Courtney R. Moore

Prostate cancer tissue usually involves either well formed glands, poorly formed glands or a combination of the two morphologies, which can be correlated with metabolic differences and tumor heterogeneity. This is particularly important for metastatic castration-resistant prostate cancer, where the heterogeneity and metabolic changes drive cancer progression and treatment refractory properties. Sortilin and syndecan-1 expression accurately define the two different morphologies in prostate cancer tissue, are critical to the process of metabolic regulation, and exhibit mechanistic/functional interactions during prostate cancer progression. As trans-membrane proteins that recycle from endocytic compartments to the cell surface, sortilin and syndecan-1 are attractive targets for therapeutic intervention that address the two major forms of prostate cancer. In this study, we describe an antibody-drug conjugate (ADC) strategy that utilizes monoclonal antibodies which bind to specific extracellular domains of these integral membrane proteins to elicit anticancer activity in prostate cancer cell lines. Anti-sortilin (clone 11H8) and anti-syndecan-1 (clone 6D11) monoclonal antibodies demonstrated high specificity for epitopes on the extracellular, N-terminal domains of these respective proteins and were effectively internalized into prostate cancer cell endocytic compartments. Monomethyl aurastatin E (MMAE)-conjugated ADCs exhibited low nanomolar cytotoxicity in LNCaP and PC-3 prostate cancer cells. Mechanistically, 11H8-MMAE and 6D11-MMAE triggered cytotoxicity and morphological alterations in androgen-sensitive and androgen-insensitive cells. However, the uptake of fluorescent labelled 11H8 and 6D11 antibodies appeared to be high, whereas the killing capacity of the MMAE-conjugated antibodies was less impressive, suggesting the need for further ADC development. These promising proof-of-concept ADCs are designed to exploit molecular and metabolic vulnerabilities in prostate cancer and may have utility for overcoming treatment resistance by simultaneously targeting different forms of the cancer.

Prostate cancer (PCa) pathogenesis relies on intercellular communication, which can involve tunnelling nanotubes (TNTs) and extracellular vesicles (EVs). TNTs and EVs have been reported to transfer critical cargo involved in cellular functions and signalling, prompting us to investigate the extent of organelle and protein transfer in PCa cells and the potential involvement of the androgen receptor. Using live cell imaging microscopy, we observed extensive formation of TNTs and EVs operating between PCa, non-malignant, and immune cells. PCa cells were capable of transferring lysosomes, mitochondria, lipids, and endoplasmic reticulum, as well as syndecan-1, sortilin, Glut1, and Glut4. In mechanistic studies, androgen-sensitive PCa cells exhibited changes in cell morphology when stimulated by R1881 treatment. Overexpression assays of a newly designed androgen receptor (AR) plasmid revealed its novel localization in PCa cellular vesicles, which were also transferred to neighbouring cells. Selected molecular machinery, thought to be involved in intercellular communication, was investigated by knockdown studies and Western blotting/immunofluorescence/scanning electron microscopy (SEM). PCa TNTs and EVs transported proteins and organelles, which may contain specialist signalling, programming, and energy requirements that support cancer growth and progression. This makes these important intercellular communication systems ideal potential targets for therapeutic intervention.

Prostate cancer remains a significant global health concern, with over 1.4 million new cases diagnosed and more than 330,000 deaths each year. The primary clinical challenge that contributes to poor patient outcomes involves the failure to accurately predict and treat at the onset of metastasis, which remains an incurable stage of the disease. This review discusses the emerging paradigm that prostate cancer metastasis is driven by a dysregulation of critical molecular machinery that regulates endosome-lysosome homeostasis. Endosome and lysosome compartments have crucial roles in maintaining normal cellular function but are also involved in many hallmarks of cancer pathogenesis, including inflammation, immune response, nutrient sensing, metabolism, proliferation, signalling, and migration. Here we discuss new insight into how alterations in the complex network of trafficking machinery, responsible for the microtubule-based transport of endosomes and lysosomes, may be involved in prostate cancer progression. A better understanding of endosome-lysosome dynamics may facilitate the discovery of novel strategies to detect and manage prostate cancer metastasis and improve patient outcomes.

Prostate cancer (PCa) pathogenesis relies on intercellular communication, which can involve tunneling nanotubes (TNTs) and extracellular vesicles (EVs). TNTs and EVs have been reported to transfer critical cargo involved in cellular function and signalling, prompting us to investigate the extent of organelle and protein transfer in PCa cells and the potential involvement of the androgen receptor. Using live cell imaging microscopy, we observed extensive formation of TNTs and EVs operating between PCa, non-malignant and immune cells. PCa cells were capable of transferring lysosomes, mitochondria, lipids and endoplasmic reticulum as well as syndecan-1, sortilin, Glut1 and Glut4. In mechanistic studies, androgen sensitive PCa cells exhibited changes in cell morphology when stimulated by R1881 treatment. Overexpression assays of a newly designed androgen receptor (AR) plasmid revealed its novel localization in PCa cellular vesicles, which were also transferred to neighbouring cells. Selected molecular machinery, thought to be involved in intercellular communication, was investigated by knockdown studies and Western blotting/immunofluorescence/scanning electron microscopy (SEM). PCa TNTs and EVs transported proteins and organelles, which may contain specialist signalling, programming and energy requirements that support cancer growth and progression. This makes these important intercellular communication systems ideal potential targets for therapeutic intervention.

Gleason scoring is used within a five-tier risk stratification system to guide therapeutic decisions for patients with prostate cancer. This study aimed to compare the predictive performance of routine H&E or biomarker-assisted ISUP (International Society of Urological Pathology) grade grouping for assessing the risk of biochemical recurrence (BCR) and clinical recurrence (CR) in patients with prostate cancer. This retrospective study was an assessment of 114 men with prostate cancer who provided radical prostatectomy samples to the Australian Prostate Cancer Bioresource between 2006 and 2014. The prediction of CR was the primary outcome (median time to CR 79.8 months), and BCR was assessed as a secondary outcome (median time to BCR 41.7 months). The associations of (1) H&E ISUP grade groups and (2) modified ISUP grade groups informed by the Appl1, Sortilin and Syndecan-1 immunohistochemistry (IHC) labelling were modelled with BCR and CR using Cox proportional hazard approaches. IHC-assisted grading was more predictive than H&E for BCR (C-statistic 0.63 vs. 0.59) and CR (C-statistic 0.71 vs. 0.66). On adjusted analysis, IHC-assisted ISUP grading was independently associated with both outcome measures. IHC-assisted ISUP grading using the biomarker panel was an independent predictor of individual BCR and CR. Prospective studies are needed to further validate this biomarker technology and to define BCR and CR associations in real-world cohorts.

Fluorescence microscopy has become a critical tool for researchers to understand biological processes at the cellular level. Micrographs from fixed and live-cell imaging procedures feature in a plethora of scientific articles for the field of cell biology, but the complexities of fluorescence microscopy as an imaging tool can sometimes be overlooked or misunderstood. This review seeks to cover the three fundamental considerations when designing fluorescence microscopy experiments: (1) hardware availability; (2) amenability of biological models to fluorescence microscopy; and (3) suitability of imaging agents for intended applications. This review will help equip the reader to make judicious decisions when designing fluorescence microscopy experiments that deliver high-resolution and informative images for cell biology.

Syntenin-1 is an essential multi-functional adaptor protein, which has multiple roles in membrane trafficking and exosome biogenesis, as well as scaffolding interactions with either the actin cytoskeleton or focal adhesions. However, how this functional multiplicity relates to syntenin-1 distribution in different endosome compartments or other intracellular locations and its underlying involvement in cancer pathogenesis have yet to be fully defined. To help facilitate the investigation of syntenin-1 biology, we developed two specific monoclonal antibodies (Synt-2C6 and Synt-3A11) to spatially distinct linear sequence epitopes on syntenin-1, which were each designed to be unique at the six-amino acid level. These antibodies produced very different intracellular staining patterns, with Synt-2C6 detecting endosomes and Synt-3A11 producing a fibrillar staining pattern suggesting a cytoskeletal localisation. Treatment of cells with Nocodazole altered the intracellular localisation of Synt-3A11, which was consistent with the syntenin-1 protein interacting with microtubules. In prostate tissue biopsies, Synt-3A11 defined atrophy and early-stage prostate cancer, whereas Synt-2C6 only showed minimal interaction with atrophic tissue. This highlights a critical need for site-specific antibodies and a knowledge of their reactivity to define differential protein distributions, interactions and functions, which may differ between normal and malignant cells.