KPC-luciferase-expressing cells elicit an anti-tumor immune response in a mouse model of pancreatic cancer.

Mouse models for the study of pancreatic ductal adenocarcinoma (PDAC) are well-established and representative of many key features observed in human PDAC. To monitor tumor growth, cancer cells that are implanted in mice are often transfected with reporter genes, such as firefly luciferase (Luc), enabling in vivo optical imaging over time. Since Luc can induce an immune response, we aimed to evaluate whether the expression of Luc could affect the growth of KPC tumors in mice by inducing immunogenicity. Although both cell lines, KPC and Luc transduced KPC (KPC-Luc), had the same proliferation rate, KPC-Luc tumors had significantly smaller sizes or were absent 13 days after orthotopic cell implantation, compared to KPC tumors. This coincided with the loss of bioluminescence signal over the tumor region. Immunophenotyping of blood and spleen from KPC-Luc tumor-bearing mice showed a decreased number of macrophages and CD4+ T cells, and an increased accumulation of natural killer (NK) cells in comparison to KPC tumor mice. Higher infiltration of CD8+ T cells was found in KPC-Luc tumors than in their controls. Moreover, the immune response against Luc peptide was stronger in splenocytes from mice implanted with KPC-Luc cells compared to those isolated from KPC wild-type mice, indicating increased immunogenicity elicited by the presence of Luc in the PDAC tumor cells. These results must be considered when evaluating the efficacy of anti-cancer therapies including immunotherapies in immunocompetent PDAC or other cancer mouse models that use Luc as a reporter for bioluminescence imaging.

OrganoIDNet: a deep learning tool for identification of therapeutic effects in PDAC organoid-PBMC co-cultures from time-resolved imaging data.

PURPOSE: Pancreatic Ductal Adenocarcinoma (PDAC) remains a challenging disease due to its complex biology and aggressive behavior with an urgent need for efficient therapeutic strategies. To assess therapy response, pre-clinical PDAC organoid-based models in combination with accurate real-time monitoring are required. METHODS: We established stable live-imaging organoid/peripheral blood mononuclear cells (PBMCs) co-cultures and introduced OrganoIDNet, a deep-learning-based algorithm, capable of analyzing bright-field images of murine and human patient-derived PDAC organoids acquired with live-cell imaging. We investigated the response to the chemotherapy gemcitabine in PDAC organoids and the PD-L1 inhibitor Atezolizumab, cultured with or without HLA-matched PBMCs over time. Results obtained with OrganoIDNet were validated with the endpoint proliferation assay CellTiter-Glo. RESULTS: Live cell imaging in combination with OrganoIDNet accurately detected size-specific drug responses of organoids to gemcitabine over time, showing that large organoids were more prone to cytotoxic effects. This approach also allowed distinguishing between healthy and unhealthy status and measuring eccentricity as organoids' reaction to therapy. Furthermore, imaging of a new organoids/PBMCs sandwich-based co-culture enabled longitudinal analysis of organoid responses to Atezolizumab, showing an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added. CONCLUSION: Optimized PDAC organoid imaging analyzed by OrganoIDNet represents a platform capable of accurately detecting organoid responses to standard PDAC chemotherapy over time. Moreover, organoid/immune cell co-cultures allow monitoring of organoid responses to immunotherapy, offering dynamic insights into treatment behavior within a co-culture setting with PBMCs. This setup holds promise for real-time assessment of immunotherapeutic effects in individual patient-derived PDAC organoids.

Colorectal cancer-associated fibroblasts inhibit effector T cells via NECTIN2 signaling.

Cancer-associated fibroblasts play a crucial role within the tumor microenvironment. However, a comprehensive characterization of CAF in colorectal cancer (CRC) is still missing. We combined scRNA-seq and spatial proteomics to decipher fibroblast heterogeneity in healthy human colon and CRC at high resolution. Analyzing nearly 23,000 fibroblasts, we identified 11 distinct clusters and verified them by spatial proteomics. Four clusters, consisting of myofibroblastic CAF (myCAF)-like, inflammatory CAF (iCAF)-like and proliferating fibroblasts as well as a novel cluster, which we named "T cell-inhibiting CAF" (TinCAF), were primarily found in CRC. This new cluster was characterized by the expression of immune-interacting receptors and ligands, including CD40 and NECTIN2. Co-culture of CAF and T cells resulted in a reduction of the effector T cell compartment, impaired proliferation, and increased exhaustion. By blocking its receptor interaction, we demonstrated that NECTIN2 was the key driver of T cell inhibition. Analysis of clinical datasets showed that NECTIN2 expression is a poor prognostic factor in CRC and other tumors. In conclusion, we identified a new class of immuno-suppressive CAF with features rendering them a potential target for future immunotherapies.

In vivo low-dose phase-contrast CT for quantification of functional and anatomical alterations in lungs of an experimental allergic airway disease mouse model.

Introduction: Synchrotron-based propagation-based imaging (PBI) is ideally suited for lung imaging and has successfully been applied in a variety of in vivo small animal studies. Virtually all these experiments were tailored to achieve extremely high spatial resolution close to the alveolar level while delivering high x-ray doses that would not permit longitudinal studies. However, the main rationale for performing lung imaging studies in vivo in small animal models is the ability to follow disease progression or monitor treatment response in the same animal over time. Thus, an in vivo imaging strategy should ideally allow performing longitudinal studies. Methods: Here, we demonstrate our findings of using PBI-based planar and CT imaging with two different detectors-MÖNCH 0.3 direct conversion detector and a complementary metal-oxide-semiconductor (CMOS) detector (Photonics Science)-in an Ovalbumin induced experimental allergic airway disease mouse model in comparison with healthy controls. The mice were imaged free breathing under isoflurane anesthesia. Results: At x-ray dose levels below those once used by commercial small animal CT devices at similar spatial resolutions, we were able to resolve structural changes at a pixel size down to 25 µm and demonstrate the reduction in elastic recoil in the asthmatic mice in cinematic planar x-ray imaging with a frame rate of up to 100 fps. Discussion: Thus, we believe that our approach will permit longitudinal small animal lung disease studies, closely following the mice over longer time spans.

Cocktail of lipophilic and hydrophilic chemotherapeutics in high-load core@shell nanocarriers to treat pancreatic tumours.

ITC/Toc@Gd2(FLP)3 core@shell nanocarriers with a chemotherapeutic cocktail of lipophilic irinotecan (ITC) as the particle core and hydrophilic fludarabine phosphate (FLP) in the particle shell are realized. They are prepared via a microemulsion approach with ITC dissolved in tocopherol (Toc) as droplet phase and stabilized by water-insoluble Gd2(FLP)3. The synthesis can be followed by zeta-potential analysis. X-ray powder diffraction, infrared spectroscopy, elemental analysis, thermogravimetry, and photometry show a drug load of 49 µg per mL ITC and 317 µg per mL FLP at a nanocarrier concentration of 1.5 mg mL-1. Size and structure are evidenced by electron microscopy, resulting in a total diameter of 45 ± 16 nm, an inner core of 40 ± 17 nm, and a shell of 3-8 nm. In vitro studies with different cancer cell lines (i.e., human melanoma/SK-Mel-28, cervical cancer/HeLa, mouse pancreatic cancer/Panc02 and KPC as well as human pancreatic cancer/Capan-1 cells) prove efficient nanocarrier uptake and promising cytostatic efficacy. Specifically for KPC cells, ITC/Toc@Gd2(FLP)3 nanocarriers show an increased efficacy, with half maximal inhibitory concentration (IC50: 4.2 µM) > 10 times lower than the free drugs (IC50: ITC: 47.7 µM, FLP: 143 µM). This points to the synergistic effect of the ITC/FLP drug cocktail in the nanocarriers and may result in a promising strategy to treat pancreatic ductal adenocarcinoma (PDAC).

Characterization of transient and progressive pulmonary fibrosis by spatially correlated phase contrast microCT, classical histopathology and atomic force microscopy.

Pulmonary fibrosis (PF) is a severe and progressive condition in which the lung becomes scarred over time resulting in pulmonary function impairment. Classical histopathology remains an important tool for micro-structural tissue assessment in the diagnosis of PF. A novel workflow based on spatial correlated propagation-based phase-contrast micro computed tomography (PBI-microCT), atomic force microscopy (AFM) and histopathology was developed and applied to two different preclinical mouse models of PF - the commonly used and well characterized Bleomycin-induced PF and a novel mouse model for progressive PF caused by conditional Nedd4-2 KO. The aim was to integrate structural and mechanical features from hallmarks of fibrotic lung tissue remodeling. PBI-microCT was used to assess structural alteration in whole fixed and paraffin embedded lungs, allowing for identification of fibrotic foci within the 3D context of the entire organ and facilitating targeted microtome sectioning of planes of interest for subsequent histopathology. Subsequently, these sections of interest were subjected to AFM to assess changes in the local tissue stiffness of previously identified structures of interest. 3D whole organ analysis showed clear morphological differences in 3D tissue porosity between transient and progressive PF and control lungs. By integrating the results obtained from targeted AFM analysis, it was possible to discriminate between the Bleomycin model and the novel conditional Nedd4-2 KO model using agglomerative cluster analysis. As our workflow for 3D spatial correlation of PBI, targeted histopathology and subsequent AFM is tailored around the standard procedure of formalin-fixed paraffin-embedded (FFPE) tissue specimens, it may be a powerful tool for the comprehensive tissue assessment beyond the scope of PF and preclinical research.

Genetic newborn screening and digital technologies: A project protocol based on a dual approach to shorten the rare diseases diagnostic path in Europe.

Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems.

Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice.

The age-related decline in immunity reduces the effectiveness of vaccines in older adults. Immunosenescence is associated with chronic, low-grade inflammation, and the accumulation of senescent cells. The latter express Bcl-2 family members (providing resistance to cell death) and exhibit a pro-inflammatory, senescence-associated secretory phenotype (SASP). Preexisting senescent cells cause many aging-related disorders and therapeutic means of eliminating these cells have recently gained attention. The potential consequences of senescent cell removal on vaccine efficacy in older individuals are still ignored. We used the Bcl-2 family inhibitor ABT-263 to investigate the effects of pre-vaccination senolysis on immune responses in old mice. Two different ovalbumin (OVA)-containing vaccines (containing a saponin-based or a CpG oligodeoxynucleotide adjuvant) were tested. ABT-263 depleted senescent cells (apoptosis) and ablated the basal and lipopolysaccharide-induced production of SASP-related factors in old mice. Depletion of senescent cells prior to vaccination (prime/boost) had little effect on OVA-specific antibody and T-cell responses (slightly reduced and augmented, respectively). We then used a preclinical melanoma model to test the antitumor potential of senolysis before vaccination (prime with the vaccine and OVA boost by tumor cells). Surprisingly, ABT-263 treatment abrogated the vaccine's ability to protect against B16 melanoma growth in old animals, an effect associated with reduced antigen-specific T-cell responses. Some, but not all, of the effects were age-specific, which suggests that preexisting senescent cells were partly involved. Hence, depletion of senescent cells modifies immune responses to vaccines in some settings and caution should be taken when incorporating senolytics into vaccine-based cancer therapies.

Spatial correlation of 2D hard-tissue histology with 3D microCT scans through 3D printed phantoms.

Hard-tissue histology-the analysis of thin two-dimensional (2D) sections-is hampered by the opaque nature of most biological specimens, especially bone. Therefore, the cutting process cannot be assigned to regions of interest. In addition, the applied cutting-grinding method is characterized by significant material loss. As a result, relevant structures might be missed or destroyed, and 3D features can hardly be evaluated. Here, we present a novel workflow, based on conventual microCT scans of the specimen prior to the cutting process, to be used for the analysis of 3D structural features and for directing the sectioning process to the regions of interest. 3D printed fiducial markers, embedded together with the specimen in resin, are utilized to retrospectively register the obtained 2D histological images into the 3D anatomical context. This not only allows to identify the cutting position, but also enables the co-registration of the cell and extracellular matrix morphological analysis to local 3D information obtained from the microCT data. We have successfully applied our new approach to assess hard-tissue specimens of different species. After matching the predicted microCT cut plane with the histology image, we validated a high accuracy of the registration process by computing quality measures namely Jaccard and Dice similarity coefficients achieving an average score of 0.90 ± 0.04 and 0.95 ± 0.02, respectively. Thus, we believe that the novel, easy to implement correlative imaging approach holds great potential for improving the reliability and diagnostic power of classical hard-tissue histology.

Effect of tissue fixation on the optical properties of structural components assessed by non-linear microscopy imaging.

Fixation methods such as formalin are commonly used for the preservation of tissue with the aim of keeping their structure as close as possible to the native condition. However, fixatives chemically interact with tissue molecules, such as collagen in the extracellular matrix (ECM) or myosin, and may thus modify their structure. Taking advantage of the second- and third-harmonic generation (SHG and THG) emission capabilities of such components, we used nonlinear two-photon microscopy (NL2PM) to evaluate the effect that preservation methods, such as chemical fixatives, have on the nonlinear capabilities of protein components within mouse tissues. Our results show that depending on the preservation technique used, the nonlinear capabilities of collagen, lipid droplets and myosin microarchitecture are strongly affected. Parameters of collagen fibers, such as density and branch points, especially in collagen-sparse regions, e.g., in kidneys, were found to be altered upon formalin fixation. Moreover, cryo-freezing drastically reduced SHG signals from myosin. Our findings provide valuable information to select the best tissue fixation method for visualization and quantification of structural proteins, such as collagen and myosin by advanced NL2PM imaging techniques. This may advance the interpretation of the role these proteins play in disease.

Longitudinal x-ray based lung function measurement for monitoring Nintedanib treatment response in a mouse model of lung fibrosis.

Lung fibrosis (LF) is a chronic progressive, incurable, and debilitating condition of the lung, which is associated with different lung disease. Treatment options are still sparse. Nintedanib, an oral tyrosine kinase inhibitor, significantly slows the LF progression. However, there is a strong need of further research and the development of novel therapies. In this study, we used a correlative set-up that combines X-ray based lung function (XLF) with microCT and whole body plethysmography (WBP) for a comprehensive functional and structural evaluation of lung fibrosis (LF) as well as for monitoring response to orally administered Nintedanib in the mouse model of bleomycin induced LF. The decline in lung function as early as one week after intratracheal bleomycin instillation was reliably detected by XLF, revealing the lowest decay rate in the LF mice compared to healthy ones. Simultaneously performed microCT and WBP measurements corroborated XLF findings by exhibiting reduced lung volume [Formula: see text] and tidal volume [Formula: see text]. In LF mice XLF also revealed profound improvement in lung function one week after Nintedanib treatment. This positive response to Nintedanib therapy was further substantiated by microCT and WBP measurements which also showed significantly improved [Formula: see text] and [Formula: see text] in the Nintedanib treated mice. By comparing the XLF data to structural features assessing the extent of fibrosis obtained by ex-vivo high-resolution synchrotron radiation-based imaging and classical histology we demonstrate that: (1) a simple low dose x-ray measurement like XLF is sensitive enough to pick up treatment response, (2) Nintedanib treatment successfully improved lung function in a bleomycin induced LF mouse model and (3) differences between the fully restored lung function and the partially reduced fibrotic burden compared to healthy and untreated mice. The presented analysis pipeline underlines the importance of a combined functional and anatomical readout to reliably measure treatment response and could easily be adapted to other preclinical lung disease models.

High-Load Gemcitabine Inorganic-Organic Hybrid Nanoparticles as an Image-Guided Tumor-Selective Drug-Delivery System to Treat Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis without effective treatment options. Thus, there is an urgent need for more effective and safe therapies. Here, inorganic-organic hybrid nanoparticles (GMP-IOH-NPs) are presented as a novel drug-delivery system for the selective delivery of extraordinarily high concentrations of gemcitabine monophosphate (GMP), not only to the primary tumor but also to metastatic sites. GMP-IOH-NPs have a composition of [ZrO]2+ [GMP]2 - with GMP as drug anion (76% of total IOH-NP mass). Multiscale fluorescence imaging confirms an efficient uptake in tumor cells, independent of the activity of the human-equilibrative-nucleoside transporter (hENT1), being responsible for gemcitabine (GEM) transport into cells and a key factor for GEM resistance. Delivering already phosphorylated GMP via GMP-IOH-NPs into tumor cells also allows the cellular resistance induced by the downregulation of deoxycytidine kinase to be overcome. GMP-IOH-NPs show high accumulation in tumor lesions and only minor liver trapping when given intraperitoneally. GMP-IOH-NPs result in a higher antitumor efficacy compared to free GEM, which is further enhanced applying cetuximab-functionalized GMP-CTX-IOH-NPs. By maximizing the therapeutic benefits with high drug load, tumor-specific delivery, minimizing undesired side effects, overcoming mechanisms of chemoresistance, and preventing systemic GEM inactivation, GMP-IOH-NPs are anticipated to have a high chance to significantly improve current PDAC-patient outcome.

Three-dimensional analysis of human pancreatic cancer specimens by phase-contrast based X-ray tomography - the next dimension of diagnosis.

BACKGROUND: The worldwide increase of pancreatic ductal adenocarcinoma (PDAC), which still has one of the lowest survival rates, requires novel imaging tools to improve early detection and to refine diagnosis. Therefore, the aim of this study was to assess the feasibility of propagation-based phase-contrast X-ray computed tomography of already paraffin-embedded and unlabeled human pancreatic tumor tissue to achieve a detailed three-dimensional (3D) view of the tumor sample in its entirety. METHODS: Punch biopsies of areas of particular interest were taken from paraffin blocks after initial histological analysis of hematoxylin and eosin stained tumor sections. To cover the entire 3.5 mm diameter of the punch biopsy, nine individual tomograms with overlapping regions were acquired in a synchrotron parallel beam configuration and stitched together after data reconstruction. Due to the intrinsic contrast based on electron density differences of tissue components and a voxel size of 1.3 µm achieved PDAC and its precursors were clearly identified. RESULTS: Characteristic tissue structures for PDAC and its precursors, such as dilated pancreatic ducts, altered ductal epithelium, diffuse immune cell infiltrations, increased occurrence of tumor stroma and perineural invasion were clearly identified. Certain structures of interest were visualized in three dimensions throughout the tissue punch. Pancreatic duct ectasia of different caliber and atypical shape as well as perineural infiltration could be contiguously traced by viewing serial tomographic slices and by applying semi-automatic segmentation. Histological validation of corresponding sections confirmed the former identified PDAC features. CONCLUSION: In conclusion, virtual 3D histology via phase-contrast X-ray tomography visualizes diagnostically relevant tissue structures of PDAC in their entirety, preserving tissue integrity in label-free, paraffin embedded tissue biopsies. In the future, this will not only enable a more comprehensive diagnosis but also a possible identification of new 3D imaging tumor markers.

Theranostic inorganic-organic hybrid nanoparticles with a cocktail of chemotherapeutic and cytostatic drugs.

Theranostic inorganic-organic hybrid nanoparticles (IOH-NPs) with a cocktail of chemotherapeutic and cytostatic drugs and a composition Gd23+[(PMX)0.5(EMP)0.5]32-, [Gd(OH)]2+[(PMX)0.74(AlPCS4)0.13]2-, or [Gd(OH)]2+[(PMX)0.70(TPPS4)0.15]2- (PMX: pemetrexed, EMP: estramustine phosphate, AlPCS4: aluminum(III) chlorido phthalocyanine tetrasulfonate, TPPS4: tetraphenylporphine sulfonate) are presented for the first time. These IOH-NPs are prepared in water (40-60 nm in size) and have a non-complex composition with outstanding drug loading (71-82% of total nanoparticle mass) of at least two chemotherapeutic or a mixture of cytostatic and photosensitizing agents. All IOH-NPs show red to deep-red emission (650-800 nm) to enable optical imaging. The superior performance of the IOH-NPs with a chemotherapeutic/cytostatic cocktail is validated based on cell-viability assays and angiogenesis studies with human umbilical vein endothelial cells (HUVEC). The synergistic anti-cancer effect of the IOH-NPs with a chemotherapeutic cocktail is shown in a murine breast-cancer cell line (pH8N8) and a human pancreatic cancer cell line (AsPC1), whereas the synergistic cytotoxic and phototoxic efficacy is verified in response to illumination of HeLa-GFP cancer cells, MTT assays with human colon cancer cells (HCT116), and normal human dermal fibroblasts (NHDF). HepG2 spheroids as 3D cell cultures prove the effective uptake of the IOH-NPs with high uniform distribution and the release of the chemotherapeutic drugs with the strong synergistic effect of the cocktail of drugs.

High resolution propagation-based lung imaging at clinically relevant X-ray dose levels.

Absorption-based clinical computed tomography (CT) is the current imaging method of choice in the diagnosis of lung diseases. Many pulmonary diseases are affecting microscopic structures of the lung, such as terminal bronchi, alveolar spaces, sublobular blood vessels or the pulmonary interstitial tissue. As spatial resolution in CT is limited by the clinically acceptable applied X-ray dose, a comprehensive diagnosis of conditions such as interstitial lung disease, idiopathic pulmonary fibrosis or the characterization of small pulmonary nodules is limited and may require additional validation by invasive lung biopsies. Propagation-based imaging (PBI) is a phase sensitive X-ray imaging technique capable of reaching high spatial resolutions at relatively low applied radiation dose levels. In this publication, we present technical refinements of PBI for the characterization of different artificial lung pathologies, mimicking clinically relevant patterns in ventilated fresh porcine lungs in a human-scale chest phantom. The combination of a very large propagation distance of 10.7 m and a photon counting detector with [Formula: see text] pixel size enabled high resolution PBI CT with significantly improved dose efficiency, measured by thermoluminescence detectors. Image quality was directly compared with state-of-the-art clinical CT. PBI with increased propagation distance was found to provide improved image quality at the same or even lower X-ray dose levels than clinical CT. By combining PBI with iodine k-edge subtraction imaging we further demonstrate that, the high quality of the calculated iodine concentration maps might be a potential tool for the analysis of lung perfusion in great detail. Our results indicate PBI to be of great value for accurate diagnosis of lung disease in patients as it allows to depict pathological lesions non-invasively at high resolution in 3D. This will especially benefit patients at high risk of complications from invasive lung biopsies such as in the setting of suspected idiopathic pulmonary fibrosis (IPF).

Basal-like mammary carcinomas stimulate cancer stem cell properties through AXL-signaling to induce chemotherapy resistance.

Basal-like breast cancer (BLBC) is the most aggressive and heterogeneous breast cancer (BC) subtype. Conventional chemotherapies represent next to surgery the most frequently employed treatment options. Unfortunately, resistant tumor phenotypes often develop, resulting in therapeutic failure. To identify the early events occurring upon the first drug application and initiating chemotherapy resistance in BLBC, we leveraged the WAP-T syngeneic mammary carcinoma mouse model and we developed a strategy combining magnetic-activated cell sorting (MACS)-based tumor cell enrichment with high-throughput transcriptome analyses. We discovered that chemotherapy induced a massive gene expression reprogramming toward stemness acquisition to tolerate and survive the cytotoxic treatment in vitro and in vivo. Retransplantation experiments revealed that one single cycle of cytotoxic drug combination therapy (Cyclophosphamide, Adriamycin and 5-Fluorouracil) suffices to induce resistant tumor cell phenotypes in vivo. We identified Axl and its ligand Pros1 as highly induced genes driving cancer stem cell (CSC) properties upon chemotherapy in vivo and in vitro. Furthermore, from our analysis of BLBC patient datasets, we found that AXL expression is also strongly correlated with CSC-gene signatures, a poor response to conventional therapies and worse survival outcomes in those patients. Finally, we demonstrate that AXL inhibition sensitized BLBC-cells to cytotoxic treatment in vitro. Together, our data support AXL as a promising therapeutic target to optimize the efficiency of conventional cytotoxic therapies in BLBC.

Crosstalk between tumor acidosis, p53 and extracellular matrix regulates pancreatic cancer aggressiveness.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with minimal treatment options and a global rise in prevalence. PDAC is characterized by frequent driver mutations including KRAS and TP53 (p53), and a dense, acidic tumor microenvironment (TME). The relation between genotype and TME in PDAC development is unknown. Strikingly, when wild type (WT) Panc02 PDAC cells were adapted to growth in an acidic TME and returned to normal pH to mimic invasive cells escaping acidic regions, they displayed a strong increase of aggressive traits such as increased growth in 3-dimensional (3D) culture, adhesion-independent colony formation and invasive outgrowth. This pattern of acidosis-induced aggressiveness was observed in 3D spheroid culture as well as upon organotypic growth in matrigel, collagen-I and combination thereof, mimicking early and later stages of PDAC development. Acid-adaptation-induced gain of cancerous traits was further increased by p53 knockout (KO), but only in specific extracellular matrix (ECM) compositions. Akt- and Transforming growth factor-ß (TGFß) signaling, as well as expression of the Na+ /H+ exchanger NHE1, were increased by acid adaptation. Whereas Akt inhibition decreased spheroid growth regardless of treatment and genotype, stimulation with TGFßI increased growth of WT control spheroids, and inhibition of TGFß signaling tended to limit growth under acidic conditions only. Our results indicate that a complex crosstalk between tumor acidosis, ECM composition and genotype contributes to PDAC development. The findings may guide future strategies for acidosis-targeted therapies.

Current Approaches for Image Fusion of Histological Data with Computed Tomography and Magnetic Resonance Imaging.

Classical analysis of biological samples requires the destruction of the tissue's integrity by cutting or grinding it down to thin slices for (Immuno)-histochemical staining and microscopic analysis. Despite high specificity, encoded in the stained 2D section of the whole tissue, the structural information, especially 3D information, is limited. Computed tomography (CT) or magnetic resonance imaging (MRI) scans performed prior to sectioning in combination with image registration algorithms provide an opportunity to regain access to morphological characteristics as well as to relate histological findings to the 3D structure of the local tissue environment. This review provides a summary of prevalent literature addressing the problem of multimodal coregistration of hard- and soft-tissue in microscopy and tomography. Grouped according to the complexity of the dimensions, including image-to-volume (2D ⟶ 3D), image-to-image (2D ⟶ 2D), and volume-to-volume (3D ⟶ 3D), selected currently applied approaches are investigated by comparing the method accuracy with respect to the limiting resolution of the tomography. Correlation of multimodal imaging could position itself as a useful tool allowing for precise histological diagnostic and allow the a priori planning of tissue extraction like biopsies.

Monitoring nanoparticle dissolution via fluorescence-colour shift.

[La(OH)]2+[ICG]-2 and [La(OH)]2+2[PTC]4- inorganic-organic hybrid nanoparticles (IOH-NPs) with indocyanine green (ICG) and perylene-3,4,9,10-tetracarboxylate (PTC) as fluorescent dye anions are used for emission-based monitoring of the dissolution of nanoparticles. Whereas ICG shows a deep red emission in the solid [La(OH)]2+[ICG]-2 IOH-NPs, the emission of PTC in the solid [La(OH)]2+2[PTC]4- IOH-NPs is completely quenched due to π-stacking. After nanoparticle dissolution, the emission of freely dissolved ICG is weak, whereas freely dissolved PTC shows intense green emission. We report on the synthesis of IOH-NPs and nanoparticle characterization as well as on the fluorescence properties and how to avoid undesirable energy transfer between different fluorescent dyes. The emission shift from red (intact solid nanoparticles) to green (freely dissolved dye anions), indicating nanoparticle dissolution, is shown for aqueous systems and verified in vitro. Based on this first proof-of-the-concept, the IOH-NP marker system can be interesting to monitor nanoparticle dissolution in cells and tissues of small animals and to evaluate cell processes and/or drug-delivery strategies.

ROBO3s: a novel ROBO3 short isoform promoting breast cancer aggressiveness.

Basal-like breast cancer (BLBC) is a highly aggressive breast cancer subtype frequently associated with poor prognosis. Due to the scarcity of targeted treatment options, conventional cytotoxic chemotherapies frequently remain the standard of care. Unfortunately, their efficacy is limited as BLBC malignancies rapidly develop resistant phenotypes. Using transcriptomic and proteomic approaches in human and murine BLBC cells, we aimed to elucidate the molecular mechanisms underlying the acquisition of aggressive and chemotherapy-resistant phenotypes in these mammary tumors. Specifically, we identified and characterized a novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s), upregulated in BLBC in response to chemotherapy and encoding for a protein variant lacking the transmembrane domain. We established an important role for the ROBO3s isoform, mediating cancer stem cell properties by stimulating the Hippo-YAP signaling pathway, and thus driving resistance of BLBC cells to cytotoxic drugs. By uncovering the conservation of ROBO3s expression across multiple cancer types, as well as its association with reduced BLBC-patient survival, we emphasize its potential as a prognostic marker and identify a novel attractive target for anti-cancer drug development.