Photoacoustic imaging for the study of oxygen saturation and total hemoglobin in bone healing and non-union formation.

Non-union formation represents a major complication in trauma surgery. Adequate vascularization has been recognized as vital for bone healing. However, the role of vascularization in the pathophysiology of non-union formation remains elusive. This is due to difficulties in studying bone microcirculation in vivo. Therefore, we herein studied in a murine osteotomy model whether photoacoustic imaging may be used to analyze vascularization in bone healing and non-union formation. We found that oxygen saturation within the callus tissue is significantly lower in non-unions compared to unions and further declines over time. Moreover, the amount of total hemoglobin (HbT) within the callus tissue was markedly reduced in non-unions. Correlation analyses showed a strong positive correlation between microvessel density and HbT, indicating that photoacoustically determined HbT is a valid parameter to assess vascularization during bone healing. In summary, photoacoustic imaging is a promising approach to study vascular function and tissue oxygenation in bone regeneration.

Inhibition of TRIF-Dependent Inflammation Decelerates Afterload-Induced Myocardial Remodeling.

Pressure-overload-induced cardiac hypertrophy represents one cause of the development of heart failure. The aim of this study is to characterize the influence of the TIR-domain-containing adapter-inducing interferon-ß (TRIF) during afterload-induced myocardial remodeling. After trans-aortic constriction (TAC), cardiac pressure overload leads to an early increase in MyD88- (Myeloid differentiation primary response gene 88) and TRIF-dependent cytokines. The maximum cytokine expression appeared within the first week and decreased to its control level within five weeks. While cardiomyocyte hypertrophy was comparable, the myocardial accumulation of the inflammatory cells was lower in TRIF-/-mice. At d7, TRIF deficiency reduced transcription factors and TRIF-dependent cytokines. Through the modulation of the TGF-ß-signaling pathway and anti-fibrotic microRNAs, TRIF was involved in the development of interstitial fibrosis. The absence of TRIF was associated with a decreased expression of proapoptotic proteins. In echocardiography and working heart analyses, TRIF deficiency slowed left-ventricular wall thickening, myocardial hypertrophy, and reduces the ejection fraction. In summary, TRIF is an important adapter protein for the release of inflammatory cytokines and the accumulation of inflammatory cells in the early stage of maladaptive cardiac remodeling. TRIF is involved in the development of cardiac fibrosis by modulating inflammatory and fibrotic signal transduction pathways.

Antagonizing Sec62 function in intracellular Ca2+ homeostasis represents a novel therapeutic strategy for head and neck cancer.

Various cancer types including head and neck squamous cell carcinomas (HNSCC) show a frequent amplification of chromosomal region 3q26 that encodes, among others, for the SEC62 gene. Located in the ER membrane, this translocation protein is known to play a critical role as a potential driver oncogene in cancer development. High SEC62 expression levels were observed in various cancer entities and were associated with a poor outcome and increased metastatic burden. Because of its intracellular localization the SEC62 protein is poorly accessible for therapeutic antibodies, therefore a functional SEC62 knockdown represents the most promising mechanism of a potential antineoplastic targeted therapy. By stimulating the Ca2+ efflux from the ER lumen and thereby increasing cellular stress levels, a functional inhibition of SEC62 bears the potential to limit tumor growth and metastasis formation. In this study, two potential anti-metastatic and -proliferative agents that counteract SEC62 function were investigated in functional in vitro assays by utilizing an immortalized human hypopharyngeal cancer cell line as well as a newly established orthotopic murine in vivo model. Additionally, a CRISPR/Cas9 based SEC62 knockout HNSCC cell line was generated and functionally characterized for its relevance in HNSCC cell proliferation and migration as well as sensitivity to SEC62 targeted therapy in vitro.

Suppression of endothelial miR-22 mediates non-small cell lung cancer cell-induced angiogenesis.

MicroRNAs (miRNAs) expressed in endothelial cells (ECs) are powerful regulators of angiogenesis, which is essential for tumor growth and metastasis. Here, we demonstrated that miR-22 is preferentially and highly expressed in ECs, while its endothelial level is significantly downregulated in human non-small cell lung cancer (NSCLC) tissues when compared to matched nontumor lung tissues. This reduction of endothelial miR-22 is possibly induced by NSCLC cell-secreted interleukin-1ß and subsequently activated transcription factor nuclear factor-κB. Endothelial miR-22 functions as a potent angiogenesis inhibitor that inhibits all of the key angiogenic activities of ECs and consequently NSCLC growth through directly targeting sirtuin 1 and fibroblast growth factor receptor 1 in ECs, leading to inactivation of AKT/mammalian target of rapamycin signaling. These findings provide insight into the molecular mechanisms of NSCLC angiogenesis and indicate that endothelial miR-22 represents a potential target for the future antiangiogenic treatment of NSCLC.

Improvement of islet transplantation by the fusion of islet cells with functional blood vessels.

Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels. These insulin-secreting organoids exhibit a significantly higher angiogenic activity compared to freshly isolated islets, cultured islets, and non-prevascularized islet organoids. This is caused by paracrine signaling between the ß-cells and the microvessels, mediated by insulin binding to its corresponding receptor on endothelial cells. In vivo, the prevascularized islet organoids are rapidly blood-perfused after transplantation by the interconnection of their autochthonous microvasculature with surrounding blood vessels. As a consequence, a lower number of islet grafts are required to restore normoglycemia in diabetic mice. Thus, prevascularized islet organoids may be used to improve the success rates of clinical islet transplantation.

Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B-cell-type diffuse large B-cell lymphoma.

It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.

Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats.

Vascularized lymph node (VLN) transfer is an emerging strategy to re-establish lymphatic drainage in chronic lymphedema. However, the biological processes underlying lymph node integration remain elusive. This study introduces an experimental approach facilitating the analysis of short-term molecular and cellular effects of ischemia/reperfusion on VLN flaps. Lymph node flaps were dissected pedicled on the lateral thoracic vessels in 44 Lewis rats. VLN flaps were exposed to 45 or 120 minutes ischemia by in situ clamping of the vascular pedicle with subsequent reperfusion for 24 hours. Flaps not exposed to ischemia/reperfusion served as controls. Lymph nodes and the perinodal adipose tissue were separately analyzed by Western blot for the expression of lymphangiogenic and angiogenic growth factors. Moreover, morphology, microvessel density, proliferation, apoptosis and immune cell infiltration of VLN flaps were further assessed by histology and immunohistochemistry. Ischemia for 120 minutes was associated with a markedly reduced cellularity of lymph nodes but not of the perinodal adipose tissue. In line with this, ischemic lymph nodes exhibited a significantly lower microvessel density and an increased expression of VEGF-D and VEGF-A. However, VEGF-C expression was not upregulated. In contrast, analyses of the perinodal adipose tissue revealed a more subtle decrease of microvessel density, while only the expression of VEGF-D was increased. Moreover, after 120 minutes ischemia, lymph nodes but not the perinodal adipose tissue exhibited significantly higher numbers of proliferating and apoptotic cells as well as infiltrated macrophages and neutrophilic granulocytes compared with non-ischemic flaps. Taken together, lymph nodes of VLN flaps are highly susceptible to ischemia/reperfusion injury. In contrast, the perinodal adipose tissue is less prone to ischemia/reperfusion injury.

Experimental induction of blunt chest trauma in mice: A modified approach with evaluation in dummies and cadavers.

Experimental chest trauma or blunt thoracic trauma using a blast wave mechanism is well established in animal models. The aim of the present study was to establish a complementary, murine experimental chest trauma model precisely defined by physical data and calculations. For this purpose, a device was developed using a dropped weight and physical properties, including velocity, energy and impact, were calculated. The device allowed for the maximum depth of impression to be measured. The device was first tested using blocks of modelling clay and was then applied to mouse cadavers. X-ray and dissection were performed to check for bone fractures and organ injuries following blunt chest traumas of increasing impact. Lesions and hemorrhages were observed in mouse cadavers which sustained a force equivalent to the energy of ~1 J.

Is adipose tissue suitable for detection of (synthetic) cannabinoids? A comparative study analyzing antemortem and postmortem specimens following pulmonary administration of JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol to pigs.

Examining fatal poisonings, chronic exposure may be reflected by the concentration in tissues known for long-term storage of drugs. Δ9-tetrahydrocannabinol (THC) persists in adipose tissue (AT), but sparse data on synthetic cannabinoids (SC) are available. Thus, a controlled pig study evaluating antemortem (AM) disposition and postmortem (PM) concentration changes of the SC 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4) as well as THC in AT was performed. The drugs were administered pulmonarily (200 µg/kg body weight) to twelve pigs. Subcutaneous (s.c.) AT specimens were collected after 15 and 30 min and then hourly up to 8 h. At the end, pigs were sacrificed and s.c., perirenal, and dorsal AT specimens were collected. The carcasses were stored at room temperature (RT; n = 6) or 4 °C (n = 6) and specimens were collected after 24, 48, and 72 h. After homogenization in acetonitrile and standard addition, LC-MS/MS was performed. Maximum concentrations were reached 0.5-2 h after administration amounting to 21 ± 13 ng/g (JWH-210), 24 ± 13 ng/g (RCS-4), and 22 ± 20 ng/g (THC) and stayed at a plateau level. Regarding the metabolites, very low concentrations of N-hydroxypentyl-RCS-4 (HO-RCS-4) were detected from 0.5 to 8 h. PM concentrations of parent compounds did not change significantly (p > 0.05) over time under both storage conditions. Concentrations of HO-RCS-4 significantly (p < 0.05) increased in perirenal AT during storage at RT. These results suggest a rapid distribution and persistence in s.c. AT. Furthermore, AT might be resistant to PM redistribution of parent compounds. However, significant PM increases of metabolite concentrations might be considered in perirenal AT.

Cancer-associated fibroblasts stimulate primary tumor growth and metastatic spread in an orthotopic prostate cancer xenograft model.

The unique microenvironment of the prostate plays a crucial role in the development and progression of prostate cancer (PCa). We examined the effects of cancer-associated fibroblasts (CAFs) on PCa progression using patient-derived fibroblast primary cultures in a representative orthotopic xenograft model. Primary cultures of CAFs, non-cancer-associated fibroblasts (NCAFs) and benign prostate hyperplasia-associated fibroblasts (BPHFs) were generated from patient-derived tissue specimens. These fibroblasts were coinjected together with cancer cells (LuCaP136 spheroids or LNCaP cells) in orthotopic PCa xenografts to investigate their effects on local and systemic tumor progression. Primary tumor growth as well as metastatic spread to lymph nodes and lungs were significantly stimulated by CAF coinjection in LuCaP136 xenografts. When NCAFs or BPHFs were coinjected, tumor progression was similar to injection of tumor cells alone. In LNCaP xenografts, all three fibroblast types significantly stimulated primary tumor progression compared to injection of LNCaP cells alone. CAF coinjection further increased the frequency of lymph node and lung metastases. This is the first study using an orthotopic spheroid culture xenograft model to demonstrate a stimulatory effect of patient-derived CAFs on PCa progression. The established experimental setup will provide a valuable tool to further unravel the interacting mechanisms between PCa cells and their microenvironment.

Time- and temperature-dependent postmortem concentration changes of the (synthetic) cannabinoids JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol following pulmonary administration to pigs.

In forensic toxicology, interpretation of postmortem (PM) drug concentrations might be complicated due to the lack of data concerning drug stability or PM redistribution (PMR). Regarding synthetic cannabinoids (SC), only sparse data are available, which derived from single case reports without any knowledge of dose and time of consumption. Thus, a controlled pig toxicokinetic study allowing for examination of PMR of SC was performed. Twelve pigs received a pulmonary dose of 200 µg/kg BW each of 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210), 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4), and Δ9-tetrahydrocannabinol via an ultrasonic nebulizer. Eight hours after, the pigs were put to death with T61 and specimens of relevant tissues and body fluids were collected. Subsequently, the animals were stored at room temperature (n = 6) or 4 °C (n = 6) and further samples were collected after 24, 48, and 72 h each. Concentrations were determined following enzymatic cleavage and solid-phase extraction by liquid-chromatography tandem mass spectrometry applying the standard addition approach. High concentrations of the parent compounds were observed in lung, liver, kidney and bile fluid/duodenum content as well as brain. HO-RCS-4 was the most prevalent metabolite detected in PM specimens. In general, changes of PM concentrations were found in every tissue and body fluid depending on the PM interval as well as storage temperature.

Apolipoprotein C3 induces inflammation and organ damage by alternative inflammasome activation.

NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.

Erythropoietin accelerates the revascularization of transplanted pancreatic islets.

BACKGROUND AND PURPOSE: Pancreatic islet transplantation is a promising therapeutic approach for Type 1 diabetes. A major prerequisite for the survival of grafted islets is a rapid revascularization after transplantation. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to promote angiogenesis. Therefore, we investigated in this study whether EPO improves the revascularization of transplanted islets. EXPERIMENTAL APPROACH: Islets from FVB/N mice were transplanted into dorsal skinfold chambers of recipient animals, which were daily treated with an intraperitoneal injection of EPO (500 IU·kg-1 ) or vehicle (control) throughout an observation period of 14 days. In a second set of experiments, animals were only pretreated with EPO over a 6-day period prior to islet transplantation. The revascularization of the grafts was assessed by repetitive intravital fluorescence microscopy and immunohistochemistry. In addition, a streptozotocin-induced diabetic mouse model was used to study the effect of EPO-pretreatment on the endocrine function of the grafts. KEY RESULTS: EPO treatment slightly accelerated the revascularization of the islet grafts. This effect was markedly more pronounced in EPO-pretreated animals, resulting in significantly higher numbers of engrafted islets and an improved perfusion of endocrine tissue without affecting systemic haematocrit levels when compared with controls. Moreover, EPO-pretreatment significantly accelerated the recovery of normoglycaemia in diabetic mice after islet transplantation. CONCLUSION AND IMPLICATIONS: These findings demonstrate that, particularly, short-term EPO-pretreatment represents a promising therapeutic approach to improve the outcome of islet transplantation, without an increased risk of thromboembolic events.

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis.

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival.

Redox signals at the ER-mitochondria interface control melanoma progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.

Distribution of the (synthetic) cannabinoids JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol following pulmonary administration to pigs.

New psychoactive substances, especially synthetic cannabinoids (SC), are gaining increasing relevance in postmortem forensic toxicology. Particularly, the interpretation of analytical results is challenging, as usually, no toxicokinetic (TK) data concerning distribution in organs and tissues are available. Thus, a controlled pig TK study allowing for examination of organ and tissue distribution of SC was performed. For this purpose, 12 pigs received a single pulmonary dose of 200 µg/kg body weight each of 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210), 2-(4-methoxyphenyl)-1-(1-pentylindole-3-yl)methanone (RCS-4), and Δ9-tetrahydrocannabinol (THC) via an ultrasonic nebulizer. Eight hours after administration, the animals were put to death by the administration of T61. Thereupon, relevant organs, important body fluids such as bile and colon content, and tissues such as muscle tissue were collected. After enzymatic hydrolysis and solid-phase extraction, analysis was performed by liquid chromatography-tandem mass spectrometry. For quantification, a standard addition method was applied. The parent compounds could be detected in every analyzed specimen with the exception of colon content. Regarding JWH-210, the kidneys and lungs are viable matrices for postmortem analysis. In terms of RCS-4, the lungs were found to be an appropriate matrix. Concerning THC, the liver, bile fluid as well as duodenum content were suitable matrices for detection. Metabolites were only detected in tissues/body fluids involved in metabolism and/or elimination. Bile fluid and duodenum content were shown, as the most appropriate specimens for quantification of metabolites.

Prevascularization of dermal substitutes with adipose tissue-derived microvascular fragments enhances early skin grafting.

Split-thickness skin grafts (STSG) are still the gold standard for the treatment of most skin defects. Hence, there is an ongoing need to improve this procedure. For this purpose, we herein analyzed dermal matrices seeded with adipose tissue-derived microvascular fragments (ad-MVF) in a bradythrophic wound model. In additional experiments, the matrices were covered with autologous STSG 10 days after implantation. Green fluorescence protein (GFP)+ ad-MVF were isolated from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and seeded onto collagen-glycosaminoglycan matrices. Non-seeded and prevascularized matrices were implanted into full-thickness skin defects on the skull of CD1 nu/nu mice for 21 days. Vascularization, lymphangiogenesis and incorporation of the matrices were analyzed using photo-acoustic imaging, trans-illumination stereomicroscopy, histology, and immunohistochemistry. The survival rate of STSG was assessed by planimetry. After 21 days, the density of microvascular and lymphatic networks was significantly higher in prevascularized matrices when compared to controls. This was associated with an improved implant integration. Moreover, prevascularization with ad-MVF allowed successful autologous skin grafting already at day 10, while coverage of non-seeded controls at day 10 resulted in STSG necrosis. In conclusion, ad-MVF represent powerful vascularization units. Seeded on dermal substitutes, they accelerate and enhance the healing of full-thickness skin defects and allow early coverage with STSG.

Treatment of SEC62 over-expressing tumors by Thapsigargin and Trifluoperazine.

Treatment with analogues of the SERCA-inhibitor Thapsigargin is a promising new approach for a wide variety of cancer entities. However, our previous studies on various tumor cells suggested resistance of SEC62 over-expressing tumors to this treatment. Therefore, we proposed the novel concept that e.g. lung-, prostate-, and thyroid-cancer patients should be tested for SEC62 over-expression, and developed a novel therapeutic strategy for a combinatorial treatment of SEC62 over-expressing tumors. The latter was based on the observations that treatment of SEC62 over-expressing tumor cells with SEC62-targeting siRNAs showed less resistance to Thapsigargin as well as a reduction in migratory potential and that the siRNA effects can be mimicked by the Calmodulin antagonist Trifluoperazine. Therefore, the combinatorial treatment of SEC62 over-expressing tumors was proposed to involve Thapsigargin and Trifluoperazine. Here, we addressed the impact of Thapsigargin and Trifluoperazine in separate and combined treatments of heterotopic tumors, induced by inoculation of human hypopharyngeal squamous cell carcinoma (FaDu)-cells into the mouse flank. Seeding of the tumor cells and/or their growth rate were significantly reduced by all three treatments, suggesting Trifluoperazine is a small molecule to be considered for future therapeutic strategies for patients, suffering from Sec62-overproducing tumors.

A novel mouse model of human prostate cancer to study intraprostatic tumor growth and the development of lymph node metastases.

BACKGROUND: In this study, we aimed to establish a versatile in vivo model of prostate cancer, which adequately mimics intraprostatic tumor growth, and the natural routes of metastatic spread. In addition, we analyzed the capability of high-resolution ultrasonography (hrUS), in vivo micro-CT (µCT), and 9.4T MRI to monitor tumor growth and the development of lymph node metastases. METHODS: A total of 5 × 105 VCaP cells or 5 × 105 cells of LuCaP136- or LuCaP147 spheroids were injected into the prostate of male CB17-SCID mice (n = 8 for each cell type). During 12 weeks of follow-up, orthotopic tumor growth, and metastatic spread were monitored by repetitive serum-PSA measurements and imaging studies including hrUS, µCT, and 9.4T MRI. At autopsy, primary tumors and metastases were harvested and examined by histology and immunohistochemistry (CK5, CK8, AMACR, AR, Ki67, ERG, and PSA). From imaging results and PSA-measurements, tumor volume doubling time, tumor-specific growth rate, and PSA-density were calculated. RESULTS: All 24 mice developed orthotopic tumors. The tumor growth could be reliably monitored by a combination of hrUS, µCT, MRI, and serum-PSA measurements. In most animals, lymph node metastases could be detected after 12 weeks, which could also be well visualized by hrUS, and MRI. Immunohistochemistry showed positive signals for CK8, AMACR, and AR in all xenograft types. CK5 was negative in VCaP- and focally positive in LuCaP136- and LuCaP147-xenografts. ERG was positive in VCaP- and negative in LuCaP136- and LuCaP147-xenografts. Tumor volume doubling times and tumor-specific growth rates were 21.2 days and 3.9 %/day for VCaP-, 27.6 days and 3.1 %/day for LuCaP136- and 16.2 days and 4.5 %/day for LuCaP147-xenografts, respectively. PSA-densities were 433.9 ng/mL per milliliter tumor for VCaP-, 6.5 ng/mL per milliliter tumor for LuCaP136-, and 11.2 ng/mL per milliliter tumor for LuCaP147-xenografts. CONCLUSIONS: By using different monolayer and 3D spheroid cell cultures in an orthotopic xenograft model, we established an innovative, versatile in vivo model of prostate cancer, which enables the study of both intraprostatic tumor growth as well as metastatic spread to regional lymph nodes. HrUS and MRI are feasible tools to monitor tumor growth and the development of lymph node metastases while these cannot be visualized by µCT.