Transcriptional Landscape of 3D vs. 2D Ovarian Cancer Cell Models.

Three-dimensional (3D) cancer models are revolutionising research, allowing for the recapitulation of an in vivo-like response through the use of an in vitro system, which is more complex and physiologically relevant than traditional monolayer cultures. Cancers such as ovarian (OvCa) are prone to developing resistance, are often lethal, and stand to benefit greatly from the enhanced modelling emulated by 3D cultures. However, the current models often fall short of the predicted response, where reproducibility is limited owing to the lack of standardised methodology and established protocols. This meta-analysis aims to assess the current scope of 3D OvCa models and the differences in the genetic profiles presented by a vast array of 3D cultures. An analysis of the literature (Pubmed.gov) spanning 2012-2022 was used to identify studies with paired data of 3D and 2D monolayer counterparts in addition to RNA sequencing and microarray data. From the data, 19 cell lines were found to show differential regulation in their gene expression profiles depending on the bio-scaffold (i.e., agarose, collagen, or Matrigel) compared to 2D cell cultures. The top genes differentially expressed in 2D vs. 3D included C3, CXCL1, 2, and 8, IL1B, SLP1, FN1, IL6, DDIT4, PI3, LAMC2, CCL20, MMP1, IFI27, CFB, and ANGPTL4. The top enriched gene sets for 2D vs. 3D included IFN-α and IFN-γ response, TNF-α signalling, IL-6-JAK-STAT3 signalling, angiogenesis, hedgehog signalling, apoptosis, epithelial-mesenchymal transition, hypoxia, and inflammatory response. Our transversal comparison of numerous scaffolds allowed us to highlight the variability that can be induced by these scaffolds in the transcriptional landscape and identify key genes and biological processes that are hallmarks of cancer cells grown in 3D cultures. Future studies are needed to identify which is the most appropriate in vitro/preclinical model to study tumour microenvironments.

Differential Regulation of Genes by the Glucogenic Hormone Asprosin in Ovarian Cancer.

Background: Ovarian cancer (OvCa) is one of the most lethal forms of gynaecological malignancy. Altered energy metabolism and increased aerobic glycolysis in OvCa are hallmarks that demand attention. The glucogenic hormone asprosin is often dysregulated in metabolic disorders such as insulin resistance, diabetes (type 2 and gestational), and preeclampsia. Despite association with metabolic disorders, its role in energy metabolism within the tumour microenvironment is yet to be explored. Here, we study the role of asprosin in OvCa using transcriptomics and expand on functional studies with clinical samples. Methods: RNA sequencing, functional gene enrichment analysis, Western blotting and ImageStream. Results: Following treatment with 100 nM of asprosin, the serous OvCa cell line, SKOV-3, displayed 160 and 173 gene regulatory changes, at 4 and 12 h respectively, when compared with control samples (p < 0.05 and Log2FC > 1). In addition to energy metabolism and glucose-related pathways, asprosin was shown to alter pathways associated with cell communication, TGF-ß signalling, and cell proliferation. Moreover, asprosin was shown to induce phosphorylation of ERK1/2 in the same in vitro model. Using liquid biopsies, we also report for novel expression of asprosin's predicted receptors OR4M1 and TLR4 in cancer-associated circulating cells; with significant reduction seen between pre-chemotherapy and end of first line chemotherapy, in addition to patients under maintenance with bevacizumab +/− olaparib for OR4M1. Conclusions: In relation to OvCa, asprosin appears to regulate numerous signalling pathways in-vitro. The prognostic potential of OR4M1 in liquid biopsies should also be explored further.

ACBD3 Bioinformatic Analysis and Protein Expression in Breast Cancer Cells.

ACBD3 overexpression has previously been found to correlate with worse prognosis for breast cancer patients and, as an incredibly diverse protein in both function and cellular localisation, ACBD3 may have a larger role in breast cancer than previously thought. This study further investigated ACBD3's role in breast cancer. Bioinformatic databases were queried to characterise ACBD3 expression and mutation in breast cancer and to investigate how overexpression affects breast cancer patient outcomes. Immunohistochemistry was carried out to examine ACBD3 location within cells and tissue structures. ACBD3 was more highly expressed in breast cancer than in any other cancer or matched normal tissue, and expression over the median level resulted in reduced relapse-free, overall, and distant metastasis-free survival for breast cancer patients as a whole, with some differences observed between subtypes. IHC analysis found that ACBD3 levels varied based on hormone receptor status, indicating that ACBD3 could be a candidate biomarker for poor patient prognosis in breast cancer and may possibly be a biomarker for ER signal reprogramming of precancerous breast tissue.

Impact of Environmentally Relevant Concentrations of Bisphenol A (BPA) on the Gene Expression Profile in an In Vitro Model of the Normal Human Ovary.

Endocrine-disrupting chemicals (EDCs), including the xenoestrogen Bisphenol A (BPA), can interfere with hormonal signalling. Despite increasing reports of adverse health effects associated with exposure to EDCs, there are limited data on the effect of BPA in normal human ovaries. In this paper, we present a detailed analysis of the transcriptomic landscape in normal Human Epithelial Ovarian Cells (HOSEpiC) treated with BPA (10 and 100 nM). Gene expression profiles were determined using high-throughput RNA sequencing, followed by functional analyses using bioinformatics tools. In total, 272 and 454 differentially expressed genes (DEGs) were identified in 10 and 100 nM BPA-treated HOSEpiCs, respectively, compared to untreated controls. Biological pathways included mRNA surveillance pathways, oocyte meiosis, cellular senescence, and transcriptional misregulation in cancer. BPA exposure has a considerable impact on 10 genes: ANAPC2, AURKA, CDK1, CCNA2, CCNB1, PLK1, BUB1, KIF22, PDE3B, and CCNB3, which are also associated with progesterone-mediated oocyte maturation pathways. Future studies should further explore the effects of BPA and its metabolites in the ovaries in health and disease, making use of validated in vitro and in vivo models to generate data that will address existing knowledge gaps in basic biology, hazard characterisation, and risk assessment associated with the use of xenoestrogens such as BPA.

Protein expression of transmembrane protease serine 4 in the gastrointestinal tract and in healthy, cancer, and SARS­CoV­2 infected lungs.

In addition to the angiotensin­converting enzyme 2 (ACE2), a number of host cell entry mediators have been identified for severe acute respiratory syndrome coronavirus­2 (SARS­CoV­2), including transmembrane protease serine 4 (TMPRSS4). The authors have recently demonstrated the upregulation of TMPRSS4 in 11 different cancers, as well as its specific expression within the central nervous system using in silico tools. The present study aimed to expand the initial observations and, using immunohistochemistry, TMPRSS4 protein expression in the gastrointestinal (GI) tract and lungs was further mapped. Immunohistochemistry was performed on tissue arrays and lung tissues of patients with non­small cell lung cancer with concurrent coronavirus disease 2019 (COVID­19) infection using TMPRSS4 antibody. The results revealed that TMPRSS4 was abundantly expressed in the oesophagus, stomach, small intestine, jejunum, ileum, colon, liver and pancreas. Moreover, the extensive TMPRSS4 protein expression in the lungs of a deceased patient with COVID­19 with chronic obstructive pulmonary disease and bronchial carcinoma, as well in the adjacent normal tissue, was demonstrated for the first time, at least to the best of our knowledge. On the whole, the immunohistochemistry data of the present study suggest that TMPRSS4 may be implicated in the broader (pulmonary and extra­pulmonary) COVID­19 symptomatology; thus, it may be responsible for the tropism of this coronavirus both in the GI tract and lungs.

Differential Expression of RAD51AP1 in Ovarian Cancer: Effects of siRNA In Vitro.

BACKGROUND: DNA double strand breaks can affect genome integrity potentially leading to cancer. RAD51-associated protein 1 (RAD51AP1), an accessory protein to RAD51, is critical for homologous recombination, a key DNA damage response pathway. Emerging studies indicate a novel role for RAD51AP1 in carcinogenesis. Here we provide additional insight into the role of RAD51AP1 in ovarian cancer (OvCa). METHODS: Gene expression and patient phenotype data were obtained from TCGA and GTEX project consortia for bioinformatics analysis. Immunohistochemistry of OvCa tissue microarray was undertaken. Functional analyses were performed in a SKOV3 OvCa cell line with down-regulation of RAD51AP1 using siRNA. RESULTS: RAD51AP1 is overexpressed at gene level in primary and recurrent OvCa compared to controls. At protein level, RAD51AP1 was up-regulated in low grade serous tumors compared to high grade OvCa. There was higher expression of RAD51AP1 in OvCa metastatic to lymph nodes compared to primary cancer samples. Gene enrichment analyses identified 12 differentially expressed genes (DEGs) related to OvCa, eight of which are also common in tissue from patients with type 2 diabetes mellitus (T2DM). CONCLUSIONS: RAD51AP1 is overexpressed in OvCa, Given the link between OvCa and T2DM, the eight-gene signature shows potential for predictive value.

Elevated Circulating Lactate Levels and Widespread Expression of Its Cognate Receptor, Hydroxycarboxylic Acid Receptor 1 (HCAR1), in Ovarian Cancer.

Background: Augmented glycolysis in cancer cells is a process required for growth and development. The Warburg effect provides evidence of increased glycolysis and lactic acid fermentation in cancer cells. The lactate end-product of glycolysis is receiving growing traction for its role as a cell signalling molecule. Ovarian cancer (OvCa) is also characterised by altered glucose metabolism. We aim to explore circulating lactate levels in patients with high-grade serous OvCa (HGSOC) and to elucidate the expression of the lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1) in OvCa. Methods: HCAR1 expression was detected in patient biopsy cores using immunohistochemistry, while lactate was measured from whole blood with a Biosen-C line clinic measuring system. Results: We noted significantly elevated lactate levels in OvCa patients (4.3 ± 1.9 mmol/L) compared with healthy controls (1.4 ± 0.6 mmol/L; p < 0.0001), with an AUC of 0.96. The HCAR1 gene is overexpressed in OvCa compared to healthy controls (p < 0.001). Using an OvCa tissue microarray (>75% expression in 100 patients), high protein expression was also recorded across all epithelial OvCa subtypes and ovarian normal adjacent tissue (NAT). Conclusions: Lactate monitoring is a simple, cost-efficient test that can offer point-of-care results. Our data suggest that the potential of circulating lactate as a screening biomarker in OvCa merits further research attention.

A pancancer overview of FBN1, asprosin and its cognate receptor OR4M1 with detailed expression profiling in ovarian cancer.

Ovarian cancer affects >295,000 women worldwide and is the most lethal of gynaecological malignancies. Often diagnosed at a late stage, current research efforts seek to further the molecular understanding of its aetiopathogenesis and the development of novel biomarkers. The present study investigated the expression levels of the glucogenic hormone asprosin [encoded by fibrillin-1 (FBN1)], and its cognate receptor, olfactory receptor 4M1 (OR4M1), in ovarian cancer. A blend of in silico open access The Cancer Genome Atlas data, as well as in vitro reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry and immunofluorescence data were used. RT-qPCR revealed expression levels of OR4M1 and FBN1 in clinical samples and in ovarian cancer cell lines (SKOV-3, PEO1, PEO4 and MDAH-2774), as well as the normal human ovarian surface epithelial cell line (HOSEpiC). Immunohistochemical staining of a tissue microarray was used to identify the expression levels of OR4M1 and asprosin in ovarian cancer samples of varying histological subtype and grade, including clear cell carcinoma, serous ovarian cancer and mucinous adenocarcinoma. Immunofluorescence analysis revealed asprosin expression in SKOV-3 and HOSEpiC cells. These results demonstrated the expression of both asprosin and OR4M1 in normal and malignant human ovarian tissues. This research invokes further investigation to advance the understanding of the role of asprosin and OR4M1 within the ovarian tumour microenvironment.

p38ß - MAPK11 and its role in female cancers.

BACKGROUND: The p38MAPK family of Mitogen Activated Protein Kinases are a group of signalling molecules involved in cell growth, survival, proliferation and differentiation. The widely studied p38α isoform is ubiquitously expressed and is implicated in a number of cancer pathologies, as are p38γ and p38δ. However, the mechanistic role of the isoform, p38ß, remains fairly elusive. Recent studies suggest a possible role of p38ß in both breast and endometrial cancer with research suggesting involvement in bone metastasis and cancer cell survival. Female tissue specific cancers such as breast, endometrial, uterine and ovary account for over 3,000,000 cancer related incidents annually; advancements in therapeutics and treatment however require a deeper understanding of the molecular aetiology associated with these diseases. This study provides an overview of the MAPK signalling molecule p38ß (MAPK11) in female cancers using an in-silico approach. METHODS: A detailed gene expression and methylation analysis was performed using datasets from cBioportal, CanSar and MEXPRESS. Breast, Uterine Endometrial, Cervical, Ovarian and Uterine Carcinosarcoma TCGA cancer datasets were used and analysed. RESULTS: Data using cBioportal and CanSAR suggest that expression of p38ß is lower in cancers: BRCA, UCEC, UCS, CESC and OV compared to normal tissue. Methylation data from SMART and MEXPRESS indicate significant probe level variation of CpG island methylation status of the gene MAPK11. Analysis of the genes' two CpG islands shows that the gene was hypermethylated in the CpG1 with increased methylation seen in BRCA, CESC and UCEC cancer data sets with a slight increase of expression recorded in cancer samples. CpG2 exhibited hypomethylation with no significant difference between samples and high levels of expression. Further analysis from MEXPRESS revealed no significance between probe methylation and altered levels of expression. In addition, no difference in the expression of BRCA oestrogen/progesterone/HER2 status was seen. CONCLUSION: This data provides an overview of the expression of p38ß in female tissue specific cancers, showing a decrease in expression of the gene in BRCA, UCEC, CESC, UCS and OV, increasing the understanding of p38ß MAPK expression and offering insight for future in-vitro investigation and therapeutic application.

COVID­19 and SARS­CoV­2 host cell entry mediators: Expression profiling of TMRSS4 in health and disease.

Severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV­2), the causative viral agent for the ongoing COVID­19 pandemic, enters its host cells primarily via the binding of the SARS­CoV­2 spike (S) proteins to the angiotensin­converting enzyme 2 (ACE2). A number of other cell entry mediators have also been identified, including neuropilin­1 (NRP1) and transmembrane protease serine 2 (TMPRSS2). More recently, it has been demonstrated that transmembrane protease serine 4 (TMPRSS4) along with TMPRSS2 activate the SARS­CoV­2 S proteins, and enhance the viral infection of human small intestinal enterocytes. To date, a systematic analysis of TMPRSS4 in health and disease is lacking. In the present study, using in silico tools, the gene expression and genetic alteration of TMPRSS4 were analysed across numerous tumours and compared to controls. The observations were also expanded to the level of the central nervous system (CNS). The findings revealed that TMPRSS4 was overexpressed in 11 types of cancer, including lung adenocarcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, thyroid carcinoma, ovarian cancer, cancer of the rectum, pancreatic cancer, colon and stomach adenocarcinoma, uterine carcinosarcoma and uterine corpus endometrial carcinoma, whilst it was significantly downregulated in kidney carcinomas, acute myeloid leukaemia, skin cutaneous melanoma and testicular germ cell tumours. Finally, a high TMPRSS4 expression was documented in the olfactory tubercle, paraolfactory gyrus and frontal operculum, all brain regions which are associated with the sense of smell and taste. Collectively, these data suggest that TMPRSS4 may play a role in COVID­19 symptomatology as another SARS­CoV­2 host cell entry mediator responsible for the tropism of this coronavirus both in the periphery and the CNS.

Co­expression of peripheral olfactory receptors with SARS­CoV­2 infection mediators: Potential implications beyond loss of smell as a COVID­19 symptom.

Severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV­2) enters into human host cells via mechanisms facilitated mostly by angiotensin­converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). New loss of smell (anosmia/hyposmia) is now recognized as a COVID­19 related symptom, which may be caused by SARS­CoV­2 infection and damage of the olfactory receptor (OR) cells in the nasal neuro­epithelium and/or central involvement of the olfactory bulb. ORs are also expressed peripherally (e.g., in tissues of the gastrointestinal and respiratory systems) and it is possible that their local functions could also be impaired by SARS­CoV­2 infection of these tissues. Using Gene Expression Profiling Interactive Analysis, The Cancer Genome Atlas, Genotype­Tissue Expression, cBioPortal and Shiny Methylation Analysis Resource Tool, we highlight the expression of peripheral ORs in both healthy and malignant tissues, and describe their co­expression with key mediators of SARS­CoV­2 infection, such as ACE2 and TMPRSS2, as well as cathepsin L (CTSL; another cellular protease mediating SARS­CoV­2 infection of host cells). A wide expression profile of peripheral ORs was noted, particularly in tissues such as the prostate, testis, thyroid, brain, liver, kidney and bladder, as well as tissues with known involvement in cardio­metabolic disease (e.g., the adipose tissue, pancreas and heart). Among these, OR51E2, in particular, was significantly upregulated in prostate adenocarcinoma (PRAD) and co­expressed primarily with TMPRSS2. Functional networks of this OR were further analysed using the GeneMANIA interactive tool, showing that OR51E2 interacts with a plethora of genes related to the prostate. Further in vitro and clinical studies are clearly required to elucidate the role of ORs, both at the olfactory level and the periphery, in the context of COVID­19.

Delivering allied health services to regional and remote participants on the autism spectrum via video-conferencing technology: lessons learned.

Context and issues: The introduction of individualised funding under the National Disability Insurance Scheme in Australia aimed to increase individual choice and control over how people received disability supports. An increase in the allied health disability workforce was anticipated; however, disability workforce sector reports have consistently indicated difficulties in attracting and retaining sufficient allied health staff to satisfy current and future demand. Autism spectrum disorder is the most prevalent primary diagnosis of participants receiving individualised funding to date and requires support staff to have specialised skills and experience. Given that overall staff attraction and retention issues are reported to be exacerbated in regional and remote areas of Australia, it is important to seek innovative ways of supporting individuals on the autism spectrum in their local communities. Technology has the potential to provide a timely and low-cost alternative that extends access to specialist services for people in remote locations. The current project aimed to identify the feasibility, essential requirements and potential barriers in delivering therapy support to regional and remote participants on the autism spectrum via video-conferencing technology. Lessons learned: A multidisciplinary team (speech pathologist, occupational therapist, psychologist and a special educator) were recruited and trained to deliver tele-therapy services to 16 participants on the autism spectrum, in collaboration with their families and local support teams. Participants resided in two northern, nine western and one southern regional area in New South Wales (NSW), Australia. There were three sets of siblings. One participant resided on remote Lord Howe Island off the coast of northern NSW. Researchers used semi-structured telephone interviews to gain insight into the program from key stakeholder groups including parents, education staff, allied health professionals and tele-therapists. A general inductive approach to data analysis was used under five project evaluation areas. The evaluation focused on five areas including: development of the tele-health delivery team, understanding the role of collaboration, examining the need for autism-specific support, establishing the need for in-person contact and identifying barriers to success. The project evaluation found that investment in staff training and support was key to building a competent tele-therapy team and delivering successful tele-therapy services under a sustainable model. For many families and support team members, collaboration was reported as an important part of the tele-therapy program, with families and teachers finding it helpful to work together with the same information. The evaluation confirmed that access to autism-specific knowledge and support was novel and regarded as beneficial for families and support teams living in regional and remote areas. There were mixed responses to the inclusion of in-person support as part of a tele-therapy service. While some families felt a tele-therapy service was no different to in-person services, other families and tele-therapists indicated that the addition of at least one in-person session would help to increase rapport. Barriers within the tele-therapy model included scheduling and local staff changes, as well as the delivery of intervention requiring physical support. Technology was not seen as a barrier in the current study. This research adds to the growing body of information supporting the use of tele-practice for geographically isolated regions. Ideally, tele-therapy should not replace in-person services; however, it is necessary when no other comparable service option is available locally. Larger scale research is needed to compare blended, online and in-person models so that an optimal ratio can be established.

Investigation of training and support needs in rural and remote disability and mainstream service providers: implications for an online training model.

Objectives It is known that there are difficulties in recruiting and retaining practitioners in rural and remote communities and that access to support and professional development can be key in breaking this cycle. Technology provides a possible solution not only for increasing access to these opportunities, but also in building community capacity to support children with autism. The aim of the present study was to investigate the current learning and support needs within rural and remote professionals prior to setting up a model of support. Methods An online survey was used to gather information from service providers in rural and remote communities on their demographics, current skills and confidence in working with clients on the autism spectrum, current supervision and professional development, identified learning and support needs, and the availability and uptake of technology for accessing professional development. Results Respondents reported below average levels of perceived confidence and skills when working with children with autism, most notably children with challenging behaviour. Half the respondents do not currently attend supervision sessions, with only 15% receiving regular supervision (fortnightly or more often), and 66% of respondents had travelled more than 3h to access professional development workshops. The majority of participants had access to technology and over half had already used this for online training. Conclusion Overall, service providers in rural and remote areas are generally not currently meeting their needs in terms of frequency of supervision and professional development. The present needs analysis identifies key areas for learning, the ideal frequency of support and the acceptability of using technology to deliver this support. This information will guide future researchers in the development of an evidence-based model that will be accessible and meaningful to its participants. What is known about the topic? It is known that there are difficulties in recruiting and retaining practitioners in rural and remote communities and that access to support and professional development can be key in breaking this cycle, which may be triggered by geographical isolation. Technology-delivered intervention and support, also known as eHealth or Telehealth, has been used successfully in the disability sector for medical rehabilitation, direct intervention, employment support and support groups, but there is little evidence as to how technology is received by and implemented with disability and mainstream service providers supporting children with autism living in remote regions. What does this paper add? This paper provides an insight into the current skills and confidence of a broad range of service providers, including educators, allied health therapists and therapy and community support workers, in working with children with autism. This paper also investigates the experience, feasibility and potential uptake of a technology-driven program of support and professional development in rural and remote Australia. Finally, this paper provides an insight into the desired frequency of training and support, as well as identified learning support needs. What are the implications for practitioners? These findings have and will continue to guide practitioners in the development of an evidence-based, technology-driven model of supporting rural and remote staff working with children with autism. Technology has the potential to provide practitioners in geographically isolated areas with access to more responsive, collaborative and individualised professional support and training. Such practice may improve the skills of practitioners and the level of support they can provide their clients with autism, with the added potential of increasing staff retention in rural and remote areas of Australia.