Eman Mohamed Othman Mosaad

Prostaglandins are involved in multiple processes important for fertility, with previous work in mice highlighting a potential role for the HSD17B12 gene in prostaglandin biosynthesis. This study aimed to determine the associations among circulating prostaglandin concentrations, a missense SNP in the HSD17B12 gene predicted to disrupt protein function, and fertility traits in first-lactation Holstein-Friesian dairy cows. We used a study population of approximately 500 animals specifically bred to have either a positive (POS, +5%) or negative (NEG, -5%) genetic merit for fertility (FertBV). Genotypes of a previously identified SNP (rs109711583) in HSD17B12 were determined, with 116 animals genotyped as AA, 215 genotyped as AG, and 153 genotyped as GG. Plasma concentrations of prostaglandin E₂ and the PGF_2α metabolite PGFM were determined at 3 time points (12 mo of age, 4 d postpartum, and 5 wk postpartum during first lactation) in a selection of animals with AA and GG genotypes from both the POS and NEG FertBV groups (n = 33-40 in each genotype for each FertBV group). Binary reproductive traits (yes or no) examined included submission for artificial breeding in the first 3 or 6 wk of the seasonal breeding period; conception to first service; conception during the first 6 wk of the breeding period; and pregnant at the end of the breeding period. Uterine health at 6 wk after calving was examined by evaluating the percentage of polymorphonuclear leukocytes following uterine cytology and by scoring vaginal discharge based on the presence of purulent material. The 3-wk submission rate was increased in animals that carried the G allele of the missense SNP in HSD17B12, but no differences were present among genotypes for 6-wk submission rate. The trait was additive, with each increase of the G allele increasing the 3-wk submission rate by 6 to 7%. We did not observe any consistent associations between SNP alleles and circulating PGE₂ concentrations; however, a complex 3-way interaction among time, fertility group, and SNP allele was present for PGFM concentrations. Plasma concentrations of PGE₂ were increased approximately 40% at 5 wk postpartum in animals that were submitted for breeding within 3 or 6 wk of the start of the breeding season, and in those that conceived during the first 6 wk of breeding, compared with those that did not. Plasma concentrations of PGFM were decreased approximately 20% in those animals that conceived to their first service and tended to be decreased in animals that were pregnant at the end of the breeding period, compared with those that were not. In summary, associations were observed between the SNP in HSD17B12 and submission rate by d 21 of the breeding season, as well as between circulating prostaglandin concentrations and fertility traits, but the SNP was not consistently linked to changes in prostaglandin concentrations. Thus, the association between submission rate by d 21 of the breeding season and the SNP in HSD17B12 were unlikely driven by changes in prostaglandins.

Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.

There is increasing appreciation that non-cancer cells within the tumour microenvironment influence cancer progression and anti-cancer drug efficacy. For metastatic prostate cancer (PCa), the bone marrow microenvironment influences metastasis, drug response, and possibly drug resistance. Using a novel microwell platform, the Microwell-mesh, we manufactured hundreds of 3D co-culture microtissues formed from PCa cells and bone marrow stromal cells. We used luciferase-expressing C42B PCa cells to enable quantification of the number of PCa cells in complex microtissue co-cultures. This strategy enabled us to quantify specific PCa cell growth and death in response to drug treatment, in different co-culture conditions. In parallel, we used Transwell migration assays to characterize PCa cell migration towards different 2D and 3D stromal cell populations. Our results reveal that PCa cell migration varied depending on the relative aggressiveness of the PCa cell lines, the stromal cell composition, and stromal cell 2D or 3D geometry. We found that C42B cell sensitivity to Docetaxel varied depending on culture geometry, and the presence or absence of different stromal cell populations. By contrast, the C42B cell response to Abiraterone Acetate was dependent on geometry, but not on the presence or absence of stromal cells. In summary, stromal cell composition and geometry influences PCa cell migration, growth and drug response. The Microwell-mesh and microtissues are powerful tools to study these complex 3D interactions.

Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.

Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.