X-gal Staining Research Articles

Osteoclast stimulation factor 1 (Ostf1) KNOCKOUT increases trabecular bone mass in mice

Osteoclast stimulation factor 1 (OSTF1) is an SH3-domain containing protein that was initially identified as a factor involved in the indirect activation of osteoclasts. It has been linked to spinal muscular atrophy in humans through its interaction with SMN1, and is one of six genes deleted in a human developmental microdeletion syndrome. To investigate the function of OSTF1, we generated an Ostf1 knockout mouse model, with exons 3 and 4 of Ostf1 replaced by a LacZ orf. Extensive X-Gal staining was performed to examine the developmental and adult expression pattern, followed by phenotyping. We show widespread expression of the gene in the vasculature of most organs and in a number of cell types in adult and embryonic mouse tissues. Furthermore, whilst SHIRPA testing revealed no behavioural defects, we demonstrate increased trabecular mass in the long bones, confirming a role for OSTF1 in bone development.

Expression of Phosphate Transporters during Dental Mineralization

The importance of phosphate (Pi) as an essential component of hydroxyapatite crystals suggests a key role for membrane proteins controlling Pi uptake during mineralization in the tooth. To clarify the involvement of the currently known Pi transporters (Slc17a1, Slc34a1, Slc34a2, Slc34a3, Slc20a1, Slc20a2, and Xpr1) during tooth development and mineralization, we determined their spatiotemporal expression in murine tooth germs from embryonic day 14.5 to postnatal day 15 and in human dental samples from Nolla stages 6 to 9. Using real-time polymerase chain reaction, in situ hybridization, immunohistochemistry, and X-gal staining, we showed that the expression of Slc17a1, Slc34a1, and Slc34a3 in tooth germs from C57BL/6 mice were very low. In contrast, Slc34a2, Slc20a1, Slc20a2, and Xpr1 were highly expressed, mostly during the postnatal stages. The expression of Slc20a2 was 2- to 10-fold higher than the other transporters. Comparable results were obtained in human tooth germs. In mice, Slc34a2 and Slc20a1 were predominantly expressed in ameloblasts but not odontoblasts, while Slc20a2 was detected neither in ameloblasts nor in odontoblasts. Rather, Slc20a2 was highly expressed in the stratum intermedium and the subodontoblastic cell layer. Although Slc20a2 knockout mice did not show enamel defects, mutant mice showed a disrupted dentin mineralization, displaying unmerged calcospherites at the mineralization front. This latter phenotypical finding raises the possibility that Slc20a2 may play an indirect role in regulating the extracellular Pi availability for mineralizing cells rather than a direct role in mediating Pi transport through mineralizing plasma cell membranes. By documenting the spatiotemporal expression of Pi transporters in the tooth, our data support the possibility that the currently known Pi transporters may be dispensable for the initiation of dental mineralization and may rather be involved later during the tooth mineralization scheme.

CXCL12-CXCR4 signalling plays an essential role in proper patterning of aortic arch and pulmonary arteries.

Chemokine CXCL12 (stromal derived factor 1: SDF1) has been shown to play important roles in various processes of cardiovascular development. In recent avian studies, CXCL12 signalling has been implicated in guidance of cardiac neural crest cells for their participation in the development of outflow tract and cardiac septum. The goal of this study is to investigate the extent to which CXCL12 signalling contribute to the development of aortic arch and pulmonary arteries in mammals. Novel Cxcl12-LacZ reporter and conditional alleles were generated. Using whole mount X-gal staining with the reporter allele and vascular casting techniques, we show that the domain branching pattern of pulmonary arteries in Cxcl12-null mice is completely disrupted and discordant with that of pulmonary veins and airways. Cxcl12-null mice also displayed abnormal and superfluous arterial branches from the aortic arch. The early steps of pharyngeal arch remodelling in Cxcl12-null mice appeared to be unaffected, but vertebral arteries were often missing and prominent aberrant arteries were present parallel to carotid arteries or trachea, similar to aberrant vertebral artery or thyroid ima artery, respectively. Analysis with computed tomography not only confirmed the results from vascular casting studies but also identified abnormal systemic arterial supply to lungs in the Cxcl12-null mice. Tie2-Cre mediated Cxcr4 deletion phenocopied the Cxcl12-null phenotypes, indicating that CXCR4 is the primary receptor for arterial patterning, whereas Cxcl12 or Cxcr4 deletion by Wnt1-Cre did not affect aortic arch patterning. CXCL12-CXCR4 signalling is essential for the correct patterning of aortic arches and pulmonary arteries during development. Superfluous arteries in Cxcl12-null lungs and the aortic arch infer a role of CXCL12 in protecting arteries from uncontrolled sprouting during development of the arterial system.

The osteogenic cell surface marker BRIL/IFITM5 is dispensable for bone development and homeostasis in mice.

BRIL (bone-restricted IFITM-like), is a short transmembrane protein expressed almost exclusively in osteoblasts. Although much is known about its bone-restricted gene expression pattern and protein biochemical and topological features, little information is available for BRIL physiological function. Two autosomal dominant forms of osteogenesis imperfecta (OI) are caused by distinct, but recurrent mutations in the BRIL gene. Yet, the underlying mechanisms by which those mutations lead to OI are still poorly understood. A previous report indicated that BRIL knockout (KO) mice had bone deformities, shortened long bones, and reproductive problems. Here we generated and systematically analyzed the skeletal phenotype of a new global Bril KO/LacZ knockin mouse model. KO mice reproduced and thrived normally up to 12 month of age. The skeletal phenotype of KO and WT littermates was assessed at embryonic (E13.5 to E18.5) and postnatal (2 days, 3 weeks, 3 months and 8 months) time-points. Embryos from E13.5 through to E18.5 showed significant X-Gal staining in all skeletal elements without any apparent patterning anomalies. Although bone deformities were never observed at any postnatal ages, minor and transient differences were noted in terms of bone length and static uCT parameters, but not systematically across all ages and genders. These changes, however, were not accompanied by significant alteration in bone material properties as assessed by a 3-point bending test. In addition, no changes were detected in circulating serum markers of bone turnover (P1NP, CTX-I, and osteocalcin). Gene expression monitoring also revealed no major impact of the loss of BRIL. Further, when mice were challenged with a surgically-induced fracture in tibia, bones repaired equally well in the KO mice as compared to WT. Finally, we showed that BRIL C-terminus is not a bona fide binding site for calcium. In conclusion, our in depth analysis suggest that skeletal patterning, bone mass accrual and remodeling in mice proceeded independent of BRIL.

Enzyme delivery using protein-stabilizing and cell-penetrating 30Kc19α protein nanoparticles

Abstract Nanoparticles (NPs) are an emerging strategy for drug delivery and have been studied for the delivery of various biomolecules, such as chemically synthesized drugs and therapeutic proteins. In particular, protein NPs are non-cytotoxic and biodegradable. Application of a full length recombinant 30Kc19 protein to human serum albumin (HSA) NPs has been shown to improve the cellular uptake and stability of the cargo enzyme. In this study, we demonstrate that drug delivery can be achieved with only the α-helix domain of the 30Kc19 protein (30Kc19α), and without the addition of HSA. Protein concentration and pH were crucial for NP generation. NPs had a uniformly spherical shape with an optimal diameter of 180–230 nm, and released β-galactosidase in a sustained manner. The 30Kc19α protein provided stability to the cargo enzyme, and helped maintain the specific activity of the enzyme. X-gal staining showed effective delivery of β-galactosidase into human dermal fibroblasts. Non-cytotoxic property of the 30Kc19α protein demonstrates that such NPs could be a resourceful tool for delivering drugs to cells.

A glycine transporter 2-Cre knock-in mouse line for glycinergic neuron-specific gene manipulation

Glycine is an inhibitory neurotransmitter in the brainstem and spinal cord. Glycine transporter 2 (GLYT2) is responsible for the uptake of extracellular glycine. GLYT2 is specifically expressed in glycinergic neurons and thus has been used as a marker of glycinergic neurons. Here, we generated GLYT2 promotor-driven Cre recombinase (Cre)-expressing mice (GLYT2-Cre knock-in mice) to develop a tool for manipulating gene expression in glycinergic neurons. Cre activity was examined by crossing the GLYT2-Cre knock-in mice with a Cre reporter mouse line, R26R, which express β-galactosidase (β-gal) in a Cre-dependent manner. X-gal staining of GLYT2-Cre/R26R double transgenic mouse brains and spinal cords revealed that the Cre activity was primarily distributed in the brainstem, cerebellum, and spinal cord. These areas are rich in glycinergic neurons. Furthermore, we performed immunohistochemistry for β-gal combined with in situ hybridization for GLYT2 in the GLYT2-Cre/R26R double transgenic mouse brains to determine whether Cre activity is specifically localized to glycinergic neurons. The β-gal protein and GLYT2 mRNAs were colocalized in the cerebellar Golgi cells, dorsal cochlear nucleus, gigantocellular reticular nucleus, spinal trigeminal nucleus, nucleus of the trapezoid body, and lateral lemniscus. More than 98% of the GLYT2 mRNA-expressing cells in these brain regions also expressed β-gal, whereas 90–98% of the β-gal-positive cells expressed the GLYT2 mRNAs. Thus, Cre activity is specifically localized to glycinergic neurons with high fidelity in the GLYT2-Cre knock-in mice. The GLYT2-Cre knock-in mouse line will be a useful tool for studying glycinergic neurons and neurotransmission.

PLAG1 deficiency impairs spermatogenesis and sperm motility in mice

Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.

A novel auditory ossicles membrane and the development of conductive hearing loss in Dmp1-null mice

Genetic mouse models are widely used for understanding human diseases but we know much less about the anatomical structure of the auditory ossicles in the mouse than we do about human ossicles. Furthermore, current studies have mainly focused on disease conditions such as osteomalacia and rickets in patients with hypophosphatemia rickets, although the reason that these patients develop late-onset hearing loss is unknown. In this study, we first analyzed Dmp1 lac Z knock-in auditory ossicles (in which the blue reporter is used to trace DMP1 expression in osteocytes) using X-gal staining and discovered a novel bony membrane surrounding the mouse malleus. This finding was further confirmed by 3-D micro-CT, X-ray, and alizarin red stained images. We speculate that this unique structure amplifies and facilitates sound wave transmissions in two ways: increasing the contact surface between the eardrum and malleus and accelerating the sound transmission due to its mineral content. Next, we documented a progressive deterioration in the Dmp1-null auditory ossicle structures using multiple imaging techniques. The auditory brainstem response test demonstrated a conductive hearing loss in the adult Dmp1-null mice. This finding may help to explain in part why patients with DMP1 mutations develop late-onset hearing loss, and supports the critical role of DMP1 in maintaining the integrity of the auditory ossicles and its bony membrane.

Liver metastasis is established by metastasis of micro cell aggregates but not single cells.

Cancer cell engraftment in the target organ is necessary to establish metastasis. Clinically, lymph node metastasis of single cells has been confirmed using cytokeratin staining. In the current study, a LacZ-labeled cancer cell line was used to visualize intrahepatic metastasis of single cells or liver micrometastasis. KM12SM-lacZ stably expressing LacZ was prepared with a highly metastatic colon cancer cell line, KM12SM. KM12SM-lacZ was injected into the spleen of nude mice and following 1 week the spleen was excised. The liver was then examined for metastasis following 1, 2 or 3 weeks. Confirmation of liver metastasis was completed by observing the grade of metastasis. Grade-1 metastasis (DNA level), human DNA in liver tissue was detected; Grade-2 metastasis (metastasis of single cells), confirmed by X-gal staining; Grade-3 metastasis (histopathological micrometastasis), diagnosed by light microscopy and Grade-4 metastasis (typical metastasis), easily detected macroscopically or by hematoxylin and eosin staining. The Grade-1 metastasis detection rates 1, 2 and 3 weeks following splenectomy were 50, 100 and 100%, respectively. Grade-2 metastasis was not detected by microscopy. The Grade-3 metastasis detection rates for 1, 2 and 3 weeks were 75, 100 and 100%, respectively. Micrometastasis was observed in the portal vein lumen and wall. The Grade-4 metastasis detection rates were 50, 100 and 100% for 1, 2 and 3 weeks respectively. Cancer cells were present in vessels surrounding the main tumor. In conclusion, a specific number of cancer cell aggregates may be necessary to establish hematogenous metastasis.

Distribution of ASIC4 transcripts in the adult wild-type mouse brain

Acid-sensing ion channel 4 (ASIC4) belongs to the ASIC gene family of neuronal proton-gated cation channels, and is the least understood subtype among the members. Previous studies of ASIC4 expression in the mammalian central nervous system have shown that ASIC4 is abundantly expressed in the spinal cord and in various brain regions, such as the cerebral cortex, the hippocampus, and the cerebellum. However, the detailed distribution of ASIC4 transcripts in mammalian brains still remains to be elucidated. In the present study, radioactive in situ hybridization histochemistry with an ASIC4-specific cRNA probe was performed on wild-type mouse brains, followed by X-gal staining experiments with Asic4-lacZ reporter mice Asic4tm1a(KOMP)Mbp. It was found that ASIC4 mRNAs were widely expressed throughout the wild-type brain, but preferentially concentrated in the olfactory bulb, the piriform cortex, the caudate putamen, the preoptic area, the paraventricular nucleus, the medial habenular nucleus, the pretectal area, the lateral geniculate nucleus, the amygdaloid complex, the superior colliculus, the interpeduncular nucleus, and the granule cell layer of the ventral hippocampus, and these results were in agreement with the X-gal-positive reactions observed in the mutant brain. In addition, X-gal staining combined with immunohistochemistry identified intense signals for ASIC4 transcriptional activity in most of the choline acetyltransferase (ChAT)-positive principal neurons located in the basal forebrain cholinergic nuclei. Our data provide useful information to speculate possible roles of ASIC4 in diverse brain functions.

A murine model of micrometastatic pancreas cancer

Objective: Pancreas cancer (PC) maintains a high mortality rate due to early metastatic spread to the liver. We have developed a murine model of metastatic PC designed to monitor the progression of a single micrometastatic (mM) cell, and are able to characterize the changes in the liver microenvironment in the presence of single mM cells. Methods: The murine PC cell line KCKO was transfected to express both Green Fluorescent Protein (GFP) and B-Galactosidase (B-Gal). Mice underwent orthotopic pancreas tumor implantation with luciferase-labeled KCKO. Seven days later, the mice underwent a second stage surgery involving primary tumor resection, splenic injection of KCKO/GFP/B-Gal, followed by hemi-splenectomy. At varying time points thereafter (24, 48, & 72 hours) the mice were sacrificed and individual liver lobes were processed. Results: Β-Gal expressing mM single tumor cells can be seen in liver tissue sections by X-gal staining. Analysis of the liver tumor microenvironment (TME) at time points after splenic injection revealed an increase in myeloid cells by flow cytometry, immunofluorescence, and immunohistochemistry. There was an up regulation of tumor-promoting and immunosuppressive genes by RT-PCR. Using CCR2−/− mice, there was a noted decrease of the myeloid cell infiltrate and mM in the liver TME, reinforcing the importance of the mobilizing signal of the CCR2–CCL2 chemokine axis. Conclusion: We developed a mouse model to recapitulate early micrometastatic PC occurrence in the liver after primary tumor resection. We can use this model to assess the progression of single cell liver mM and associated changes in the liver TME.

Survivin a radiogenetic promoter for glioblastoma viral gene therapy independently from CArG motifs

BackgroundRadiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation.AimTo select a potent radiation inducible promoter in the context of brain tumors and to investigate if CArG radio responsive motifs or other elements in the promoter nucleotide sequences can correlate to its response to radiation.MethodsTo select initial candidates for promoter inducible elements, the levels of mRNA expression of six different promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by five different promoters; CMV, VEGF, FLT-1, DR5 and survivin were constructed. Glioma cell lines were infected with different multiplicity of infection of the (promoter) Ad or CMV Ad. Cells were then exposed to a range of radiation (0–12 Gy) at single fraction. Fluorescent microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. Different glioma cell lines and normal astrocytes were infected with Ad survivin and exposed to radiation. The promoters were analyzed for presence of CArG radio-responsive motifs and CCAAT box consensus using NCBI blast bioinformatics software.ResultsRadiotherapy increases the expression of gene expression by 1.25–2.5 fold in different promoters other than survivin after 2 h of radiation. RNA analysis was done and has shown an increase in copy number of tenfold for survivin. Most importantly cells treated with RT and Ad Luc driven by survivin promoter showed a fivefold increase in expression after 2 Gy of radiation in comparison to non-irradiated cells. Presence or absence of CArG motifs did not correlate with promoter response to radiation. Survivin with the best response to radiation had the lowest number of CCAAT box.ConclusionSurvivin is a selective potent radiation inducible promoter for glioblastoma viral gene therapy and this response to radiation could be independent of CArG motifs.

Development of an efficient screening system to identify novel bone metabolism-related genes using the exchangeable gene trap mutagenesis mouse models

Despite numerous genetic studies on bone metabolism, understanding of the specific mechanisms is lacking. We developed an efficient screening system to identify novel genes involved in bone metabolism using mutant mouse strains registered with the Exchangeable Gene Trap Clones (EGTC) database. From 1278 trap clones in the EGTC database, 52 candidate lines were selected in the first screening, determined based on “EST profile”, “X-gal”, “Related article”, and “Novel gene”. For the second screening, bone morphometric analysis, biomechanical strength analysis, bone X-gal staining, etc. were performed on candidate lines. Forty-two male trap lines (80.8%) showed abnormalities with either bone morphometric analysis or biomechanical strength analysis. In the screening process, X-gal staining was significantly efficient (P = 0.0057). As examples, Lbr and Nedd4 trap lines selected using the screening system showed significant bone decrease and fragility, suggesting a relationship with osteoblast differentiation. This screening system using EGTC mouse lines is extremely efficient for identifying novel genes involved in bone metabolism. The gene trap lines identified as abnormal using this screening approach are highly likely to trap important genes for bone metabolism. These selected trap mice will be valuable for use as novel bio-resources in bone research.

The Citrus Flavanone Naringenin Protects Myocardial Cells against Age-Associated Damage.

In recent years, the health-promoting effects of the citrus flavanone naringenin have been examined. The results have provided evidence for the modulation of some key mechanisms involved in cellular damage by this compound. In particular, naringenin has been revealed to have protective properties such as an antioxidant effect in cardiometabolic disorders. Very recently, beneficial effects of naringenin have been demonstrated in old rats. Because aging has been demonstrated to be directly related to the occurrence of cardiac disorders, in the present study, the ability of naringenin to prevent cardiac cell senescence was investigated. For this purpose, a cellular model of senescent myocardial cells was set up and evaluated using colorimetric, fluorimetric, and immunometric techniques. Relevant cellular senescence markers, such as X-gal staining, cell cycle regulator levels, and the percentage of cell cycle-arrested cells, were found to be reduced in the presence of naringenin. In addition, cardiac markers of aging-induced damage, including radical oxidative species levels, mitochondrial metabolic activity, mitochondrial calcium buffer capacity, and estrogenic signaling functions, were also modulated by the compound. These results suggested that naringenin has antiaging effects on myocardial cells.

Direct orthotopic implantation of hepatic organoids

BackgroundLiver organoids show potential for development as a tissue replacement therapy for patients with end-stage liver disease, but efficient methods for introducing organoids into host livers have not been established. In this study, we aimed to develop a surgical technique to implant hepatic organoids into the liver and assess their engraftment. MethodsDonor hepatocytes were isolated from ROSA26 C57BL/6 mice, so that engrafted cells, when implanted into wild-type mice, could be easily identified by X-gal staining. Hepatic organoids were generated by three-dimensional culture in rotating wall vessel bioreactors. We qualitatively and quantitatively compared organoid engraftment to that of single-cell hepatocyte transplants. In addition, we determined the effect of adding stellate cells to hepatocytes to form co-aggregated organoids and the effect of partial hepatectomy of the host liver on organoid engraftment. ResultsDirect orthotopic implantation of hepatic organoids within a hepatotomy site resulted in local engraftment of exogenous hepatocytes with limited durability. Hepatocyte-stellate cell organoids produced more extracellular matrix but did not significantly improve engraftment compared with hepatocyte-alone organoids. Partial hepatectomy of the host liver led to significantly decreased engraftment of organoids. Survival of organoids was limited by the presence of apoptotic hepatocytes within organoids as early as 1 h after implantation. Organoids eventually became necrotic and elicited a chronic inflammatory giant cell reaction similar to a foreign body response. ConclusionsWith additional organoid and host factor optimization, direct orthotopic implantation of hepatic organoids may be an approach to introduce large numbers of exogenous hepatocytes into recipient livers.

Inactivation of bone morphogenetic protein 1 (Bmp1) and tolloid-like 1 (Tll1) in cells expressing type I collagen leads to dental and periodontal defects in mice.

Bone morphogenetic protein 1 (BMP1) and tolloid-like 1 (TLL1) belong to the BMP1/tolloid-like proteinase family, which cleaves secretory proteins. The constitutive deletion of the Bmp1 or Tll1 genes causes perinatal or embryonic lethality in mice. In this study, we first studied the β-galactosidase activity in mice in which an IRES-lacZ-Neo cassette was inserted in the intron of either the Bmp1 or the Tll1 gene; the β-galactosidase activities were used to reflect the expression of endogenous Bmp1 and Tll1, respectively. Our X-gal staining results showed that the odontoblasts in the tooth and cells in the periodontal ligament express both Bmp1 and Tll1. We then created Bmp1 flox/flox and Tll1 flox/flox mice by removing the IRES-lacZ-Neo cassette. By breeding 2.3kb Col1a1-Cre mice with the Bmp1 flox/flox and Tll1 flox/flox mice, we further generated Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice in which both Bmp1 and Tll1 were inactivated in the Type I collagen-expressing cells. We employed X-ray radiography, histology and immunohistochemistry approaches to characterize the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice. Our results showed that the molars of the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice had wider predentin, thinner dentin and larger pulp chambers than those of the normal controls. The dentinal tubules of the molars in the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice appeared disorganized. The level of dentin sialophosphoprotein in the molars of the 6-week-old Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice was lower than in the normal controls. The periodontal ligaments of the Col1a1-Cre;Bmp1 flox/flox ;Tll1 flox/flox mice were disorganized and had less fibrillin-1. Our findings indicate that the proteinases encoded by Bmp1 and Tll1 genes play essential roles in the development and maintenance of mouse dentin and periodontal ligaments.

Regulation and function of bone morphogenetic protein signaling in colonic injury and inflammation.

In this study we report a series of novel observations that underscore the importance of bone morphogenetic protein (BMP) signaling in the regulation of colonic homeostasis during the development of injury and inflammation. In particular, we present evidence that BMP signaling mitigates the response of the colonic epithelium to injury and inflammation and that cytokines, such as TNF-α and IL-1β, inhibit the expression of BMP-4.

Abstract A133: Micrometastatic progression of pancreas cancer: Murine model

Abstract Introduction: Pancreas cancer remains one the most lethal malignancies in the world and the high rate of metastatic progression contributes to its lethality. Up to 80% of patients with no clinical evidence of distant metastasis upon initial diagnosis recur with distant metastatic lesions even after surgical resection of their pancreas tumor. The critical drivers of metastatic dissemination and immune escape are not yet well understood, and a model to recapitulate the progression of pancreas cancer to its metastatic spread to the liver is not available. Understanding the biology of this and being able to predict which patients are at risk for metastatic reoccurrence would be a great treatment advance. Here we present a murine model to reflect human pancreas cancer disease progression. Following resection of the primary pancreas tumor, we observe arrival of early micrometastatic tumor cell spread in liver and monitor its progression to full metastatic lesion development. During this tumor progression, we are able to monitor the tumor microenvironment. Methods: A murine model was developed to recapitulate early micrometastatic occurrence in the liver after primary tumor resection. The murine pancreatic adenocarcinoma cell line KCKO was retro-virally transfected with a LacZ gene, containing the genetic expression for Green Fluorescent Protein (GFP) and β-Galactosidase. Clonal selection for a stable GFP expression was done by flow cytometry. For experiments, eight to ten week-old C57BL/6 mice underwent orthotopic primary tumor implantation via injection into the tail of the pancreas with 1×105 luciferase-labeled KCKO cells suspended in a 1:1 PBS to Matrigel (Corning) mixture. Following tumor implantation, mice were monitored for primary tumor growth with bioluminescent imaging. After six days from the date of orthotopic implantation, the mice were anesthetized and underwent the second stage surgery involving primary tumor resection, injection of 1×105 LacZ-transfected KCKO cells into the lower pole of the spleen, followed by hemi-splenectomy. At varying time points thereafter (i.e. 24 hours, 48 hours, 72 hours, 96 hours), the mice were sacrificed following intracardiac perfusion with PBS, and individual liver lobes were taken for processing for flow cytometry, formalin fixation-paraffin embedment, and frozen section medium embedment. Results: Analysis of the liver tumor microenvironment (TME) was performed to elucidate the spatial-temporal relationship between the tumor cells and stromal cells, which we determined were predominantly immune cells. β-Galactosidase expressing micrometastatic tumor cells could be clearly delineated in the liver by X-gal staining of frozen sections. Immunohistochemistry staining with anti-GFP and anti β-Galactosidase further confirmed the presence of the micrometastatic tumor cells. Furthermore, IHC staining was used to localize immune (CD45, CD11b, GR1, Ly6C, Ly6G, FoxP3, CD4, CD8) and non-immune cells (CD31, Collagen, SMA). Flow cytometry was used to characterize tumor and non-tumor cell presence across the time of metastatic progression. Conclusion: We developed a clinically relevant murine model of micrometastatic pancreas cancer that we used to assess the formation of liver metastasis and associated changes in the liver TME, many of which promote tumor progression. Understanding the complex biology behind this process will lead to improved understanding of how tumor-associated changes in the subsequent site of metastasis promote metastasis formation. Citation Format: Ankit Patel, Brian Belt, Nathania Figueroa, Sapna Patel, Aditi Murthy, Kelli Connolly, Scott Gerber, David Linehan. Micrometastatic progression of pancreas cancer: Murine model [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A133.

Expression of the reporter LacZ driven by human dentin sialophosphoprotein promoter in human dental mesenchymal cells

The expression of dentin sialophosphoprotein (DSPP) is the marker for cells differentiated into odontoblasts. This study attempted to analyze the DSPP promoter and build the reporter LacZ expression system driven by this promoter, which allows convenient and quick detection of odontoblast cells. First, we separated the human dental mesenchymal cells in which the expression of DSPP can be effectively induced by dexamethasone. Second, four 5' flanking regions of human DSPP gene (- 4 000-+54, -2 500-+54, -1 447-+54 and -1 027-+54) were analyzed, the results showed that the highest promoter activity lied in the -2 500-+54 region. The promoter activity is reduced when the 5' flanking region was extended from -2 500 to -4 000 bp upstream from the transcription start site; The promoter activity are also decreased when the 5' flanking regions were shorted from -2 500 to -1 447 bp and from -1 447 to -1 027 bp, indicating that potential suppresser elements are lied in the region between -4 000 and -2 500 bp and potential activator elements are lied in the region between -2 500 and -1 027 bp. Then we constructed the lentiviral report vector phDSPP-LacZ containing the -2 500-+ 54 promoter region in front of the LacZ gene. The expression of LacZ was detected using X-Gal staining in both human dental mesenchymal cells and immortalized human dental mesenchymal cells infected with phDSPP-LacZ. The phDSPP-LacZ lentiviral vector may provide a more convenient method to detect the expression of DSPP in human odontogenic differentiation, tooth development and tooth regeneration studies.

Abstract 589: Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model

Abstract Osteosarcoma (OS) is a malignancy of bone and five-year survival rates are still at a low 20% if metastatic disease is present. Metastatic OSs most frequently metastasize to the lung. TNF-α is a powerful cytokine already shown to be active against many cancers. Nevertheless, TNF-α is highly toxic at clinically relevant concentrations. To overcome this limitation, targeted forms of TNF-α can be used. In this study, targeting is achieved through linking TNF-α to the antibody fragment F8, targeting the EDA-domain of fibronectin. To test the efficacy of F8-TNF-α against OS, we sought to investigate the effects of F8-TNF-α in an orthotopic syngeneic OS model. In addition to evaluating compound efficacy, we also tested whether the route of administration (systemic versus local application) can even further increase treatment efficacy. Ultimately, we aimed at measuring treatment success by assessing different stages of OS progression, especially metastatic disease. To show clinical relevance, we performed immunofluorescence (IF) of EDA on primary human OS samples. In a second step, we evaluated drug efficacy in a syngeneic OS mouse model. K7M2L2 OS cells, stably infected to express the marker proteins lacZ and mCherry, were orthotopically injected into the tibia of mice. Once primary tumors were detectable, the mice were treated with either PBS or F8-TNF-α. The compound was given either systemically (intravenously (i.v.), tail vein) or locally (intraarterial (i.a.), femoral artery). Four days after the last treatment, all mice were sacrificed, terminal blood sampling was performed and blood samples were analyzed for the presence of circulating tumor cells (CTCs) via FACS/mCherry fluorescence. Following X-gal staining, metastases on the surface of lungs were counted. EDA and infiltration of immune cells (CD4+, CD8+, natural killer (NK) cells, CD19+) were assessed histologically. Using IF, we detected strong expression of EDA in primary tumors as well as bone and lung metastases from human OS patients. Preliminary results from our treatment study showed a significant decrease in the number of lung metastases as well as the number of CTCs in mice treated with i.a. F8-TNF-α while the treatment was well tolerated by the mice. The reduction of lung metastases could, at least in part, be explained by a strong expression of EDA in the lung metastases. In addition, significant increases in lung-infiltrating immune cells were observed after administration of F8-TNF-α (i.v. F8- TNF-α: increased CD4+ T-cells; i.a. F8-TNF-α: increased NK cells). Although significant reductions in numbers of metastases and CTCs were detected, F8-TNF-α did not significantly impair the growth of the primary tumors. In conclusion, our study demonstrates a reduction of CTCs as well as lung metastases after administration of F8-TNF-α and thus, provides a rationale for further studying the anti-metastatic effects of this compound in a clinically relevant model of OS. Citation Format: Bernhard Robl, Sander Martijn Botter, Aleksandar Boro, Dario Neri, Bruno Fuchs. Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 589.