaculture areas using the conventional spectrum, shape, or texture information. Here, we proposed an object-based method combining multi-scale segmentation and object-based neighbor features to delineate existing coastal aquaculture areas. We adopted the multi-scale segmentation to generate semantically meaningful image objects for different land cover classes, and then utilized the object-based neighbor features for classification. Our results show that the proposed approach effectively identified different types of coastal aquaculture areas, with 96% overall accuracy. It also performed much better than other conventional methods (e.g., single-scale based classification with conventional features) with higher classification accuracy. Our results also suggest that the multi-scale segmentation and neighbor features can obviously improve the classification performance for the extraction of cage culture areas and raft culture areas, respectively. Our developed approach lays a solid foundation for intelligent monitoring and management of coastal ecosystems.