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.

Metastatic melanoma remains a fatal disease to many worldwide, even after the breakthrough introduction of targeted therapies such as BRAF inhibitors and immune checkpoint blockade therapies such as CTLA‐4 and PD‐1 inhibitors. With advances in our understanding of this disease, as well as the increasing data gathered from patient studies, the significance of the host immune response to cancer progression and response to treatment is becoming clear. More specifically, the presence of intratumoral CD8⁺ cytotoxic T‐cells correlates with better prognosis whereas the accumulation of monocytes/macrophages and neutrophils in the tumour is often associated with worse prognosis. Access and infiltration of circulating leukocytes into the tumour is governed by adhesion molecules and chemokines expressed by the endothelial cells of the vasculature. This review focuses on the adhesion molecules and chemokines which control the homing of CD8⁺ cytotoxic T‐cells, monocytes and neutrophils to peripheral tissues, including tumours. We discuss the role of these leukocyte subsets in regulating melanoma growth, and detail the mechanisms used by tumours to selectively recruit or exclude these leukocytes for their own advantage. In doing so, we bring to light an underappreciated component of tumour biology which should be considered in combination with current treatments to selectively alter the leukocyte composition of tumours and ultimately enhance treatment outcome.

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-x^Plt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.

Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occur-ring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop bio-markers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient samples, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC-assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy sample cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient samples. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the po-tential to transform clinical pathology practice by stand-ardising the interpretation of the tissue morphology.

The presence of intraductal carcinoma of the prostate (IDCP) correlates with late-stage disease and poor outcomes for patients with prostatic adenocarcinoma, but the accurate and reliable staging of disease severity remains challenging. Immunohistochemistry (IHC) has been utilised to overcome problems in assessing IDCP morphology, but the current markers have only demonstrated limited utility in characterising the complex biology of this lesion. In a retrospective study of a cohort of patients who had been diagnosed with IDCP, we utilised IHC on radical prostatectomy sections with a biomarker panel of Appl1, Sortilin and Syndecan-1, to interpret different architectural patterns and to explore the theory that IDCP occurs from retrograde spread of high-grade invasive prostatic adenocarcinoma. Cribriform IDCP displayed strong Appl1, Sortilin and Syndecan-1 labelling patterns, while solid IDCP architecture had high intensity Appl1 and Syndecan-1 labelling, but minimal Sortilin labelling. Notably, the expression pattern of the biomarker panel in regions of IDCP was similar to that of adjacent invasive prostatic adenocarcinoma, and also comparable to prostate cancer showing perineural and vascular invasion. The Appl1, Sortilin, and Syndecan-1 biomarker panel in IDCP provides evidence for the model of retrograde spread of invasive prostatic carcinoma into ducts/acini, and supports the inclusion of IDCP into the five-tier Gleason grading system.

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.