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Residual β cell function can positively affect diabetes regulation in type 1 diabetes (T1D), but details on residual β cell mass distribution in T1D is largely lacking in a whole organ context. Implementing an optical 3D imaging pipeline, we generated a complete account of the remaining β cells throughout an entire human late onset T1D pancreas at a microscopic resolution. Our data show that most of the residual β cells were present as scattered individual cells or as punctated clusters of individual β cells, spatially separated from each other and all other endocrine cell types. Compared to islet-associated β cells, extra-islet β cells appeared in a substantially higher relative abundance in the head region of the T1D pancreas. This 3D depiction of an entire T1D pancreas shows that individual β cells may be preserved and/or formed in a highly regionalized manner, potentially reflecting key aspects of disease dynamics, advocating for increased focus on extra-islet islet β cells in attempts to develop strategies for pancreatic β cell preservation in T1D.

Background: Lymphatic metastasis is strongly associated with poor prognosis. Although the chemokine receptor CCR7 is a well-established promoter of lymphatic dissemination, its prognostic relevance remains weak. We show that tumour cell plasticity, defined by podoplanin (PDPN)-expression and promoted by hypoxia, intersects with CCR7 function in triple-negative breast cancer (TNBC).

Methods: In vivo and in vitro studies using a CCR7-expressing TNBC mouse model were combined with transcriptomic profiling. Human relevance was assessed using scRNA-seq datasets from cell lines and primary tumours, as well as METABRIC breast cancer cohorts.

Results: A PDPN-defined tumour cell mesenchymal shift, promoted by hypoxia, was required for efficient CCR7-driven lymphatic metastasis and tumour progression. PDPN-expression was linked to tumour cell collagen-expression and suppression of interferon-signalling, features associated with an immune-cold microenvironment. PDPN-expression with effects on interferon and collagen programmes was observed across murine and human TNBC cell lines and correlated with hypoxia signatures in primary TNBC, mirroring murine findings. In METABRIC, a high combined CCR7–PDPN score predicted poor survival in lymph node–positive patients, whereas either marker alone lacked prognostic value

Conclusions: PDPN is a tumour cell-associated biomarker of plasticity in TNBC, revealing synergy between hypoxia-induced mesenchymal phenotypic shifts and CCR7 in promoting lymphatic dissemination and poor prognosis. (Figure presented.)

Although major advances have been made in the field of mesoscopic imaging and associated tissue clearing protocols, these applications are greatly challenged when applied to imaging of pancreatic ductal adenocarcinoma (PDAC) tissue. Most importantly, penetration of labelling agents, typically antibodies, can be drastically reduced from the characteristically dense PDAC stroma. To circumvent this issue, we present a method by which machine learning assisted segmentation is applied to resolve the 3D PDAC microarchitecture from autofluorescence (AF) based light-sheet fluorescence microscopy (LSFM) scans. Hereby, PDAC tissue features could be studied in 3D space without the need for labelling or sectioning. In this proof of principle study, we applied this imaging pipeline on surgical specimens from five PDAC patients and normal pancreatic tissue, generating mosaics of cm3-sized tissue discs at micrometre resolution, each on scanning depths corresponding to thousands of standard pathological 2D tissue sections. Using this method, we generated 3D volumes for quantification of blood vasculature, neoplastic epithelium, islets of Langerhans and stromal components. We further showcase the potential for downstream 2D histochemical and immunohistochemical analysis of scanned specimen. As such, the method may facilitate studies of metastatic routes, vessel microarchitecture, islet phenotypes, and spatial relationships in the PDAC tumour microenvironment.

To facilitate the understanding of how a complex organ such as the pancreas is formed, this chapter illustrates the general anatomical dynamics of pancreas morphogenesis that occur during development in mice (and in humans where relevant). By applying recent advances in optical imaging techniques, including optical projection tomography and light sheet fluorescence microscopy (LSFM), this chapter presents a full image series demonstrating pancreatic bud formation and growth, as well as key morphological events that result in murine and human organs that are anatomically quite different. Further, it is now well established that pancreas development is governed by complex gene regulatory networks, where the timing and duration of gene expression, as well as the degree of molecular interactions are critical. Where appropriate, these key molecular determinants in inductive processes or other events are discussed in relation to pancreas organogenesis. Finally, this chapter describes the spatial and quantitative distribution of insulin as an example of pancreatic endocrine structure-function relationships, where lobular islet heterogeneity in the adult pancreata of mice and humans are evaluated and discussed.

Pancreatic islets of Langerhans play a pivotal role in regulating blood glucose homeostasis, but critical information regarding their mass, distribution and composition is lacking within a whole organ context. Here, we apply a 3D imaging pipeline to generate a complete account of the insulin-producing islets throughout the human pancreas at a microscopic resolution and within a maintained spatial 3D context. These data show that human islets are far more heterogenous than previously accounted for with regards to their size distribution and cellular make up. By deep tissue 3D imaging, this in-depth study demonstrates that 50% of the human insulin-expressing islets are virtually devoid of glucagon-producing α-cells, an observation with significant implications for both experimental and clinical research.

We previously demonstrated that the lungs of deceased COVID-19 patients were filled with a clear hydrogel consisting of hyaluronan (HA). In this translational study, we investigated the role of HA at all stages of COVID-19 disease to map the consequences of elevated HA on morbidity and identify the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced HA production. A reduced alveolar surface area was observed in the lungs of deceased COVID-19 patients compared to healthy controls, as visualized by a 3D rendering of lung morphology using light-sheet fluorescence microscopy. We confirmed the presence of HA in lung biopsies and found large quantities of proinflammatory fragmented HA. The association of systemic HA in blood plasma and disease severity was assessed in patients with mild (WHO Clinical Progression Scale, WHO-CPS, 1–5) and severe COVID-19 (WHO-CPS, 6–9) during the acute and convalescent phases and related to lung function. We found that systemic levels of HA were high during acute COVID-19 disease, remained elevated during convalescence, and were associated with a reduced diffusion capacity. In vitro 3D-lung models, differentiated from primary human bronchial epithelial cells, were used to study the effects of SARS-CoV-2 infection on HA metabolism, and transcriptomic analyses revealed a dysregulation of HA synthases and hyaluronidases, both contributing to increased HA in apical secretions. Furthermore, corticosteroid treatment reduced the inflammation and downregulated HA synthases. Our findings demonstrate that HA plays a role in COVID-19 morbidity and that sustained elevated HA concentrations may contribute to long-term respiratory impairment.