ELUCIDATING THE ROLE OF CD44 EXPRESSION ON MESENCHYMAL STEM CELLS WITHIN THE TUMOR MICROENVIRONMENT

The tumor microenvironment is comprised of a vast array of heterogeneous cells including both normal and neoplastic cells. The tumor stroma recruitment process has been exploited for an effective gene delivery technique using bone marrow derived MSC. Targeted migration of the MSC toward the tumor microenvironment, while successful, is not yet fully understood. This study was designed to assess the role of CD44 in the migration of MSC toward the tumor microenvironment and to determine the implications of CD44-deficient MSC within the tumor stroma. Inhibition of MSC migration was evaluated through a variety of methods in vitro and in vivo including CD44 receptor knockdown, CD44 antagonists, CD44 neutralizing antibodies and small molecule inhibitor of matrix metalloproteinases. Blocking CD44 signaling through MMP inhibition was characterized by lack of intracellular domain cleavage and lead to the decrease in Twist gene expression. A functional relationship between CD44 and Twist expression was confirmed by chromatin immunoprecipitation. Next, a series of murine tumor models were used to examine the role of CD44 deficient stroma within the tumor microenvironment. Labeled transgenic CD44 knockout (KO) MSC or wild type (WT) C57/B6 MSC were used to analyze the stromal incorporation within murine breast carcinomas (EO771 and 4T1). Subsequent tumors were analyzed for vessel formation (CD31), and the presence of tumor associated fibroblast (TAF) markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific protein (FSP). The tumors with CD44KO MSC cells had less vessel formation than the tumors with WT MSC. The lack of fibroblastic TAF population as defined by FAP/FSP expression by the CD44KO MSC admixed tumors suggest that the bone marrow derived population of MSC were unable to contribute to the fibroblastic stromal population. Subsequently, a bone marrow transplantation experiment confirmed the endogenous migratory deficiencies of the CD44KO bone marrow derived stromal cells toward the tumor microenvironment in vivo. WT mice with CD44KO bone marrow had less CD44KOderived tumor stroma compared to mice with WT bone marrow. These results indicate that CD44 is crucial to stromal cell migration and incorporation to the tumor microenvironment as TAF.

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

Antibodies targeting hepatoma-derived growth factor as a novel strategy in treating lung cancer.

Hepatoma-derived growth factor (HDGF) is overexpressed in lung cancer and the overexpression correlates with aggressive biological behaviors and poor clinical outcomes. We developed anti-HDGF monoclonal antibodies and tested their antitumor activity in lung cancer xenograft models. We also determined biological effects in tumors treated with the antibody alone or in combination with bevacizumab/avastin (an anti-vascular endothelial growth factor antibody) and/or gemcitabine (a chemotherapeutic agent). We found the anti-HDGF was effective to inhibit tumor growth in non-small cell lung cancer xenograft models. In the A549 model, compared with control IgG, tumor growth was substantially inhibited in animals treated with anti-HDGF antibodies, particularly HDGF-C1 (P = 0.002) and HDGF-H3 (P = 0.005). When HDGF-H3 was combined with either bevacizumab or gemcitabine, we observed enhanced tumor growth inhibition, particularly when the three agents were used together. HDGF-H3-treated tumors exhibited significant reduction of microvessel density with a pattern distinctive from the microvessel reduction pattern observed in bevacizumab-treated tumors. HDGF-H3-treated but not bevacizumab-treated tumors also showed a significant increase of apoptosis. Interestingly, many of the apoptotic cells in HDGF-H3-treated tumors are stroma cells, suggesting that the mechanism of the antitumor activity is, at least in part, through disrupting formation of tumor-stroma structures. Our results show that HDGF is a novel therapeutic target for lung cancer and can be effectively targeted by an antibody-based approach.

Incidental detection of an occult oral malignancy with autofluorescence imaging: a case report.

BACKGROUND: Autofluorescence imaging is used widely for diagnostic evaluation of various epithelial malignancies. Cancerous lesions display loss of autofluorescence due to malignant changes in epithelium and subepithelial stroma. Carcinoma of unknown primary site presents with lymph node or distant metastasis, for which the site of primary tumour is not detectable. We describe here the use of autofluorescence imaging for detecting a clinically innocuous appearing occult malignancy of the palate which upon pathological examination was consistent with a metastatic squamous cell carcinoma. CASE DESCRIPTION: A submucosal nodule was noted on the right posterior hard palate of a 59-year-old white female during clinical examination. Examination of this lesion using a multispectral oral cancer screening device revealed loss of autofluorescence at 405 nm illumination. An excisional biopsy of this nodule, confirmed the presence of a metastatic squamous cell carcinoma. Four years ago, this patient was diagnosed with metastatic squamous cell carcinoma of the right mid-jugular lymph node of unknown primary. She was treated with external beam irradiation and remained disease free until current presentation. CONCLUSION: This case illustrates the important role played by autofluorescence tissue imaging in diagnosing a metastatic palatal tumour that appeared clinically innocuous and otherwise would not have been biopsied.

The Role of Cancer-Associated Fibroblasts in Lung Tumorigenesis

The extracellular milieu is rich in growth factors that drive tumor progression,but the mechanisms that govern tumor cell sensitivity to those ligands have notbeen fully defined. In this study, we address this question in mice that developmetastatic lung adenocarcinomas through the suppression of the microRNA-200 (miR-200) family. Cancer-associated fibroblasts (CAF) enhance tumorgrowth and invasion by secreting VEGF-A that binds to VEGFR1, a processrequired for tumor growth and metastasis in mice and correlated with a poorprognosis in lung adenocarcinoma patients. In this study, we discovered thatmiR-200 blocked CAF-induced tumor cell invasion by directly targetingVEGFR1 in tumor cells. In the context of previous studies, our findings suggestthat the miR-200 family is a point of convergence for diverse biologic processesthat regulate tumor cell proliferation, invasion, and metastasis; its target genesixdrive epithelial-to-mesenchymal transition (ZEB1 and ZEB2) and promotesensitivity to a potent tumor growth factor emanating from the microenvironment(VEGFR1). Clinical trials should focus not only on the role of VEGFR1 inangiogenesis but also on the expression and activation of VEGFR1 in tumorcells by stromal sources of VEGF-A in the tumor microenvironment as a targetfor metastasis prevention.

ROLE OF PROSTAGLANDIN E2 IN THE REGULATION OF PANCREATIC STELLATE CELLS HYPER ACTIVITY ASSOCIATED WITH PANCREATIC CANCER

Pancreatic cancer is one of the most lethal type of cancer due to its high metastasis rate and resistance to chemotherapy. Pancreatic fibrosis is a constant pathological feature of chronic pancreatitis and the hyperactive stroma associated with pancreatic cancer. Strong evidence supports an important role of cyclooxygenase-2 (COX-2) and COX-2 generated prostaglandin E2 (PGE2) during pancreatic fibrosis. Pancreatic stellate cells (PSC) are the predominant source of extracellular matrix production (ECM), thus being the key players in both diseases. Given this background, the primary objective is to delineate the role of PGE2 on human pancreatic stellate cells (PSC) hyper activation associated with pancreatic cancer. This study showed that human PSC cells express COX-2 and synthesize high levels of PGE2. PGE2 stimulated PSC migration and invasion; expression of extra cellular matrix (ECM) genes and tissue degrading matrix metallo proteinases (MMP) genes. I further identified the PGE2 EP receptor responsible for mediating these effects on PSC. Using genetic and pharmacological approaches I identified the receptor required for PGE2 mediates PSC hyper activation. Treating PSC with Specific antagonists against EP1, EP2 and EP4, demonstrated that blocking EP4 receptor only, resulted in a complete reduction of PGE2 mediated PSC activation. Furthermore, siRNA mediated silencing of EP4, but not other EP receptors, blocked the effects of PGE2 on PSC fibrogenic activity. Further examination of the downstream pathway modulators revealed that PGE2 stimulation of PSC involved CREB and not AKT pathway. The regulation of PSC by PGE2 was further investigated at the molecular level, with a focus on COL1A1. Collagen I deposition by PSC is one of the most important events in pancreatic cancer. I found that PGE2 regulates PSC through activation of COL1A1 expression and transcriptional activity. Downstream of PGE2, silencing of EP4 receptor caused a complete reduction of COL1A1 expression and activity supporting the role of EP4 mediated stimulation of PSC. Taken together, this data indicate that PGE2 regulates PSC via EP4 and suggest that EP4 can be a better therapeutic target for pancreatic cancer to reduce the extensive stromal reaction, possibly in combination with chemotherapeutic drugs can further kill pancreatic cancer cells.

The development and characterization of two types of chronic responses in irradiated mouse colon

The hypothesis to be tested is that there are two distinct types of chronic responses in irradiated normal tissues, each resulting from damage to different cell populations in the tissue. The first is a sequala of chronic epithelial depletion in which the tissue's integrity cannot be maintained, i.e. a "consequential" chronic response. The other response is due to cell loss in the connective tissue and/or vascular stroma, i.e. a "primary" chronic response. The purpose of this study was to test the hypothesis in the murine colon by first, establishing a model of each chronic response and then, by determining whether the responses differed in timing of expression, histology, and expression of specific collagen types. The model of late damage used was colonic obstructions/strictures induced by a single dose of 27 Gy ("consequential" response) and two equal doses of 14.75 Gy (t = 10 days) ("primary" response). "Consequential" lesions appeared as early as 5 weeks after 27 Gy and were characterized by a deep mucosal ulceration and a thickened fibrotic serosa containing excessive accumulations of collagen types I and III. Both types were commingled in the scar at the base of the ulcer. Fibroblasts were synthesizing pro-collagen types I and III mRNA 10 weeks prior to measurable increases in collagen. A significant decrease in the ratio of collagen types I:III was associated with the "consequential" response at 4-5 months post-irradiation. The "primary" response, on the other hand, did not appear until 40 weeks after the split dose even though the total dose delivered was approximately the same as that for the "consequential" response. The "primary" response was characterized with an intact mucosa and a thickened fibrotic submucosa which contained excessive amounts of only collagen type I. An increased number of fibroblasts were synthesizing pro-collagen type I mRNA nearly 25 weeks before collagen type I levels were increased. The "primary" response lesion had a significantly elevated collagen type I:III ratio at 10-13 months post-irradiation. These data show a clear difference between the two chronic response and suggest that not all chronic responses share a common pathogenesis, but depend on the cell population in the tissue that is damaged. ^

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^

Development of prostate cancer gene therapy

Osseous metastases account for most of the morbidity and mortality associated with prostate cancer, for which there are currently no effective therapies. In the skeletal metastatic environment, neoplastic prostatic epithelial cells interact in a bidirectional stimulatory manner with osteoblastic stromal cells. Similarly, the presence of osteoblastic cells is essential for the survival and maintenance of intraosseous prostate cancer cells. In this thesis, I have developed novel gene therapy strategies for the treatment of androgen-independent human prostate cancers in experimental animal models. First, Ad-CMV-p53, a recombinant adenovirus (Ad) containing p53 tumor suppressor gene driven by the universal cytomegalovirus promoter, was effective in inhibiting prostate cancer cell growth, and direct intratumoral injections of Ad-CMV-p53 resulted in tumor regression. Second, because prostate cancer cells as well as osteoblastic cells produce osteocalcin (OC), OC promoter mediated tissue/tumor specific toxic gene therapy is developed to interrupt stromal-epithelial communications by targeting both cell types. Ad-OC-TK, a recombinant Ad containing the herpes simplex virus thymidine kinase (TK) gene driven by the OC promoter, was generated to inhibit the growth of osteoblastic osteosarcoma with prodrug acyclovir (ACV). Ad-OC-TK/ACV also inhibited the growth of prostate cancer cells and suppressed the growth of subcutaneous and intraosseous prostate tumor. In order to combine treatment modalities to maximize tumor cell-kill with minimized host toxicities, Ad-OC-TK/ACV was applied in combination with low dose methotrexate to eradicate osteoblastic osteosarcoma. In targeting of micrometastatic disease, intravenous Ad-OC-TK/ACV treatment resulted in significant tumor nodule reduction and prolonged the survival of animals harboring osteosarcoma lung metastases without significant host toxicity. Ad-OC-TK is a rational choice for the treatment of prostate cancer skeletal metastasis because OC is uniformly detected in both primary and metastatic human prostate cancer specimens by immunohistochemistry. Ad-OC-TK/ACV inhibits the growth not only of prostate cancer cells but also of their supporting bone stromal cells. Targeting both prostate cancer epithelium and its supporting stroma may be most efficacious for the treatment of prostate cancer osseous metastases. ^

THE EFFECT OF RELAXIN ON BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS OF RAT MYOMETRIAL CELLS IN CULTURE (CYCLIC-AMP, CELL SHAPE CHANGE)

Relaxin is able to inhibit spontaneous, oxytocin-and prostaglandin-driven uterine contractions. The intracellular mechanism of action of relaxin on uterine relaxation had previously been studied using isometrically suspended uterine strips. Since uterine strips contain stroma as well as myometrium, the changes in biochemical parameters induced by relaxin treatment may not occur in the same cell types responsible for the physical changes. In these studies, cultures of enriched populations of rat myometrial cells were used to investigate the effect of relaxin on biochemical and morphological parameters which are related to relaxation.^ Under optimal culture conditions (initial plating density 1 - 1.5 x 10('6)cells/ml, 3 ml/35 mm dish, 2 days culture), enzymatically isolated rat myometrial cells were able to respond to relaxin with cAMP elevation. Relaxin elevated cAMP levels in the presence but not the absence of 0.1 mM methylisobutylxanthine or 0.4 um forskolin in a time- and concentration-dependent manner. In contrast, isoproterenol was able to elevate cAMP levels in the presence and absence of 0.1 mM methylisobutylxanthine.^ Oxytocin treatment caused a decrease in mean cell length and area of myometrial cells in culture which could be considered analogous to contraction. Under optimal culture conditions, relaxin increased myometrial cell length and area (i.e. analogous to relaxation) of oxytocin-treated cells in a time- and concentration-dependent manner. Other relaxants such as isoproterenol and dibutyryl cAMP also increased cell length and area of oxytocin - treated myometrial cells in culture.^ Under optimal culture conditions, relaxin decreased myosin light chain kinase activity in a time-and concentration-dependent manner by increasing the K(,50) of the enzyme for calmodulin (CaM), i.e. decreasing the affinity of the enzyme for CaM. The decrease in the affinity of myosin light chain kinase for CaM may be due to the phosphorylation of the enzyme by cAMP-dependent protein kinase. Relaxin also decreased the Ca('2+)(.)CaM-independent myosin light chain kinase activity to a lesser extent than that of the Ca('2+)(.)CaM-dependent enzyme activity. This was not attributable to a decrease in the affinity of the enzyme for myosin in myometrial cells in culture, in contrast to the finding of such a change following relaxin treatment of uterine strips. Further studies are required to clarify this point.^ There was a temporal association between the effects of relaxin on elevation of cAMP levels in the presence of 0.4 uM forskolin, increase in cell length and decrease in myosin light chain kinase activity. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

The role of LMX1B and PITX2 in anterior segment development and adult ocular function

Several congenital syndromes associated with anterior segment (AS) anomalies can lead to impaired vision and glaucoma, such as nail-patella syndrome (NPS), caused by mutations in the LIM homeodomain transcription factor LMX1B and Axenfeld-Rieger's syndrome (ARS), caused by mutations in the bicoid-related homeodomain transcription factor PITX2. Targeted mutations in lmx1b and pitx2 and RNA in situ analysis reveal that both genes are required for AS development and are co-expressed within the periocular mesenchyme, suggesting they participate in a shared genetic pathway. Lmx1b homozygous mutants display iris and corneal stroma hypoplasia, and defects in ciliary body formation. In contrast, pitx2 homozygous mutants exhibit a more severe phenotype: the AS chamber, corneal endothelium, and extraocular muscles (EOM) fail to develop. The absence of EOM in pitx2 mutants suggests pitx2 acts upstream of lmx1b, or that other lmx1b family members, such as lmx1a, can compensate for lmx1b function. Lmxla/lmx1b double homozygous mutants have a reduced capacity to generate EOM, implying that lmx1 gene products have a redundant function in EOM development and that lmx1 family members may act downstream of pitx2. However, analysis of pitx2 expression in the AS tissues of lmx1b mutants and reciprocal studies of lmx1b expression in pitx2 mutants indicate that these genes do not function in a simple linear pathway. Instead, lmx1b and pitx2 may regulate a shared set of downstream targets or both genes may work in parallel transcribing unique targets required for a common biological process. Ultrastructural analysis of lmx1b and pitx2 mutant corneas indicates that collagen fibrillogenesis is perturbed, revealing a common role for both genes in the deposition of extracellular matrix. Furthermore, lmx1b/pitx2 double heterozygotes develop corneal opacities not observed in single heterozygotes demonstrating that lmx1b and pitx2 genetically interact. Data suggests that defects in the basement membrane of the corneal endothelium underlie the opacities observed in double heterozygotes. Additionally, double heterozygotes develop anterior synechias that occlude the trabecular meshwork, potentially blocking aqueous humor drainage. These data suggest that lmx1b and pitx2 are responsible for ECM deposition in multiple cell types and imply that such defects may contribute to the glaucomas observed in NPS and ARS patients. ^

New aspects of estrogen action in the endometrium: Reciprocal interplay with retinoic acid pathway and a novel estrogen-regulated gene

The uterine endometrium is a major target for the estrogen. However, the molecular basis of estrogen action in the endometrium is largely unknown. I have used two approaches to study the effects of estrogen on the endometrium. One approach involved the study of the interaction between estrogen and retinoic acid (RA) pathways in the endometrium. I have demonstrated that estrogen administration to rodents and estrogen replacement therapy (ERT) in postmenopausal women selectively induced the endometrial expression of retinaldehyde dehydrogenase II (RALDH2), a critical enzyme of RA biosynthesis. RALDH2 was expressed exclusively in the stromal cells, especially in the stroma adjacent to the luminal and glandular epithelia. The induction of RALDH2 by estrogen required estrogen receptor and occurred via a direct increase in RALDH2 transcription. Among the three RA receptors, estrogen selectively induced the expression of RARα. In parallel, estrogen also increased the utilization of all-trans retinol (the substrate for RA biosynthesis) and the expression of two RA-regulated marker genes, cellular retinoic acid binding protein II (CRABP2) and tissue transglutaminase (tTG) in the endometrium. Thus estrogen coordinately upregulated both the production and signaling of RA in both the rodent and human endometrium. This coordinate upregulation of RA system appeared to play a role in counterbalancing the stimulatory effects of estrogen on the endometrium, since the depletion of endogenous RA in mice led to an increase in estrogen-stimulated stromal proliferation and endometrial Akt phosphorylation. In addition, I have also used a systematic approach (DNA microarray) to categorize genes and pathways affected by the ERT in the endometrium of postmenopausal women and identified a novel estrogen-regulated gene EIG121. EIG121 was exclusively expressed in the glandular epithelial cells of the endometrium and induced by estrogen in vivo and in cultured cell lines. Compared with the normal endometrium, EIG121 was highly overexpressed in type 1 endometrial cancer, but profoundly suppressed in type 2 endometrial tumors. Taken together, these studies suggested that estrogen regulates the expression of many genes of both the pro-proliferative and anti-proliferative pathways and the abnormality of these pathways may increase the risks for estrogen-dependent endometrial hyperplasia and endometrial cancer. ^

Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

SFRP4 regulation of Wnt7a signaling and cellular proliferation in the endometrium

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication and thus mediate hormone action. Characterization of specific Wnt signaling components in the endometrium was performed using cellular localization studies and evaluating hormone effects in a rat model. Wnt7a was expressed in the luminal epithelium, whereas the extracellular Wnt modulator, SFRP4, was localized to the endometrial stroma. SFRP4 expression is significantly decreased in endometrial carcinoma and aberrant Wnt7a signaling has been shown to cause uterine defects and contribute to the onset of disease. The specific Fzds and SFRPs that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of Wnt7a and SFRP4 in the endometrium has not been addressed. A survey of all Wnt signaling proteins expressed in the endometrium was conducted and Fzd5 and Fzd10 were identified as two receptors capable of transducing the Wnt7a signal. Biologically active recombinant Wnt7a and SFRP4 proteins were purified for quantitative biochemical studies. In Ishikawa cells, Wnt7a binding to Fzd5 activated β-catenin/canonical Wnt signaling and increased cellular proliferation. Wnt7a signaling mediated by Fzd10 induced a non-canonical/JNK-responsive pathway. SFRP4 suppressed Wnt7a action in both an autocrine and paracrine manner. Treatment with SFRP4 protein and overexpression of SFRP4 inhibited endometrial cancer cell growth and induced apoptosis in vitro. A split-eGFP complementation assay was developed to visually detect Wnt7a-Fzd interactions and subsequent pathway activation in cells. By employing a unique ELISA-based protein-protein binding technique, it was demonstrated that Wnt7a binds to SFRP4 and Fzd5 with equal nanomolar affinity. The development of these novel biological tools could lead to a better understanding of Wnt-protein interactions and the identification of new modulators of Wnt signaling. This study supports a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent upon the Fzd repertoire of the cell and can be regulated by SFRP4. The potential tumor suppressor function of SFRP4 suggests it may serve as a therapeutic target for endometrial carcinoma. ^

Fluorophotometry is a sensitive method/technique to evaluate epithelial permeability in murine experimental dry eye

Purpose. Fluorophotometry is a well validated method for assessing corneal permeability in human subjects. However, with the growing importance of basic science animal research in ophthalmology, fluorophotometry’s use in animals must be further evaluated. The purpose of this study was to evaluate corneal epithelial permeability following desiccating stress using the modified Fluorotron Master™. ^ Methods. Corneal permeability was evaluated prior to and after subjecting 6-8 week old C57BL/6 mice to experimental dry eye (EDE) for 2 and 5 days (n=9/time point). Untreated mice served as controls. Ten microliters of 0.001% sodium fluorescein (NaF) were instilled topically into each mouse’s left eye to create an eye bath, and left to permeate for 3 minutes. The eye bath was followed by a generous wash with Buffered Saline Solution (BSS) and alignment with the Fluorotron Master™. Seven corneal scans using the Fluorotron Master were performed during 15 minutes (1 st post-wash scans), followed by a second wash using BSS and another set of five corneal scans (2nd post-wash scans) during the next 15 minutes. Corneal permeability was calculated using data calculated with the FM™ Mouse software. ^ Results. When comparing the difference between the Post wash #1 scans within the group and the Post wash #2 scans within the group using a repeated measurement design, there was a statistical difference in the corneal fluorescein permeability of the Post-wash #1 scans after 5 days (1160.21±108.26 vs. 1000.47±75.56 ng/mL, P<0.016 for UT-5 day comparison 8 [0.008]), but not after only 2 days of EDE compared to Untreated mice (1115.64±118.94 vs. 1000.47±75.56 ng/mL, P>0.016 for UT-2 day comparison [0.050]). There was no statistical difference between the 2 day and 5 day Post wash #1 scans (P=.299). The Post-wash #2 scans demonstrated that EDE caused a significant NaF retention at both 2 and 5 days of EDE compared to baseline, untreated controls (1017.92±116.25, 1015.40±120.68 vs. 528.22±127.85 ng/mL, P<0.05 [0.0001 for both]). There was no statistical difference between the 2 day and 5 day Post wash #2 scans (P=.503). The comparison between the Untreated post wash #1 with untreated post wash #2 scans using a Paired T-test showed a significant difference between the two sets of scans (P=0.000). There is also a significant difference between the 2 day comparison and the 5 day comparison (P values = 0.010 and 0.002, respectively). ^ Conclusion. Desiccating stress increases permeability of the corneal epithelium to NaF, and increases NaF retention in the corneal stroma. The Fluorotron Master is a useful and sensitive tool to evaluate corneal permeability in murine dry eye, and will be a useful tool to evaluate the effectiveness of dry eye treatments in animal-model drug trials.^