Rapid microwave-stimulated fixation of entire prostatectomy specimens

Conventional fixation of large solid surgical specimens is a slow process. Consequently, autolytic damage to tissues may occur if the fixative does not reach the central part of the specimen in time. However, as there is also a time relationship between formalin fixation and antigen masking, fixation for too long can also be detrimental. In seeking the optimum balance for fixation, microwave irradiation might be of assistance. This study set out to evaluate methods for fixing entire prostate glands within a brief period of time, using microwave-stimulated formalin fixation. The results show that entire prostates can be optimally fixed if formalin is present throughout the tissue as the temperature is increased by microwave irradiation. This is achieved by injecting the fixative into the prostate at multiple sites immediately following prostatectomy. The technique described ensures standardization of a critical step during tissue processing, leading to uniform microscopic results with both routine and immunohistochemical stains. It is a simple, rapid method, suitable for routine diagnostic use. Using this modified approach, DNA of much larger sizes can be extracted from paraffin-embedded material, which could expand the possibilities for molecular analysis

Intermediate Cells in Human Prostate Epithelium Are Enriched in Proliferative Inflammatory Atrophy

Within the human prostate epithelium four cell populations can be discriminated based on their expression of keratins (K). Basal cells express high levels of K5 and K14, as well as p63, whereas they have very low levels of androgen receptor, prostate-specific antigen (PSA), K8, and K18. Luminal secretory cells lack p63, K5, and K14 but express high levels of K8, K18, androgen receptor, and PSA. Additionally, cells have been identified with a keratin phenotype intermediate between basal and luminal cells that co-express high levels of K5 and K18 (K5/18) as well as hepatocyte growth factor receptor c-MET. Although intermediate cells have been proposed as precursor cells of prostate cancer, their biology is ill defined. Epithelial cells in proliferative inflammatory atrophy (PIA) appear to be cycling rapidly as indicated by expression of Ki-67, and morphological transitions have been identified between PIA and high-grade prostate intraepithelial neoplasia. Many of the atrophic epithelial luminal cells in PIA are candidates for intermediate cells based in part on weak expression of PSA and androgen receptor, high levels of K8/18, and lack of p63. The objective of this study was to further clarify the phenotype of the proposed intermediate cells in PIA and to quantitatively determine the level in which these intermediate cells preferentially occur in PIA lesions. Intermediate cells were immunohistochemically demonstrated using antibodies to K5, K14, K18, and c-MET. Using radical prostatectomy specimens (n = 15) the area fraction of intermediate cells in normally differentiated prostate epithelium and PIA were quantified by a grid point counting method. Atrophic luminal cells of PIA lesions expressed K5 in 39.2 ± 7.4% of cells compared to 2.4 ± 2.3% in normal epithelium (P < 0.00001). By contrast, K14 was only expressed in 3.0 ± 3.2% of the luminal cells. Previous studies have shown that virtually 100% of these atrophic luminal cells are strongly positive for K8/18. c-MET was present in 44.1 ± 14.1% of luminal cells in PIA but only in 2.1 ± 2.8% of luminal cells in normal epithelium (P < 0.00001). To unambiguously determine whether intermediate luminal cells in PIA show increased proliferative activity and decreased p27(kip1) expression, double-staining immunofluorescence of Ki-67 and K5, as well as p27(Kip1) and K5 was performed. Luminal cells in PIA often co-expressed K5 and Ki-67. Although p27(Kip1) was strongly expressed in K5-negative differentiated cells in normal epithelium, p27(Kip1) staining was absent in many of the K5-positive cells in the luminal compartment of PIA. We conclude that cells phenotypically intermediate between basal and secretory cells are enriched in PIA lesions. The finding of a large number of highly proliferating intermediate cells in PIA provides further support that these cells may serve as preferred target cells in prostate carcinogenesis

Polyisocyanide hydrogels as a tunable platform for mammary gland organoid formation

In the last decade, organoid technology has developed as a primary research tool in basic biological and clinical research. The reliance on poorly defined animal-derived extracellular matrix, however, severely limits its application in regenerative and translational medicine. Here, a well-defined, synthetic biomimetic matrix based on polyisocyanide (PIC) hydrogels that support efficient and reproducible formation of mammary gland organoids (MGOs) in vitro is presented. Only decorated with the adhesive peptide RGD for cell binding, PIC hydrogels allow MGO formation from mammary fragments or from purified single mammary epithelial cells. The cystic organoids maintain their capacity to branch for over two months, which is a fundamental and complex feature during mammary gland development. It is found that small variations in the 3D matrix give rise to large changes in the MGO: the ratio of the main cell types in the MGO is controlled by the cell–gel interactions via the cell binding peptide density, whereas gel stiffness controls colony formation efficiency, which is indicative of the progenitor density. Simple hydrogel modifications will allow for future introduction and customization of new biophysical and biochemical parameters, making the PIC platform an ideal matrix for in depth studies into organ development and for application in disease models

Precision oncology using organoids of a secretory carcinoma of the salivary gland treated with TRK-inhibitors

•We describe the first NTRK3 fusion positive organoid model for secretory carcinoma;•The model was used to in vitro predict the in vivo drug response to larotrectinib;•The importance of thorough characterization of organoids was illustrated;•Characterization is required prior to extrapolating in vitro findings to the clinic. The use of anticancer drugs targeting specific molecular tumor characteristics is rapidly increasing in clinical practice, but selecting patients to benefit from these remains a challenge. It has been suggested that organoid cultures would be ideally suited to test drug responses in vitro. Here we describe and characterize in depth a case of ETV6-NTRK3 gene fusion-positive secretory carcinoma of the salivary glands and corresponding organoid cultures that responded and subsequently acquired resistance to TRK targeting therapy with larotrectinib. This case-culture-characterization illustrates the advances made in precision oncology, but also exposes important caveats in using organoids to predict treatment response