Spectrally selective bSSFP using off-resonant RF excitations permits high spatiotemporal resolution 3D metabolic imaging of hyperpolarized [1-13 C]Pyruvate-to-[1-13 C]lactate conversion.

PURPOSE: To develop a high spatiotemporal resolution 3D dynamic pulse sequence for preclinical imaging of hyperpolarized [1-13 C]pyruvate-to-[1-13 C]lactate metabolism at 7T. METHODS: A standard 3D balanced SSFP (bSSFP) sequence was modified to enable alternating-frequency excitations. RF pulses with 2.33 ms duration and 900 Hz FWHM were placed off-resonance of the target metabolites, [1-13 C]pyruvate (by approximately -245 Hz) and [1-13 C]lactate (by approximately 735 Hz), to selectively excite those resonances. Relatively broad bandwidth (compared to those metabolites' chemical shift offset) permits a short TR of 6.29 ms, enabling higher spatiotemporal resolution. Bloch equation simulations of the bSSFP response profile guided the sequence parameter selection to minimize spectral contamination between metabolites and preserve magnetization over time. RESULTS: Bloch equation simulations, phantom studies, and in vivo studies demonstrated that the two target resonances could be cleanly imaged without substantial bSSFP banding artifacts and with little spectral contamination between lactate and pyruvate and from pyruvate hydrate. High spatiotemporal resolution 3D images were acquired of in vivo pyruvate-lactate metabolism in healthy wild-type and endogenous pancreatic tumor-bearing mice, with 1.212 s acquisition time per single-metabolite image and (1.75 mm)3 isotropic voxels with full mouse abdomen 56 × 28 × 21 mm3 FOV and fully-sampled k-space. Kidney and tumor lactate/pyruvate ratios of two consecutive measurements in one animal, 1 h apart, were consistent. CONCLUSION: Spectrally selective bSSFP using off-resonant RF excitations can provide high spatio-temporal resolution 3D dynamic images of pyruvate-lactate metabolic conversion.

Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes.

BACKGROUND & AIMS: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. METHODS: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. RESULTS: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. CONCLUSIONS: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.

Functional biomarkers derived from computed tomography and magnetic resonance imaging differentiate PDAC subgroups and reveal gemcitabine-induced hypo-vascularization.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a molecularly heterogeneous tumor entity with no clinically established imaging biomarkers. We hypothesize that tumor morphology and physiology, including vascularity and perfusion, show variations that can be detected by differences in contrast agent (CA) accumulation measured non-invasively. This work seeks to establish imaging biomarkers for tumor stratification and therapy response monitoring in PDAC, based on this hypothesis. METHODS AND MATERIALS: Regional CA accumulation in PDAC was correlated with tumor vascularization, stroma content, and tumor cellularity in murine and human subjects. Changes in CA distribution in response to gemcitabine (GEM) were monitored longitudinally with computed tomography (CT) Hounsfield Units ratio (HUr) of tumor to the aorta or with magnetic resonance imaging (MRI) ΔR1 area under the curve at 60 s tumor-to-muscle ratio (AUC60r). Tissue analyses were performed on co-registered samples, including endothelial cell proliferation and cisplatin tissue deposition as a surrogate of chemotherapy delivery. RESULTS: Tumor cell poor, stroma-rich regions exhibited high CA accumulation both in human (meanHUr 0.64 vs. 0.34, p < 0.001) and mouse PDAC (meanAUC60r 2.0 vs. 1.1, p < 0.001). Compared to the baseline, in vivo CA accumulation decreased specifically in response to GEM treatment in a subset of human (HUr -18%) and mouse (AUC60r -36%) tumors. Ex vivo analyses of mPDAC showed reduced cisplatin delivery (GEM: 0.92 ± 0.5 mg/g, vs. vehicle: 3.1 ± 1.5 mg/g, p = 0.004) and diminished endothelial cell proliferation (GEM: 22.3% vs. vehicle: 30.9%, p = 0.002) upon GEM administration. CONCLUSION: In PDAC, CA accumulation, which is related to tumor vascularization and perfusion, inversely correlates with tumor cellularity. The standard of care GEM treatment results in decreased CA accumulation, which impedes drug delivery. Further investigation is warranted into potentially detrimental effects of GEM in combinatorial therapy regimens.

CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice.

Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.

Microvascular dysfunction in the course of metabolic syndrome induced by high-fat diet.

BACKGROUND: Metabolic syndrome (MetS) is associated with increased risk of cardiovascular disease (CVD). One important feature underlying the pathophysiology of many types of CVD is microvascular dysfunction. Although components of MetS are themselves CVD risk factors, the risk is increased when the syndrome is considered as one entity. We aimed to characterize microvascular function and some of its influencing factors in the course of MetS development. METHODS: Development of MetS in C57BL/6 mice on a high-fat diet (HFD, 51% of energy from fat) was studied. The initial phase of MetS (I-MetS) was defined as the first 2 weeks of HFD feeding, with the fully developed phase occurring after 8 weeks of HFD. We characterized these phases by assessing changes in adiposity, blood pressure, and microvascular function. All data are presented as mean ± standard error (SEM). Differences between cumulative dose-response curves of myograph experiments were calculated using non-linear regression analysis. In other experiments, comparisons between two groups were made with Student's t-test. Comparisons between more than two groups were made using one-way ANOVA with Tukey post-hoc test. A probability value <0.05 was considered statistically significant. RESULTS: I-MetS mice presented with weight gain, blood pressure elevation, and microvascular dysfunction characterized by augmented vasoconstriction. This finding, contrary to those in mice with fully developed MetS, was not associated with endothelial dysfunction, insulin resistance, or systemic inflammation. In the initial phase, perivascular adipose tissue showed no sign of inflammation and had no influence on the pattern of vasoconstriction. These findings suggest that the onset of hypertension in MetS is strongly influenced by vascular smooth muscle cell dysfunction and independent of important factors known to influence microvascular function and consequently blood pressure levels. CONCLUSION: We identified in I-MetS the occurrence of isolated augmented vasoconstriction along with blood pressure elevation, but not the presence of classical MetS components known to influence microvascular function. These findings increase our understanding of the pathophysiology of CVD risk associated with MetS.

Early requirement of Rac1 in a mouse model of pancreatic cancer.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease without effective chemopreventive or therapeutic approaches. Although the role of oncogenic Kras in initiating development of PDAC is well established, downstream targets of aberrant Ras signaling are poorly understood. Acinar-ductal metaplasia (ADM) appears to be an important prerequisite for development of pancreatic intraepithelial neoplasia (PanIN), a common precursor to PDAC. RAS-related C3 botulinum substrate 1 (Rac1), which controls actin reorganization, can be activated by Ras, is up-regulated in several human cancers, and is required for cerulein-induced morphologic changes in acini. We investigated effects of loss of Rac1 in Kras-induced pancreatic carcinogenesis in mice. METHODS: Using a Cre/lox approach, we deleted Rac1 from pancreatic progenitor cells in different mouse models of PDAC and in mice with cerulein-induced acute pancreatitis. Acinar epithelial explants of mutant mice were used to investigate the role of Rac1 in vitro. RESULTS: Rac1 expression increased in mouse and human pancreatic tumors, particularly in the stroma. Deletion of Rac1 in Kras(G12D)-induced PDAC in mice reduced formation of ADM, PanIN, and tumors and significantly prolonged survival. Pancreatic epithelial metaplasia was accompanied by apical-basolateral redistribution of F-actin, along with basal expression of Rac1. Acinar epithelial explants that lacked Rac1 or that were incubated with inhibitors of actin polymerization had a reduced ability to undergo ADM in 3-dimensional cultures. CONCLUSIONS: In mice, Rac1 is required for early metaplastic changes and neoplasia-associated actin rearrangements in development of pancreatic cancer. Rac1 might be developed as a diagnostic marker or therapeutic target for PDAC.

Functional noninvasive detection of glycolytic pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) lacks effective treatment options beyond chemotherapy. Although molecular subtypes such as classical and QM (quasi-mesenchymal)/basal-like with transcriptome-based distinct signatures have been identified, deduced therapeutic strategies and targets remain elusive. Gene expression data show enrichment of glycolytic genes in the more aggressive and therapy-resistant QM subtype. However, whether the glycolytic transcripts are translated into functional glycolysis that could further be explored for metabolic targeting in QM subtype is still not known. METHODS: We used different patient-derived PDAC model systems (conventional and primary patient-derived cells, patient-derived xenografts (PDX), and patient samples) and performed transcriptional and functional metabolic analysis. These included RNAseq and Illumina HT12 bead array, in vitro Seahorse metabolic flux assays and metabolic drug targeting, and in vivo hyperpolarized [1-13C]pyruvate and [1-13C]lactate magnetic resonance spectroscopy (HP-MRS) in PDAC xenografts. RESULTS: We found that glycolytic metabolic dependencies are not unambiguously functionally exposed in all QM PDACs. Metabolic analysis demonstrated functional metabolic heterogeneity in patient-derived primary cells and less so in conventional cell lines independent of molecular subtype. Importantly, we observed that the glycolytic product lactate is actively imported into the PDAC cells and used in mitochondrial oxidation in both classical and QM PDAC cells, although more actively in the QM cell lines. By using HP-MRS, we were able to noninvasively identify highly glycolytic PDAC xenografts by detecting the last glycolytic enzymatic step and prominent intra-tumoral [1-13C]pyruvate and [1-13C]lactate interconversion in vivo. CONCLUSION: Our study adds functional metabolic phenotyping to transcriptome-based analysis and proposes a functional approach to identify highly glycolytic PDACs as candidates for antimetabolic therapeutic avenues.

Correlation of in vivo imaging to morphomolecular pathology in translational research: challenge accepted.

Correlation of in vivo imaging to histomorphological pathology in animal models requires comparative interdisciplinary expertise of different fields of medicine. From the morphological point of view, there is an urgent need to improve histopathological evaluation in animal model-based research to expedite translation into clinical applications. While different other fields of translational science were standardized over the last years, little was done to improve the pipeline of experimental pathology to ensure reproducibility based on pathological expertise in experimental animal models with respect to defined guidelines and classifications. Additionally, longitudinal analyses of preclinical models often use a variety of imaging methods and much more attention should be drawn to enable for proper co-registration of in vivo imaging methods with the ex vivo morphological read-outs. Here we present the development of the Comparative Experimental Pathology (CEP) unit embedded in the Institute of Pathology of the Technical University of Munich during the Collaborative Research Center 824 (CRC824) funding period together with selected approaches of histomorphological techniques for correlation of in vivo imaging to morphomolecular pathology.

Hyperpolarized 13C Spectroscopy with Simple Slice-and-Frequency-Selective Excitation.

Hyperpolarized 13C nuclear magnetic resonance spectroscopy can characterize in vivo tissue metabolism, including preclinical models of cancer and inflammatory disease. Broad bandwidth radiofrequency excitation is often paired with free induction decay readout for spectral separation, but quantification of low-signal downstream metabolites using this method can be impeded by spectral peak overlap or when frequency separation of the detected peaks exceeds the excitation bandwidth. In this work, alternating frequency narrow bandwidth (250 Hz) slice-selective excitation was used for 13C spectroscopy at 7 T in a subcutaneous xenograft rat model of human pancreatic cancer (PSN1) to improve quantification while measuring the dynamics of injected hyperpolarized [1-13C]lactate and its metabolite [1-13C]pyruvate. This method does not require sophisticated pulse sequences or specialized radiofrequency and gradient pulses, but rather uses nominally spatially offset slices to produce alternating frequency excitation with simpler slice-selective radiofrequency pulses. Additionally, point-resolved spectroscopy was used to calibrate the 13C frequency from the thermal proton signal in the target region. This excitation scheme isolates the small [1-13C]pyruvate peak from the similar-magnitude tail of the much larger injected [1-13C]lactate peak, facilitates quantification of the [1-13C]pyruvate signal, simplifies data processing, and could be employed for other substrates and preclinical models.

Qualitative and Quantitative Comparison of Respiratory Triggered Reduced Field-of-View (FOV) Versus Full FOV Diffusion Weighted Imaging (DWI) in Pancreatic Pathologies.

RATIONALE AND OBJECTIVES: To investigate the effects of a reduced field-of-view (rFOV) acquisition in diffusion-weighted magnetic resonance imaging of the pancreas. MATERIALS AND METHODS: We enrolled 153 patients who underwent routine clinical MRI work-up including respiratory-triggered diffusion-weighted single-shot echo-planar imaging (DWI) with full field-of-view (fFOV, 3 × 3 × 4 mm3 voxel size) and reduced field-of-view (rFOV, 2.5 × 2.5 × 3 mm3 voxel size) for suspected pancreatic pathology. Two experienced radiologists were asked to subjectively rate (Likert Scale 1-4) image quality (overall image quality, lesion conspicuity, anatomical detail, artifacts). In addition, quantitative image parameters were assessed (apparent diffusion coefficient, apparent signal to noise ratio, apparent contrast to noise ratio [CNR]). RESULTS: All subjective metrics of image quality were rated in favor of rFOV DWI images compared to fFOV DWI images with substantial-to-high inter-rater reliability. Calculated ADC values of normal pancreas, pancreatic pathologies and reference tissues revealed no differences between both sequences. Whereas the apparent signal to noise ratio was higher in fFOV images, apparent CNR was higher in rFOV images. CONCLUSION: rFOV DWI provides higher image quality and apparent CNR values, favorable in the analysis of pancreatic pathologies.

Hyperpolarized 13C pyruvate magnetic resonance spectroscopy for in vivo metabolic phenotyping of rat HCC.

The in vivo assessment of tissue metabolism represents a novel strategy for the evaluation of oncologic disease. Hepatocellular carcinoma (HCC) is a high-prevalence, high-mortality tumor entity often discovered at a late stage. Recent evidence indicates that survival differences depend on metabolic alterations in tumor tissue, with particular focus on glucose metabolism and lactate production. Here, we present an in vivo imaging technique for metabolic tumor phenotyping in rat models of HCC. Endogenous HCC was induced in Wistar rats by oral diethyl-nitrosamine administration. Peak lactate-to-alanine signal ratios (L/A) were assessed with hyperpolarized magnetic resonance spectroscopic imaging (HPMRSI) after [1-13C]pyruvate injection. Cell lines were derived from a subset of primary tumors, re-implanted in nude rats, and assessed in vivo with dynamic hyperpolarized magnetic resonance spectroscopy (HPMRS) after [1-13C]pyruvate injection and kinetic modelling of pyruvate metabolism, taking into account systemic lactate production and recirculation. For ex vivo validation, enzyme activity and metabolite concentrations were spectroscopically quantified in cell and tumor tissue extracts. Mean peak L/A was higher in endogenous HCC compared to non-tumorous tissue. Dynamic HPMRS revealed higher pyruvate-to-lactate conversion rates (kpl) and lactate signal in subcutaneous tumors derived from high L/A tumor cells, consistent with ex vivo measurements of higher lactate dehydrogenase (LDH) levels in these cells. In conclusion, HPMRS and HPMRSI reveal distinct tumor phenotypes corresponding to differences in glycolytic metabolism in HCC tumor tissue.

Combined DCE-MRI- and FDG-PET enable histopathological grading prediction in a rat model of hepatocellular carcinoma.

PURPOSE: To test combined dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and 18F-FDG positron emission tomography (FDG-PET)-derived parameters for prediction of histopathological grading in a rat Diethyl Nitrosamine (DEN)-induced hepatocellular carcinoma (HCC) model. METHODS: 15 male Wistar rats, aged 10 weeks were treated with oral DEN 0.01 % in drinking water and monitored until HCCs were detectable. DCE-MRI and PET were performed consecutively on small animal scanners. 38 tumors were identified and manually segmented based on HCC-specific contrast enhancement patterns. Grading (G2/3: 24 tumors, G1:14 tumors) alongside other histopathological parameters, tumor volume, contrast agent and 18F-FDG uptake metrics were noted. Class imbalance was addressed using SMOTE and collinearity was removed using hierarchical clustering and principal component analysis. A logistic regression model was fit separately to the individual parameter groups (DCE-MRI-derived, PET-derived, tumor volume) and the combined parameters. RESULTS: The combined model using all imaging-derived parameters achieved a mean ± STD sensitivity of 0.88 ± 0.16, specificity of 0.70 ± 0.20 and AUC of 0.90 ± 0.03. No correlation was found between tumor grading and tumor volume, morphology, necrosis, extracellular matrix, immune cell infiltration or underlying liver fibrosis. CONCLUSION: A combination of DCE-MRI- and 18F-FDG-PET-derived parameters provides high accuracy for histopathological grading of hepatocellular carcinoma in a relevant translational model system.

MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.

Implementing cell-free DNA of pancreatic cancer patient-derived organoids for personalized oncology.

One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.

Oncogenic KRas-induced Increase in Fluid-phase Endocytosis is Dependent on N-WASP and is Required for the Formation of Pancreatic Preneoplastic Lesions.

Fluid-phase endocytosis is a homeostatic process with an unknown role in tumor initiation. The driver mutation in pancreatic ductal adenocarcinoma (PDAC) is constitutively active KRasG12D, which induces neoplastic transformation of acinar cells through acinar-to-ductal metaplasia (ADM). We have previously shown that KRasG12D-induced ADM is dependent on RAC1 and EGF receptor (EGFR) by a not fully clarified mechanism. Using three-dimensional mouse and human acinar tissue cultures and genetically engineered mouse models, we provide evidence that (i) KRasG12D leads to EGFR-dependent sustained fluid-phase endocytosis (FPE) during acinar metaplasia; (ii) variations in plasma membrane tension increase FPE and lead to ADM in vitro independently of EGFR; and (iii) that RAC1 regulates ADM formation partially through actin-dependent regulation of FPE. In addition, mice with a pancreas-specific deletion of the Neural-Wiskott-Aldrich syndrome protein (N-WASP), a regulator of F-actin, have reduced FPE and impaired ADM emphasizing the in vivo relevance of our findings. This work defines a new role of FPE as a tumor initiating mechanism.

Co-clinical Assessment of Tumor Cellularity in Pancreatic Cancer.

Purpose: Tumor heterogeneity is a hallmark of pancreatic ductal adenocarcinoma (PDAC). It determines tumor biology including tumor cellularity (i.e., amount of neoplastic cells and arrangement into clusters), which is related to the proliferative capacity and differentiation and the degree of desmoplasia among others. Given the close relation of tumor differentiation with differences in progression and therapy response or, e.g., the recently reported protective role of tumor stroma, we aimed at the noninvasive detection of PDAC groups, relevant for future personalized approaches. We hypothesized that histologic differences in PDAC tissue composition are detectable by the noninvasive diffusion weighted- (DW-) MRI-derived apparent diffusion coefficient (ADC) parameter.Experimental design: PDAC cellularity was quantified histologically and correlated with the ADC parameter and survival in genetically engineered mouse models and human patients.Results: Histologic analysis showed an inverse relationship of tumor cellularity and stroma content. Low tumor cellularity correlated with a significantly prolonged mean survival time (PDAClow = 21.93 months vs. PDACmed = 12.7 months; log-rank P < 0.001; HR = 2.23; CI, 1.41-3.53). Multivariate analysis using the Cox regression method confirmed tumor cellularity as an independent prognostic marker (P = 0.034; HR = 1.73; CI, 1.04-2.89). Tumor cellularity showed a strong negative correlation with the ADC parameter in murine (r = -0.84; CI, -0.90- -0.75) and human (r = -0.79; CI, -0.90 to -0.56) PDAC and high preoperative ADC values correlated with prolonged survival (ADChigh = 41.7 months; ADClow = 14.77 months; log rank, P = 0.040) in PDAC patients.Conclusions: This study identifies high tumor cellularity as a negative prognostic factor in PDAC and supports the ADC parameter for the noninvasive identification of PDAC groups. Clin Cancer Res; 23(6); 1461-70. ©2016 AACR.

Tumor Uptake of Anti-CD20 Fabs Depends on Tumor Perfusion.

Antibodies have become an established treatment modality in cancer therapy during the last decade. However, these treatments often suffer from an insufficient and heterogeneous response despite validated antigen or target receptor expression in the tumor. In fact, therapeutic success depends on both the presence of the tumor antigen and its accessibility by the antibody. In search of a suitable preclinical animal model to evaluate the mechanisms of tumor heterogeneity and hemodynamics, we characterized two exemplary non-Hodgkin lymphoma subtypes with comparable CD20 expression and metabolism, SUDHL-4 and Granta-519, using multimodal imaging techniques. METHODS: To investigate in vivo biodistribution, two differently modified αCD20 antigen-binding fragments (Fab), prepared by PASylation with a 200-residue polypeptide tag comprising Pro, Ala, and Ser (PAS200) and by fusion with an albumin-binding domain (ABD), were radiolabeled with 125I and intravenously injected into immunocompromised mice bearing corresponding xenografts. RESULTS: Validation with 18F-FDG revealed a similar distribution in vital tumor tissue 1 h after injection. However, large differences in tumor uptake were observed when the CD20-specific radiotracers 125I-Fab-ABD and 125I-Fab-PAS200 were applied (respective percentages injected dose per gram at 24 h after injection: 12.3 and 2.4 for Granta-519 vs. 5.8 and 1.2 for SUDHL-4). Three-dimensional light-sheet fluorescence microscopy with Cy5-Fab-PAS200 confirmed better tracer extravasation in the Granta-519 tumors. Moreover, dynamic contrast-enhanced (DCE) MRI revealed significantly reduced perfusion in the SUDHL-4 tumors. CONCLUSION: Tracer uptake was highly dependent on local tumor perfusion and Fab permeation in the SUDHL-4 and Granta-519 tumors. Thus, the SUDHL-4 xenograft offers an excellent model for investigating the influence of therapies affecting tumor angiogenesis.

Membranous CD24 drives the epithelial phenotype of pancreatic cancer.

Surface CD24 has previously been described, together with CD44 and ESA, for the characterization of putative cancer stem cells in pancreatic ductal adenocarcinoma (PDAC), the most fatal of all solid tumors. CD24 has a variety of biological functions including the regulation of invasiveness and cell proliferation, depending on the tumor entity and subcellular localization. Genetically engineered mouse models (GEMM) expressing oncogenic KrasG12D recapitulate the human disease and develop PDAC. In this study we investigate the function of CD24 using GEMM of endogenous PDAC and a model of cerulein-induced acute pancreatitis. We found that (i) CD24 expression was upregulated in murine and human PDAC and during acute pancreatitis (ii) CD24 was expressed exclusively in differentiated PDAC, whereas CD24 absence was associated with undifferentiated tumors and (iii) membranous CD24 expression determines tumor subpopulations with an epithelial phenotype in grafted models. In addition, we show that CD24 protein is stabilized in response to WNT activation and that overexpression of CD24 in pancreatic cancer cells upregulated ß-catenin expression augmenting an epithelial, non-metastatic signature. Our results support a positive feedback model according to which (i) WNT activation and subsequent ß-catenin dephosphorylation stabilize CD24 protein expression, and (ii) sustained CD24 expression upregulates ß-catenin expression. Eventually, membranous CD24 augments the epithelial phenotype of pancreatic tumors. Thus we link the WNT/ß-catenin pathway with the regulation of CD24 in the context of PDAC differentiation.