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Document describing important findings related to the main manuscript.

Document describing important mouse lines, treatment protocols, antibodies and primers used in the present study.

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations.Significance:This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

PDF file - 790K, S Table 1. Primary and secondary antibodies used in immunostaining and Western Blotting experiments S Table 2. Antibodies used for immunoprecipitation experiments S Table 3. Primers used in real time RT-PCR and PCR of the ChIP experiments S Figure 1: The expression pattern of Sirt1 and beta-catenin changes in Caerulein induced pancreatitis. S Figure 2: The expression pattern of Sirt1 and beta-catenin, but not Dbc1, changes in pancreatic duct ligation. S Figure 3: In an in vitro ADM model, Sirt1 undergoes transient changes in subcellular localisation without changes in expression levels. S Figure 4: Deletion of functional Sirt1 in Pdx1-Cre;Sirt1ex4lox/lox pancreas impacts on acinar cell differentiation during ADM. S Figure 5: Acinar cell differentiation is not altered during ADM in Sirt1 transgenic (SirtTg) pancreas. S Figure 6: Nicotinamide represses ADM of human cells in culture. S Figure 7: Sirt1 co-localizes and interacts with beta-Catenin in acinar cells. S Figure 8: Nicotinamide increases beta-catenin/Wnt signaling in ADM cultures. S Figure 9: Dbc1 expression is decreased in PDAC but not in chronic pancreatitis.

PDF file - 1.7MB, This file contain Supplementary figures 1 to 8. Supplementary Figure 1: MMR expression on distinct cell types present in s.c. TS/A and 3LL-R tumor suspensions. Supplementary Figure 2: Anti-MMR differentially labels TAM subsets in s.c. TS/A tumor sections Supplementary Figure 3. SPR competition studies with anti-MMR Nbs cl1 and cl3 on immobilized mouse MMR protein Supplementary Figure 4. Anti-MMR Nb cl1 binding to distinct cell types in liver single-cell suspensions Supplementary Figure 5. TAM subpopulations from 3LL-R tumors grown s.c. in MMR-KO mice. Supplementary Figure 6. Flow cytometric analyses of WT and CCR2-KO s.c. 3LL-R tumors. Supplementary Figure 7. Effect of mono- and bivalent anti-MMR Nb on immune cell activation in vitro. Supplementary Figure 8. Effect of mono- and bivalent anti-MMR Nb on immune cell activation in vivo

PDF file - 125K, This file contains Supplementary Methods, used for experiments only shown in the Supplementary Figures

AVI file - 102K, Full 3D reconstruction of whole body SPECT/CT images visualizing MMR+ cells specifically in a subcutaneous TS/A tumor

PDF file - 69K, This file contains Supplementary Tables Supplementary Table 1. Sequences of primers Supplementary Table 2. SPR kinetic and equilibrium parameters for anti-MMR Nanobodies and bivalent Nanobody cl1 derivatives. Supplementary Table 3. Uptake values of 99mTc-labeled anti-MMR Nb cl1, cl3 and Nb BCII10 in naive WT and MMR-KO mice, based on dissection at 1 hour post injection. Supplementary Table 4. Uptake values of 99mTc-labeled anti-MMR Nb in s.c. 3LL-R tumor-bearing WT or CCR2-KO mice, based on dissection at 3 hours post injection Supplementary Table 5. Uptake values of 99mTc-labeled bivalent anti-MMR Nbs in naive WT and MMR-KO mice, based on dissection at 1 hour post injection

AVI file - 1.9MB, Full 3D reconstruction of whole body SPECT/CT images visualizing MMR+ cells specifically in spontaneous MMTV-PyMT tumors

MOV file - 1MB, Full 3D reconstruction of whole body SPECT/CT images visualizing MMR+ cells specifically in an orthotopic TS/A tumor

Microglia and border-associated macrophages (BAMs) are brain-resident self-renewing cells. Here, we examined the fate of microglia, BAMs, and recruited macrophages upon neuroinflammation and through resolution. Upon infection, Trypanosoma brucei parasites invaded the brain via its border regions, triggering brain barrier disruption and monocyte infiltration. Fate mapping combined with single-cell sequencing revealed microglia accumulation around the ventricles and expansion of epiplexus cells. Depletion experiments using genetic targeting revealed that resident macrophages promoted initial parasite defense and subsequently facilitated monocyte infiltration across brain barriers. These recruited monocyte-derived macrophages outnumbered resident macrophages and exhibited more transcriptional plasticity, adopting antimicrobial gene expression profiles. Recruited macrophages were rapidly removed upon disease resolution, leaving no engrafted monocyte-derived cells in the parenchyma, while resident macrophages progressively reverted toward a homeostatic state. Long-term transcriptional alterations were limited for microglia but more pronounced in BAMs. Thus, brain-resident and recruited macrophages exhibit diverging responses and dynamics during infection and resolution.

Objective: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. Design: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. Results: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. Conclusion: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models., SCOPUS: ar.j, info:eu-repo/semantics/published

Purpose The combination of standard-of-care radiation therapy (RT) with immunotherapy is moving to the mainstream of non-small cell lung cancer treatment. Multiple preclinical studies reported on the CD8+ T cell stimulating properties of RT, resulting in abscopal therapeutic effects. A literature search demonstrates that most preclinical lung cancer studies applied subcutaneous lung tumor models. Hence, in-depth immunologic evaluation of clinically relevant RT in orthotopic lung cancer models is lacking. Methods and Materials We studied the therapeutic and immunologic effects of low-dose fractionated RT on lungs from C57BL/6 mice, challenged 2 weeks before with firefly luciferase expressing Lewis lung carcinoma cells via the tail vein. Low-dose fractionation was represented by 4 consecutive daily fractions of image guided RT at 3.2 Gy. Results We showed reduced lung tumor growth upon irradiation using in vivo bioluminescence imaging and immunohistochemistry. Moreover, significant immunologic RT-induced changes were observed in irradiated lungs and in the periphery (spleen and blood). First, a significant decrease in the number of CD8+ T cells and trends toward more CD4+ and regulatory T cells were seen after RT in all evaluated tissues. Notably, only in the periphery did the remaining CD8+ T cells show a more activated phenotype. In addition, a significant expansion of neutrophils and monocytes was observed upon RT locally and systemically. Locally, RT increased the influx of tumor-associated macrophages and conventional type 2 dendritic cells, whereas the alveolar macrophages and conventional type 1 DCs dramatically decreased. Functionally, these antigen-presenting cells severely reduced their CD86 expression, suggesting a reduced capacity to induce potent immunity. Conclusions Our results imply that low-dose fractionated RT of tumor-bearing lung tissue shifts the immune cell balance toward an immature myeloid cell dominating profile. These data argue for myeloid cell repolarizing strategies to enhance the abscopal effects in patients with non-small cell lung cancer treated with fractionated RT.

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. Significance: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed in mice since the availability of human cells is scarce.Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized.RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in ‘Pathways of cancer’ with prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death and suppressed acinar cell dedifferentiation by limited ERK signaling.In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.

SUMMARYObjectiveAn aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) exists, driven by ΔNp63. In other epithelia, ΔNp63+basal cells have stem cell capacity and can be at the origin of tumors. In the pancreas, basal cells have not been identified.DesignWe assessed basal cell markers in human and mouse pancreas, chronic pancreatitis and PDAC, and developed a 3D imaging protocol (FLIP-IT) to study sizeable samples at single cell resolution. We generated organoid cultures of ducts from Sox9-eGFP reporter mice.ResultsIn normal human pancreas, rare ΔNp63+cells exist in ducts that expand in chronic pancreatitis. ΔNp63+cells express KRT19 and canonical basal markers (KRT5, KRT14 and S100A2) but lack markers of duct cells such as CA19.9 and SOX9. In addition, ΔNp63+cells pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views of the ductal tree in formalin fixed paraffin embedded samples show that basal cells are localized on the basal membrane of medium to large ducts and expand as multilayer dome-like structures in chronic pancreatitis. In mice, ΔNp63 expression is induced when culturing organoids from Sox9-low ductal cells but could not be found in normal pancreas nor in models of pancreatitis or pancreatic cancer.ConclusionWe discovered a novel ductal cell population in normal human pancreas similar to basal cells in other tissues. Using FLIP-IT, we provide unprecedented 3D visualization of these cells in archival clinical specimens. ΔNp63+cells may play an important role in pancreatic tissue regeneration and cancer.SUMMARY BOXWhat is already known about this subject?ΔNp63 has a central role in determining the basal-like subtype of pancreatic ductal adenocarcinoma (PDAC).Different to other tissues with basal cancers, the normal pancreas reportedly does not contain (ΔNp63-expressing) basal cells.Current protocols face severe limitations for marker-based identification and 3D imaging of individual (rare) cells in archival pancreatic samples.What are the new findings?We report a rare and atypical pancreatic duct cell that expresses ΔNp63, other basal cell markers and g.i. stem cell markers.The number of these basal cells increases in diseases such as chronic pancreatitis and pancreatic cancer.We provide an easy to implement protocol for 3D clearing and high-resolution imaging of sizeable samples of (fresh or FFPE) human pancreas or an entire mouse pancreas.Except after culturing medium to large ducts as organoids, we fail to detect basal cells in mouse experimental pancreatic models.How might it impact on clinical practice in the foreseeable future?Extrapolating from knowledge in other organs, basal cells in the pancreas may have a stem cell/progenitor role, including in diseases such as (basal) pancreatic cancer.Use of the 3D imaging protocol in archival clinical specimens will allow unprecedented insights in pancreatic histopathology.For above mentioned diseases, we caution for findings in experimental mouse models that may not (fully) recapitulate the etiopathogenesis.

Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-seq to map the glioblastoma immune landscape in mouse tumors and in patients with newly diagnosed disease or recurrence. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia- or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia- and monocyte-derived TAMs were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors, but were outnumbered by monocyte-derived TAMs following recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions. ispartof: NATURE NEUROSCIENCE vol:24 issue:4 pages:595-610 ispartof: location:United States status: published

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Scope Aspalathin, the main polyphenolic phytochemical of rooibos (Aspalathus linearis), has been attributed with health promoting properties, including a glucose lowering effect that can prove interesting for application as nutraceutical or therapeutic in (pre-)diabetics. Preservation of β cell mass in the pancreas is considered a key issue for diabetes prevention or treatment, therefore the aim is to investigate whether aspalathin also has β cell cytoprotective potential. Methods and results Rat pancreatic islets and the β cell line Insulinoma 1E (INS1E) are studied in vitro after exposure to various cytotoxic agents, namely streptozotocin (STZ), hydrogen peroxide, or chronic high glucose. The effect of aspalathin on cell survival and apoptosis is studied. Expression of relevant cytoprotective genes is analyzed by qRT-PCR and proteins by Western blot. Aspalathin is found to protect β cells against cytotoxicity and apoptosis. This is associated with increased translocation of nuclear factor erythroid 2-related factor 2 (NRF2) and expression of its antioxidant target genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo-1), and superoxide dismutase 1 (Sod1). Conclusion It is proposed that aspalathin protects β cells against glucotoxicity and oxidative stress by increasing the expression of NRF2-regulated antioxidant enzymes. This indicates that aspalathin is an interesting β cell cytoprotectant.

OBJECTIVE: Targeting the glucagon-like peptide-1 receptor with radiolabeled exendin is a very promising method to noninvasively determine the β cell mass in the pancreas, which is needed to unravel the pathophysiology of type 1 and type 2 diabetes. The present study aimed to explore the effects of both hyperglycemia and insulitis on the uptake of exendin in a spontaneous type 1 diabetes mouse model, nonobese diabetic (NOD) mice. METHODS: NOD mice (n = 75, 7-21 weeks old) were injected intravenously with [111In]In-DTPA-exendin-3, and single-photon emission computed tomography (SPECT) images were acquired 1 h pi. The pancreatic accumulation of [111In]In-DTPA-exendin-3 was quantified in vivo using SPECT and by ex vivo counting and correlated to the β cell mass (BCM). The influence of insulitis and hyperglycemia on the exendin uptake was assessed. RESULTS: The pancreas could be visualized longitudinally using SPECT. A linear correlation was found between the BCM (%) and pancreatic uptake (%ID/g) as measured by ex vivo counting (Pearson r = 0.64, p < 0.001), which was not affected by either insulitis (Pearson r = 0.66, p = 0.83) or hyperglycemia (Pearson r = 0.57, p = 0.51). Biodistribution and ex vivo autoradiography revealed remaining [111In]In-DTPA-exendin-3 uptake in the pancreas despite total ablation of BCM. CONCLUSIONS: Despite hyperglycemia and severe insulitis, we have found a good correlation between BCM and pancreatic exendin uptake, even in a suboptimal model with relatively high background activity.

Kras mutations are not sufficient to induce precancerous lesions in the pancreas, unlike in other organs such as lungs. Pancreatic intraepithelial neoplasia (PanIN) appear when mutated Kras is associated with pancreatitis. Since acinar cells are thought to be the cell origin of PanIN and pancreatic ductal adenocarcinoma (PDAC), our aim is to understand how acinar cells initially resist to Kras mutations and how pancreatitis enables mutated Kras to promote pancreatic neoplasia. Pancreatitis and associated inflammation were induced by injections of cerulein in tamoxifen-treated Elastase-CreER/LSLKrasG12D mice, in which mutated Kras expression is induced exclusively in acinar cells. RNAseq analysis on FACS-sorted acinar cells revealed that Kras expression and several cancer-associated pathways were rapidly induced in the presence of inflammation. In situ hybridization, RTqPCR, and Western blot (WB) experiments confirmed that Kras and/or KrasG12D mRNA and protein were significantly increased in acinar cells after cerulein treatment. To further explore the control of Kras expression, we generated a novel mouse model in which a citrine gene (a variant of GFP) is fused in frame with the endogenous Kras gene, allowing to detect Kras on tissue sections using a GFP antibody. We demonstrated by WB that the protein expression of Kras, KrasG12D and citrine-Kras fusion was similarly regulated in response to cerulein treatment, suggesting the involvement of the same regulatory elements on all three alleles. The ability of citrine-Kras fusion to bind GTP and translocate to the plasma membrane in acini was not altered compared to wild-type Kras protein. Little or no citrine-Kras was visualized in acinar cells of tamoxifen-treated Elastase-CreER/LSLKrasG12D/citrine-Kras mice. However, a 30-fold increase of citrine-Kras at the cell membrane of neoplastic lesions was detected after cerulein treatment. This indicates that inflammation is required for expression and plasma membrane targeting of Kras and KrasG12D in metaplastic acini. Importantly, protein expression of FT-α/β, Rce1 and ICMT, three enzymes necessary for Kras targeting to the membrane was greatly increased in murine acinar cells during pancreatitis. Basal FT-α/β, Rce1 and ICMT levels were found to be very low in normal mouse and human pancreata, compared to other organs, and their expression increased in primary mouse and human acini grown under conditions inducing the formation of preneoplastic lesions. In addition, computational analysis of a PDAC cohort (n=178) from The Cancer Genome Atlas (TCGA) revealed a positive correlation between expression of KrasG12D and its regulating enzymes. We conclude that Kras and KrasG12D are normally not present in acinar cells in vivo and that inflammation induces a coordinated program driving their expression and translocation to plasma membrane. Our findings provide a first clue to the long-standing observation that in several organs, Kras mutations, alone, are not sufficient to affect tissue homeostasis. Citation Format: Mohamad Nabil Assi, Jean-Nicolas Lodewyckx, Younes Achouri, Claude Gérard, Nicolas Dauguet, Isabelle Houbracken, Yves Heremans, Donatienne Tyteca, Ilse Rooman, Luc Bouwens, Frédéric Lemaigre, Patrick Jacquemin. Inflammation enables pancreatic acinar cells to overcome resistance to oncogenic Kras by increasing its expression and plasma membrane localization [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A07.

The incidence of type 2 diabetes (T2D) increases dramatically worldwide and has created an enormous health care burden. Obesity, dyslipidemia and insulin resistance are major risk factors for the development of T2D, but the major factor leading to the disease is failure of the insulin-producing beta cell mass to compensate for increasing insulin demands of the body. Progression of the disease further diminishes the beta cell mass as a result of lipotoxicity and glucotoxicity for which beta cells are particularly sensitive. Hence, treatment aiming to prevent beta cell loss or increase the number of beta cells could inhibit diabetes progression or lead to restoration of normal metabolism. Whereas current and new antidiabetic drugs are mainly targeting insulin secretion and action or glucose uptake, newer interventions must be found that prevent beta cell loss or increase beta cell number. The targets for this are beta cell proliferation, neogenesis and survival. This review examines major evidence from animal experiments suggesting that it is feasible to regulate the beta cell mass by bioactive compounds like growth factors, cytokines, hormones, phytochemicals and small molecules. Often the mode of action remains unclear due to inadequate methods to assess the effects of the compounds on the beta cell dynamics. Furthermore, a major challenge is to identify compounds with sufficient specificity in order to avoid unwanted effects on other cell types. Provided such safety issues can be solved, this may provide a curative approach for diabetes treatment.

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-βH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Objective Previous studies demonstrated that circulating microRNA-375 (miR-375) is a suitable plasma biomarker for real-time detection of beta cell death. The present study evaluated the use of this biomarker to assess the beta cytoprotective effect of phenylpropenoic acid glucoside (PPAG), which was previously demonstrated to protect beta cells against various types of injury, and of exendin-4, which is an established antidiabetic drug. Methods PPAG or exendin-4 were administered in mice treated with streptozotocin (STZ) to acutely induce beta cell death. Beta cell mass and apoptotic death were measured in pancreatic tissue sections. Circulating miR-375 was measured in blood plasma by RT-qPCR. The release of miR-375 was also measured in vitro by MIN-6 beta cells. Results Administration of STZ resulted in measurable circulating levels of miR-375, a decrease in beta cell mass and increase in frequency of apoptotic beta cells. In vitro, there was a good correlation between miR-375 release and the extent of beta cell death. Treatment of mice with PPAG or exendin-4 significantly attenuated STZ-induced loss of beta cell mass and beta cell apoptosis, and normalized the blood level of miR-375. Conclusions These findings show the potential use of serological miR-375 measurements to evaluate the beta cytoprotective effect of (potential) antidiabetic drugs in vivo.

Objective Epithelial-mesenchymal transition may interfere with the differentiation of cultured pancreatic acinar cells toward endocrine cells. Therefore, it will be important to investigate into detail the reprogramming of human pancreatic acinar cells toward a mesenchymal phenotype: the association with acinoductal transdifferentiation, the influence of cell adhesion, and the regulation behind this process. Methods Human exocrine cells, isolated from donor pancreata, were cultured in suspension or as monolayers. Non-genetic lineage tracing, using labeled ulex europaeus agglutinin 1 lectin, was performed, and the role of the transforming growth factor (TGF-β) superfamily was investigated. Results After 7 days in monolayer culture, the human acinar cells coexpressed the mesenchymal marker vimentin and the ductal marker Sox9. However, when the human exocrine cells were cultured in suspension, epithelial-mesenchymal transition was not observed. The spontaneous transition of the human acinar cells toward a ductal and mesenchymal phenotype was decreased by inhibition of the TGF-β and activin signaling pathways. Conclusions The human acinar cells spontaneously undergo TGF-β- regulated reprogramming in the monolayer culture. These observations are helpful to develop culture methods for the in vitro reprogramming of pancreatic exocrine to endocrine cells. They are also of potential interest for studies on exocrine acinar cells in the development of pancreatic cancer.

Contains fulltext : 157000.pdf (Publisher’s version ) (Closed access) PURPOSE: In order to evaluate future beta cell tracers in vivo, we aimed to develop a standardized in vivo method allowing semiquantitative measurement of a prospective beta cell tracer within the pancreas. PROCEDURES: 2-[(123)I]Iodo-L-phenylalanine ([(123)I]IPA) and [Lys(40)([(111)In]DTPA)]exendin-3 ([(111)In]Ex3) pancreatic uptake and biodistribution were evaluated using SPECT, autoradiography, and an ex vivo biodistribution study in a controlled unilaterally nephrectomized mouse beta cell depletion model. Semiquantitative measurement of the imaging results was performed using [(123)I]IPA to delineate the pancreas and [(111)In]Ex3 as a beta cell tracer. RESULTS: The uptake of [(123)I]IPA was highest in the pancreas. Aside from the kidneys, the uptake of [(111)In]Ex3 was highest in the pancreas and lungs. Autoradiography showed only uptake of [(111)In]Ex3 in insulin-expressing cells. Semiquantitative measurement of [(111)In]Ex3 in the SPECT images based on the delineation of the pancreas with [(123)I]IPA showed a high correlation with the [(111)In]Ex3 uptake data of the pancreas obtained by dissection. A strong positive correlation was observed between the relative insulin positive area and the pancreas-to-blood ratios of [(111)In]Ex3 uptake as determined by counting with a gamma counter and the semiquantitative analysis of the SPECT images. CONCLUSIONS: [(123)I]IPA is a promising tracer to delineate pancreatic tissue on SPECT images. It shows a high uptake in the pancreas as compared to other abdominal tissues. This study also demonstrates the feasibility and accuracy to measure the beta cell mass in vivo in an animal model of diabetes.

Purpose Molecular imaging has the potential to provide quantitative information about specific biological aspects of developing atherosclerotic lesions. This requires the generation of reliable, highly specific plaque tracers. This study reports a new camelid single-domain antibody fragment (sdAb) targeting the Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a biomarker for the detection and molecular phenotyping of vulnerable atherosclerotic plaques. Procedures A camelid sdAb was generated and selected for high affinity binding to LOX-1. Ex vivo biodistribution and in vivo single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in wild-type mice and in fat-fed atherosclerotic apolipoprotein E-deficient mice with 99mTc-labeled sdAbs. Gamma-counting and autoradiography analyses were performed on dissected aorta segments with different degrees of plaque burden. The specificity of the LOX-1-targeting sdAb was evaluated by blocking with unlabeled sdAb or by comparison with a nontargeting 99mTc-labeled control sdAb. Results We generated a sdAb binding LOX-1 with a KD of 280 pM?±?62 pM affinity. After 99mTc-labeling, the tracer had radiochemical purity higher then 99 % and retained specificity in in vitro binding studies. Tracer blood clearance was fast with concomitant high kidney retention. At 3 h after injection, uptake in tissues other than plaques was low and not different than background, suggesting a restricted expression pattern of LOX-1. Conversely, uptake in aortic segments increased with plaque content and was due to specific LOX-1 binding. In vivo SPECT/CT imaging 160 min after injection in atherosclerotic mice confirmed specific targeting of LOX-1-expressing aortic plaques. Conclusions The LOX-sdAb specifically targets LOX-1-expressing atherosclerotic plaques within hours after injection. The possibility to image LOX-1 rapidly after administration combined with the favourable biodistribution of a sdAb are beneficial for molecular phenotyping of atherosclerotic plaques and the generation of a future prognostic tracer.

An accurate and noninvasive tracer able to detect molecular events underlying the development of rheumatoid arthritis (RA) would be useful for RA diagnosis and drug efficacy assessment. A complement receptor of the Ig superfamily (CRIg) is expressed on synovial macrophages of RA patients, making it an interesting target for molecular imaging of RA. We aim to develop a radiotracer for the visualization of CRIg in a mouse model for RA using radiolabeled single-domain variable antibody VHH fragments (Nanobodies). Methods: Quantitative polymerase chain reaction was used to locate CRIg expression in mice with collagen-induced arthritis (CIA). A Nanobody, NbV4m119, was generated to specifically target CRIg. Flow cytometry, phosphorimaging, and confocal microscopy were used to confirm NbVm119 binding to CRIg-positive cells. SPECT (SPECT/CT) was used to image arthritic lesions in the inflamed paws of 29 mice using 99mTc-NbV4m119 Nanobody. Results: CRIg is constitutively expressed in the liver and was found to be upregulated in synovial tissues of CIA mice. SPECT/CT imaging revealed that 99mTc-NbV4m119 specifically targeted CRIg-positive liver macrophages in naive wild-type but not in CRIg−/− (CRIg knockout) mice. In CIA mice, 99mTc-NbV4m119 accumulation in arthritic lesions increased according to the severity of the inflammation. In the knees of mice with CIA, 99mTc-NbV4m119 was found to accumulate even before the onset of macroscopic clinical symptoms. Conclusion: SPECT/CT imaging with 99mTc-NbV4m119 visualizes joint inflammation in CIA. Furthermore, imaging could predict which mice will develop clinical symptoms during CIA. Consequently, imaging of joint inflammation with CRIg-specific Nanobodies offers perspectives for clinical applications in RA patients.

The endocrine pancreas represents an interesting arena for regenerative medicine and cell therapeutics. One of the major pancreatic diseases, diabetes mellitus is a metabolic disorder caused by having an insufficient number of insulin-producing β cells. Replenishment of β cells by cell transplantation can restore normal metabolic control. The shortage in donor pancreata has meant that the demand for transplantable β cells has outstripped the supply, which could be met by using alternative sources of stem cells. This situation has opened up new areas of research, such as cellular reprogramming and in vivo β-cell regeneration. Pluripotent stem cells seem to be the best option for clinical applications of β-cell regeneration in the near future, as these cells have been demonstrated to represent an unlimited source of functional β cells. Although compelling evidence shows that the adult pancreas retains regenerative capacity, it remains unclear whether this organ contains stem cells. Alternatively, specialized cell types within or outside the pancreas retain plasticity in proliferation and differentiation. Cellular reprogramming or transdifferentiation of exocrine cells or other types of endocrine cells in the pancreas could provide a long-term solution.

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.

Embryonic stem cells (ESCs) have the potential to be used as unlimited resources for tissue replacement therapy, thereby compensating for organ donor shortage. To reach this goal, the molecular principles governing early differentiation events in the developing embryo need to be addressed, understood and properly implemented in vitro. Studies carried out in several vertebrate models have established that Nodal/Activin A, BMP, WNT and FGF signaling pathways regulate early embryo development and that these pathways are similarly used during germ layer formation by cultured ESCs. However, differences have also been identified in the way these pathways function or interact in mouse vs. human ESCs, making it sometimes difficult to extrapolate findings from one system to the other. In this review, we discuss and compare the role of the relevant signaling pathways and their crosstalk during undifferentiated growth and during the endoderm differentiation of mouse and human ESCs.

Objective Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalathus linearis) extract had anti-hyperglycemic activity and significant protective effects on the pancreatic beta cell mass in a chronic diet-induced diabetes model. The present study evaluated the cytoprotective effect of the phytochemical on beta cells exposed to acute cell stress. Methods Synthetically prepared PPAG was administered orally in mice treated with a single dose of streptozotocin to acutely induce beta cell death and hyperglycemia. Its effect was assessed on beta cell mass, proliferation and apoptotic cell death. Its cytoprotective effect was also studied in vitro on INS-1E beta cells and on human pancreatic islet cells. Results Treatment with the phytochemical PPAG protected beta cells during the first days after the insult against apoptotic cell death, as evidenced by TUNEL staining, and prevented loss of expression of anti-apoptotic protein BCL2 in vivo. In vitro, PPAG protected INS-1E beta cells from streptozotocin-induced apoptosis and necrosis in a BCL2-dependent and independent way, respectively, depending on glucose concentration. PPAG also protected human pancreatic islet cells against the cytotoxic action of the fatty acid palmitate. Conclusions These findings show the potential use of PPAG as phytomedicine which protects the beta cell mass exposed to acute diabetogenic stress., SCOPUS: ar.j, info:eu-repo/semantics/published

Objective Acinar cells display plasticity in vitro and in vivo and can activate a variety of differentiation programmes that may contribute to pancreatic diseases. The aims were to determine: (1) the differentiation potential of acinar cells under conditions which favour stem cell survival, and (2) its relationship to the phenotypes acquired by pancreatic epithelial cells in chronic pancreatitis. Design Murine acinar cells were cultured in suspension and their molecular phenotype was characterised by qRT-PCR, chromatin immunoprecipitation, immunocytochemistry and global transcriptome analysis. These findings were compared to the changes occurring in experimental chronic pancreatitis induced by pancreatic duct ligation and chronic caerulein administration. Results Acinar cells in suspension culture acquired a dedifferentiated phenotype characteristic of pancreatic embryonic progenitors, consisting of the co-expression of Ptf1a and Pdx1, presence of an embryonic-type PTF1 transcriptional complex, activation of the Notch pathway, and expression of additional pancreatic progenitor cell markers such as CpA1, Sox9 and Hnf1b. A senescence programme, associated with activation of Ras and ERK signalling, limited the proliferative capacity of the cells. A similar progenitor-like phenotype with activation of a senescence programme was observed in experimental chronic pancreatitis induced by pancreatic duct ligation or repeated caerulein administration, with the concomitant and differential activation of proliferation and senescence in distinct cell populations. Conclusions Acinar cells dedifferentiate into an embryonic progenitor-like phenotype upon suspension culture. This is associated with the activation of a senescence programme. Both processes take place in experimental chronic pancreatitis where senescence may contribute to limit tumour progression.

Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers.We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays.After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na(+)-K(+)-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2gammaa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2gammaa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2gammaa and loss of insulin-positive cells.We propose human FXYD2gammaa as a novel beta cell-specific biomarker.