Targeting pancreatic cancer with combinatorial treatment of CPI-613 and inhibitors of lactate metabolism.

Pancreatic cancer is the fourth leading cause of cancer death, with a 5-year survival rate of 10%. A stagnant high mortality rate over the last decades highlights the need for innovative therapeutic approaches. Pancreatic tumors pursue an altered metabolism in order to maintain energy generation under low nutrient influx and hypoxic conditions. Targeting these metabolic strategies might therefore be a reasonable therapeutic approach for pancreatic cancer. One promising agent is CPI- 613, a potent inhibitor of two enzymes of the tricarboxylic acid cycle. The present study evaluated the anti-cancerous efficacy of CPI-613 in combination with galloflavin, a lactate dehydrogenase inhibitor or with alpha-cyano-4-hydroxycinnamic acid, an inhibitor of monocarboxylate transporters. The efficacy of both combination therapies was tested in vitro on one human and two murine pancreatic cancer cell lines and in vivo in an orthotopic pancreatic cancer model. Tumor progression was evaluated by MRI and 18F-FDG PET-CT. Both combinatorial treatments demonstrated in vitro a significant inhibition of pancreatic cancer cell proliferation and induction of cell death. In contrast to the in vitro results, both combination therapies did not significantly reduce tumor growth in vivo. The in vitro results suggest that a combined inhibition of different metabolic pathways might be a promising approach for cancer therapy. However, the in vivo experiments indicate that applying a higher dosage or using other drugs targeting these metabolic pathways might be more promising.

An optimized 3D-printed perfusion bioreactor for homogeneous cell seeding in bone substitute scaffolds for future chairside applications.

A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailor-made bioreactor design. A perfusion system for efficient, homogeneous, and rapid seeding with human adipogenic stem cells in bone substitute scaffolds was designed. Variants concerning medium inlet and outlet port geometry, i.e. cylindrical or conical diffuser, cell concentration, perfusion mode and perfusion rates were simulated in silico. Cell distribution during perfusion was monitored by dynamic [18F]FDG micro-PET/CT and validated by laser scanning microscopy with three-dimensional image reconstruction. By iterative feedback of the in silico and in vitro experiments, the homogeneity of cell distribution throughout the scaffold was optimized with adjustment of flow rates, cell density and perfusion properties. Finally, a bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode. During this short period of time, the cells initially adhere within the entire scaffold and stay viable. After two weeks, the formation of several cell layers was observed, which was associated with an osteogenic differentiation process. This newly designed bioreactor may be considered as a prototype for chairside application.

Multimodal Imaging Techniques to Evaluate the Anticancer Effect of Cold Atmospheric Pressure Plasma.

BACKGROUND: Skin cancer is the most frequent cancer worldwide and is divided into non-melanoma skin cancer, including basal cell carcinoma, as well as squamous cell carcinoma (SCC) and malignant melanoma (MM). METHODS: This study evaluates the effects of cold atmospheric pressure plasma (CAP) on SCC and MM in vivo, employing a comprehensive approach using multimodal imaging techniques. Longitudinal MR and PET/CT imaging were performed to determine the anatomic and metabolic tumour volume over three-weeks in vivo. Additionally, the formation of reactive species after CAP treatment was assessed by non-invasive chemiluminescence imaging of L-012. Histological analysis and immunohistochemical staining for Ki-67, ApopTag®, F4/80, CAE, and CD31, as well as protein expression of PCNA, caspase-3 and cleaved-caspase-3, were performed to study proliferation, apoptosis, inflammation, and angiogenesis in CAP-treated tumours. RESULTS: As the main result, multimodal in vivo imaging revealed a substantial reduction in tumour growth and an increase in reactive species after CAP treatment, in comparison to untreated tumours. In contrast, neither the markers for apoptosis, nor the metabolic activity of both tumour entities was affected by CAP. CONCLUSIONS: These findings propose CAP as a potential adjuvant therapy option to established standard therapies of skin cancer.

Anatomical MRI and [18F]FDG PET/CT imaging of Schistosoma mansoni in a NMRI mouse model.

Schistosomiasis represents one of the most devastating worm parasitosis in the world. Current diagnostic methods are insufficient to determine the infection grade and the disease related organ damage. We herein investigated whether discrimination of infection grade and its correlation to liver damage could be accurately performed by multimodal imaging in a mouse model of Schistosoma mansoni infection. Therefore, groups of uninfected and infected mice underwent MRI and [18F]FDG PET/CT imaging. Anatomical MRI images were used for liver volumetry and for quantification of hepatic granulomas. For PET/CT images a volume of interest based analyses were employed to calculate the [18F]FDG uptake in liver, portal vein, spleen and abdomen. Herein, we demonstrate that the combined use of [18F]FDG-PET/CT and MRI represents an appropriate diagnostic tool for Schistosoma mansoni infection, but fails to discriminate the infection grade and the linked organ damage. Only the splenic [18F]FDG uptake in the 25 cercariae group (5.68 ± 0.90%ID/cc) and 50 cercariae group (4.98 ± 1.43%ID/cc) was significantly higher compared to the control group (2.13 ± 0.69%ID/cc). Nevertheless, future multimodal imaging studies with new radiopharmaceuticals could build a highly sensitive and specific basis for the diagnosis and evaluation of organ damage of schistosomiasis.

[68Ga]-NODAGA-RGD Positron Emission Tomography (PET) for Assessment of Post Myocardial Infarction Angiogenesis as a Predictor for Left Ventricular Remodeling in Mice after Cardiac Stem Cell Therapy.

Angiogenesis plays a central role in the healing process following acute myocardial infarction. The PET tracer [68Ga]-NODAGA-RGD, which is a ligand for the αvß3 integrin, has been investigated for imaging angiogenesis in the process of healing myocardium in both animal and clinical studies. It´s value as a prognostic marker of functional outcome remains unclear. Therefore, the aim of this work was to establish [68Ga]-NODAGA-RGD for imaging angiogenesis in the murine infarct model and evaluate the tracer as a predictor for cardiac remodeling in the context of cardiac stem cell therapy. [68Ga]-NODAGA-RGD PET performed seven days after left anterior descending coronary artery (LAD) occlusion in 129S6 mice showed intense tracer accumulation within the infarct region. The specificity was shown in a sub-group of animals by application of the competitive inhibitor cilengitide prior to tracer injection in a subgroup of animals. Myocardial infarction (MI) significantly reduced cardiac function and resulted in pronounced left ventricular remodeling after three weeks, as measured by cardiac MRI in a separate group. Cardiac induced cells (CiC) that were derived from mESC injected intramyocardially in the therapy group significantly improved left ventricular ejection fraction (LVEF). Surprisingly, CiC transplantation resulted in significantly lower tracer accumulation seven days after MI induction. Accordingly, we successfully established the PET tracer [68Ga]-NODAGA-RGD for the assessment of αvß3 integrin expression in the healing process after MI in the mouse model. Yet, our results indicate that the mere extent of angiogenesis following MI does not serve as a sufficient prognostic marker for functional outcome.

18F-FDG PET-Based Imaging of Myocardial Inflammation Predicts a Functional Outcome Following Transplantation of mESC-Derived Cardiac Induced Cells in a Mouse Model of Myocardial Infarction.

Cellular inflammation following acute myocardial infarction has gained increasing importance as a target mechanism for therapeutic approaches. We sought to investigate the effect of syngeneic cardiac induced cells (CiC) on myocardial inflammation using 18F-FDG PET (Positron emission tomography)-based imaging and the resulting effect on cardiac pump function using cardiac magnetic resonance (CMR) imaging in a mouse model of myocardial infarction. Mice underwent permanent left anterior descending coronary artery (LAD) ligation inducing an acute inflammatory response. The therapy group received an intramyocardial injection of 106 CiC into the border zone of the infarction. Five days after myocardial infarction, 18F-FDG PET was performed under anaesthesia with ketamine and xylazine (KX) to image the inflammatory response in the heart. Flow cytometry of the mononuclear cells in the heart was performed to analyze the inflammatory response. The effect of CiC therapy on cardiac function was determined after three weeks by CMR. The 18F-FDG PET imaging of the heart five days after myocardial infarction (MI) revealed high focal tracer accumulation in the border zone of the infarcted myocardium, whereas no difference was observed in the tracer uptake between infarct and remote myocardium. The CiC transplantation induced a shift in 18F-FDG uptake pattern, leading to significantly higher 18F-FDG uptake in the whole heart, as well as the remote area of the heart. Correspondingly, high numbers of CD11+ cells could be measured by flow cytometry in this region. The CiC transplantation significantly improved the left ventricular ejection function (LVEF) three weeks after myocardial infarction. The CiC transplantation after myocardial infarction leads to an improvement in pump function through modulation of the cellular inflammatory response five days after myocardial infarction. By combining CiC transplantation and the cardiac glucose uptake suppression protocol with KX in a mouse model, we show for the first time, that imaging of cellular inflammation after myocardial infarction using 18F-FDG PET can be used as an early prognostic tool for assessing the efficacy of cardiac stem cell therapies.

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Early short-term PXT3003 combinational therapy delays disease onset in a transgenic rat model of Charcot-Marie-Tooth disease 1A (CMT1A).

The most common type of Charcot-Marie-Tooth disease is caused by a duplication of PMP22 leading to dysmyelination, axonal loss and progressive muscle weakness (CMT1A). Currently, no approved therapy is available for CMT1A patients. A novel polytherapeutic proof-of-principle approach using PXT3003, a low-dose combination of baclofen, naltrexone and sorbitol, slowed disease progression after long-term dosing in adult Pmp22 transgenic rats, a known animal model of CMT1A. Here, we report an early postnatal, short-term treatment with PXT3003 in CMT1A rats that delays disease onset into adulthood. CMT1A rats were treated from postnatal day 6 to 18 with PXT3003. Behavioural, electrophysiological, histological and molecular analyses were performed until 12 weeks of age. Daily oral treatment for approximately 2 weeks ameliorated motor deficits of CMT1A rats reaching wildtype levels. Histologically, PXT3003 corrected the disturbed axon calibre distribution with a shift towards large motor axons. Despite dramatic clinical amelioration, only distal motor latencies were improved and correlated with phenotype performance. On the molecular level, PXT3003 reduced Pmp22 mRNA overexpression and improved the misbalanced downstream PI3K-AKT / MEK-ERK signalling pathway. The improved differentiation status of Schwann cells may have enabled better long-term axonal support function. We conclude that short-term treatment with PXT3003 during early development may partially prevent the clinical and molecular manifestations of CMT1A. Since PXT3003 has a strong safety profile and is currently undergoing a phase III trial in CMT1A patients, our results suggest that PXT3003 therapy may be a bona fide translatable therapy option for children and young adolescent patients suffering from CMT1A.

Chemo-immunotherapy improves long-term survival in a preclinical model of MMR-D-related cancer.

BACKGROUND: Mismatch Repair Deficiency (MMR-D)-related tumors are highly immunogenic and constitute ideal vaccination targets. In a proof-of-concept study delayed tumorigenesis and prolonged survival has been shown in a clinically-relevant mouse model for MMR-D-related diseases (=MLH1 knock out mice). To refine this approach, vaccination was combined with immune modulatory low-dose chemotherapy to polarize immune regulatory subtypes. METHODS: Mice (prophylactic: 8-10 weeks; therapeutic: > 36 weeks) received a single injection of cyclophosphamide (CPX, 120 mg/kg bw, i.p.) or gemcitabine (GEM, 100 mg/kg bw, i.p.) prior to vaccination (lysate of a gastrointestinal tumor allograft, 10 mg/kg bw, n = 9 mice/group). The vaccine was given repetitively (10 mg/kg bw, s.c., 4 x / once a week, followed by monthly boosts) until tumor formation or progression. Tumor growth ([18F] FDG PET/CT imaging) and immune responses were monitored (flow cytometry, IFNγ ELISpot). The microenvironment was analyzed by immunofluorescence. RESULTS: Prophylactic application of GEM + lysate delayed tumorigenesis compared to lysate monotherapy and CPX-pre-treatment (median time of onset: 53 vs. 47 vs. 48 weeks). 33% of mice even remained tumor-free until the experimental endpoint (= 65 weeks). This was accompanied by long-term effect on cytokine plasma levels; splenic myeloid derived suppressor cells (MDSC) as well as regulatory T cell numbers. Assessment of tumor microenvironment from GEM + lysate treated mice revealed low numbers of MDSCs, but enhanced T cell infiltration, in some cases co-expressing PD-L1. Therapeutic chemo-immunotherapy (GEM + lysate) had minor impact on overall survival (median time: 12 (GEM + lysate) vs. 11.5 (lysate) vs. 3 weeks (control)), but induced complete remission in one case. Dendritic and T cell infiltrates increased in both treatment groups. Reactive T cells specifically recognized MLH1-/- tumor cells in IFNγ ELISpot, but lacked response towards NK cell targets YAC-1. CONCLUSIONS: Combined chemo-immunotherapy impairs tumor onset and growth likely attributable to modulation of immune responses. Depleting or 're-educating' immunosuppressive cell types, such as MDSC, may help moving a step closer to combat cancer.

Blocking Autophagy in Cancer-Associated Fibroblasts Supports Chemotherapy of Pancreatic Cancer Cells.

In this study we evaluated the interaction of pancreatic cancer cells, cancer-associated fibroblasts, and distinct drugs such as α-cyano-4-hydroxycinnamate, metformin, and gemcitabine. We observed that α-cyano-4-hydroxycinnamate as monotherapy or in combination with metformin could significantly induce collagen I deposition within the stromal reaction. Subsequently, we demonstrated that cancer-associated fibroblasts impaired the anti-proliferation efficacy of α-cyano-4-hydroxycinnamate, metformin and gemcitabine. Interestingly, inhibition of autophagy in these fibroblasts can augment the anti-proliferation effect of these chemotherapeutics in vitro and can reduce the tumor weight in a syngeneic pancreatic cancer model. These results suggest that inhibiting autophagy in cancer-associated fibroblasts may contribute to strategies targeting cancer.

Short-Term Effects of Microglia-Specific Mitochondrial Dysfunction on Amyloidosis in Transgenic Models of Alzheimer's Disease.

Reduction of mitochondrial activity is a subtle and early event in the pathogenesis of Alzheimer's disease. Mitochondrial damage and consequentially enhanced production of reactive oxygen species is particularly occurring in the vicinity of amyloid plaques. Since all cells are affected by mitochondrial damage, analyses of cell type-specific effects are challenging. To study the impact of mitochondrial alterations on microglial activity in a homogeneous genetic background, we generated bone marrow chimeras of irradiated 46-days-old APP-transgenic mice. For reconstitution, bone marrow from CX3CR1-eGFP mice with mitochondria of either non-obese diabetic or C57BL/6J animals was utilized. Successful reconstitution was evident in 100-day-old animals, by the presence of eGFP-positive cells in liver and spleen. In the brain, one-third of IBA1-positive microglia cells were newly recruited eGFP-expressing cells. Although donor-derived microglia were equally located in the proximity of amyloid plaques, no difference was observed in either the amyloid level, total number, or microglial coverage of plaques. These results indicate that during this brief and early phase of amyloid deposition, beneficial mitochondrial alterations in the newly recruited third of microglial cells were not sufficient to affect the amyloidosis in APP-transgenic mice.

Cellular vaccination of MLH1-/- mice - an immunotherapeutic proof of concept study.

Mismatch-repair deficiency (MMR-D) is closely linked to hypermutation and accordingly, high immunogenicity. MMR-D-related tumors thus constitute ideal vaccination targets for both therapeutic and prophylactic approaches. Herein, the prophylactic and therapeutic impact of a cellular vaccine on tumor growth and tumor-immune microenvironment was studied in a murine MLH1-/- knockout mouse model. Prophylactic application of the lysate (+/- CpG ODN 1826) delayed tumor development, accompanied by increased levels of circulating T cell numbers. Therapeutic application of the vaccine prolonged overall survival (median time: 11.5 (lysate) and 12 weeks (lysate + CpG ODN) vs. 3 weeks (control group), respectively) along with reduced tumor burden, as confirmed by PET/CT imaging and immune stimulation (increased CD3+CD8+ T - and NK cell numbers, reduced levels of TIM-3+ cells in both treatment groups). Coding microsatellite analysis of MMR-D-related target genes revealed increased mutational load upon vaccination (total mutation frequency within 28 genes: 28.6% vaccine groups vs. 14.9% control group, respectively). Reactive immune cells recognized autologous tumor cells, but also NK cells target YAC-1 in IFNγ ELISpot and, even more importantly, in functional kill assays. Assessment of tumor microenvironment revealed infiltration of CD8+ T-cells and granulocytes, but also upregulation of immune checkpoint molecules (LAG-3, PD-L1). The present study is the first reporting in vivo results on a therapeutic cellular MMR-D vaccine. Vaccination-induced prolonged survival was achieved in a clinically-relevant mouse model for MMR-D-related diseases by long-term impairment of tumor growth and this could be attributed to re-activated immune responses.

[68Ga]pentixafor for CXCR4 imaging in a PC-3 prostate cancer xenograft model - comparison with [18F]FDG PET/CT, MRI and ex vivo receptor expression.

PURPOSE: The aim was to characterize the properties of [68Ga]Pentixafor as tracer for prostate cancer imaging in a PC-3 prostate cancer xenograft mouse model and to investigate its correlation with [18F]FDG PET/CT, magnetic resonance imaging (MRI) and ex vivo analyses. METHODS: Static [68Ga]Pentixafor and [18F]FDG PET as well as morphological/ diffusion weighted MRI and 1H MR spectroscopy was performed. Imaging data were correlated with ex vivo biodistribution and CXCR4 expression in PC-3 tumors (immunohistochemistry (IHC), mRNA analysis). Flow cytometry was performed for evaluation of localization of CXCR4 receptors (in vitro PC-3 cell experiments). RESULTS: Tumor uptake of [68Ga]Pentixafor was significantly lower compared to [18F]FDG. Ex vivo CXCR4 mRNA expression of tumors was shown by PCR. Only faint tumor CXCR4 expression was shown by IHC (immuno reactive score of 3). Accordingly, flow cytometry of PC-3 cells revealed only a faint signal, cell membrane permeabilisation showed a slight signal increase. There was no significant correlation of [68Ga]Pentixafor tumor uptake and ex vivo receptor expression. Spectroscopy showed typical spectra of prostate cancer. CONCLUSION: PC-3 tumor uptake of [68Ga]Pentixafor was existent but lower compared to [18F]FDG. No significant correlation of ex vivo tumor CXCR4 receptor expression and [68Ga]Pentixafor tumor uptake was shown. CXCR4 receptor expression on the surface of PC-3 cells was existent but rather low possibly explaining the limited [68Ga]Pentixafor tumor uptake; receptor localization in the interior of PC-3 cells is presumable as shown by cell membrane permeabilisation. Further studies are necessary to define the role of [68Ga]Pentixafor in prostate cancer imaging.

Application of in vivo imaging techniques to monitor therapeutic efficiency of PLX4720 in an experimental model of microsatellite instable colorectal cancer.

OBJECTIVES: Patient-derived tumor cell lines are a powerful tool to analyze the sensitivity of individual tumors to specific therapies in mice. An essential prerequisite for such an approach are reliable quantitative techniques to monitor tumor progression in vivo. METHODS: We have employed HROC24 cells, grown heterotopically in NMRI Foxn1nu mice, as a model of microsatellite instable colorectal cancer to investigate the therapeutic efficiencies of 5'-fluorouracil (5'-FU) and the mutant BRAF inhibitor PLX4720, a vemurafenib analogue, by three independent methods: external measurement by caliper, magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) with 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG). RESULTS: Repeated measure ANOVA by a general linear model revealed that time-dependent changes of anatomic tumor volumes measured by MRI differed significantly from those of anatomic volumes assessed by caliper and metabolic volumes determined by PET/CT. Over the investigation period of three weeks, neither 5'-FU, PLX4720 nor a combination of both drugs affected the tumor volumes. Also, there was no drug effect on the apparent diffusion constant (ADC) value as detected by MRI. Interestingly, however, PET/CT imaging showed that PLX4720-containing therapies transiently reduced the standardized uptake value (SUV), indicating a temporary response to treatment. CONCLUSIONS: 5'-FU and PLX4720 were largely ineffective with respect to HROC24 tumor growth. Tumoral uptake of 18F-FDG, as expressed by the SUV, proved as a sensitive indicator of small therapeutic effects. Metabolic imaging by 18F-FDG PET/CT is a suitable approach to detect effects of tumor-directed therapies early and even in the absence of morphological changes.

One Year Follow-Up Risk Assessment in SKH-1 Mice and Wounds Treated with an Argon Plasma Jet.

Multiple evidence in animal models and in humans suggest a beneficial role of cold physical plasma in wound treatment. Yet, risk assessment studies are important to further foster therapeutic advancement and acceptance of cold plasma in clinics. Accordingly, we investigated the longterm side effects of repetitive plasma treatment over 14 consecutive days in a rodent full-thickness ear wound model. Subsequently, animals were housed for 350 days and sacrificed thereafter. In blood, systemic changes of the proinflammatory cytokines interleukin 1ß and tumor necrosis factor α were absent. Similarly, tumor marker levels of α-fetoprotein and calcitonin remained unchanged. Using quantitative PCR, the expression levels of several cytokines and tumor markers in liver, lung, and skin were found to be similar in the control and treatment group as well. Likewise, histological and immunohistochemical analysis failed to detect abnormal morphological changes and the presence of tumor markers such as carcinoembryonic antigen, α-fetoprotein, or the neighbor of Punc11. Absence of neoplastic lesions was confirmed by non-invasive imaging methods such as anatomical magnetic resonance imaging and positron emission tomography-computed tomography. Our results suggest that the beneficial effects of cold plasma in wound healing come without apparent side effects including tumor formation or chronic inflammation.

Genomic background-related activation of microglia and reduced ß-amyloidosis in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is by far the most common neurodegenerative disease. AD is histologically characterized not only by extracellular senile plaques and vascular deposits consisting of ß-amyloid (Aß) but also by accompanying neuroinflammatory processes involving the brain's microglia. The importance of the microglia is still in controversial discussion, which currently favors a protective function in disease progression. Recent findings by different research groups highlighted the importance of strain-specific and mitochondria-specific genomic variations in mouse models of cerebral ß-amyloidosis. Here, we want to summarize our previously presented data and add new results that draw attention towards the consideration of strain-specific genomic alterations in the setting of APP transgenes. We present data from APP-transgenic mice in commonly used C57Bl/6J and FVB/N genomic backgrounds and show a direct influence on the kinetics of Aß deposition and the activity of resident microglia. Plaque size, plaque deposition rate and the total amount of Aß are highest in C57Bl/6J mice as compared to the FVB/N genomic background, which can be explained at least partially by a reduced microglia activity towards amyloid deposits in the C57BL/6J strain.