ROBO1 Expression in Metastasizing Breast and Ovarian Cancer: SLIT2-induced Chemotaxis Requires Heparan Sulfates (Heparin).

BACKGROUND: The members of the slit homolog (SLIT) and roundabout homolog (ROBO) families have emerged as important signaling molecules in tumor metastasis. This study analyzed their role in regulating breast cancer (BC) cell motility and chemotaxis and assessed expression of ROBO1 in brain metastases (BMs) of breast, lung, and colon cancer, and in peritoneal metastases (PMs) of ovarian cancer. MATERIALS AND METHODS: The BC cell line MDA-MB231 was subjected to scratch, motility, and chemotaxis assays using heparin and a purified recombinant N-terminal SLIT2 fragment. Protein expression was assessed in primary tumors and metastases by immunohistochemistry. RESULTS: Exposure to SLIT2 induced MDA-MB231 cell motility, but no significant chemotaxis without the presence of heparin. ROBO1 was expressed in 4/5 primary BC and in 18/21 BC-derived BM samples; 7/9 BM primary lung cancer samples also stained positive. In contrast, BMs from colorectal cancer were negative for ROBO1. Primary ovarian cancer and ovarian PM showed ROBO1 expression in 0/6 and in only 2/6 samples, respectively, whereas SLIT2 was observed in 1/6 primary cancer and in 6/6 PMs samples. CONCLUSION: SLIT2 can induce BC cell motility and chemotaxis, but the latter requires the presence of heparin. BM expression of ROBO1 is a common feature of some, but not all cancer types. SLIT2 expression appears to be a general feature of ovarian cancer-derived PMs.

Influence of preadipocyte-conditioned medium on the proliferation and invasive potential of breast cancer cell lines in vitro.

PURPOSE: Autologous transplantation of adipose tissue into the breast is commonly performed in clinical practice, but its oncological safety has not been established. METHODS: We conducted an in vitro study to assess the influence of factors released by adipose-derived stem cells (ASCs), from multiple source tissues and harvested using different techniques, on proliferation and invasiveness of two breast cancer cell lines. RESULTS: Fat specimens of 66 donors (57 female, 9 male) were collected and 44 ASC cultures were established. ASC conditioning of the medium (CM) increased the proliferation of MCF-7 cells (178.4 ± 62.8%; P < 0.001), whereas MDA-MB321 proliferation was decreased (87.3 ± 15.3%; P = 0.032). We observed increased cell migration (174.0 ± 62.8%; P = 0.002), but not cell invasion (1.28 ± 0.51; P = 0.14) in MDA-MB231. Migration and invasion of MCF-7 cells were not affected by exposure to ASC-CM. For MCF-7 cell migration, lower BMI (< 25 kg/m2) was associated with increased migration, both in univariate (P = 0.015) and multivariate (P = 0.039) analyses. Regarding the cytokine secretome, proliferation of MCF-7 was positively correlated with levels of eotaxin 1 and insulin-like growth factor-binding protein 3 in the CM, and inversely correlated with levels of interleukin 1ß and transforming growth factor ß-3. In case of MDA-MB231, granulocyte colony-stimulating factor, angiogenin, eotaxin 1 and 3, neutrophil activating peptide 2, and neurotrophin-3 were positively correlated with proliferation. CONCLUSIONS: We conclude that fat tissue transplantation increases proliferation and migration, but not invasion, of breast cancer cells. These findings are consistent with clinical data regarding the safety of autologous fat transplantation in breast cancer patients.

Factors influencing yield and neuronal differentiation of mesenchymal stem cells from umbilical cord blood and matrix.

AIM: Umbilical cord blood and Wharton's jelly (WJ) are potential sources of mesenchymal stem cells (MSCs). We investigated whether harvesting and donor characteristics affected yield and neuronal differentiation, and compared human umbilical cord blood (hUCB) and WJ-derived MSCs regarding neuronal differentiation and cytokine secretion. MATERIALS & METHODS: MSCs were analyzed by immunoblotting after seven days of differentiation; cytokine protein arrays were used to analyze conditioned medium. RESULTS: Birth weight and blood/anticoagulant ratio influenced MSC yield per unit blood volume, but not maternal and gestational age, delivery mode or fetal gender. Expression of the early neuronal differentiation marker nestin was unaffected by these variables. hUCB- and WJ-derived MSC secrete distinct cytokine profiles. CONCLUSION: Cell yield is affected by certain donor characteristics. hUCB- and WJ-derived MSCs may serve distinct therapeutic niches.

Spread of endometriosis to pelvic sentinel lymph nodes: gene expression analysis.

OBJECTIVE: Endometriotic spread to the lymphatic system has been described, but little is known about the molecular events and changes in gene expression associated with this process. We sought to determine the expression levels of a panel of 28 genes in samples of primary endometriosis lesions (EL), isolated endometriotic-like cells (IELC)-positive pelvic sentinel lymph nodes (PSLN), and IELC-negative PSLN, in order to identify candidate genes that may play a role in this process. STUDY DESIGN: Quantitative real-time PCR and immunohistochemistry (IHC) of primary EL and PSLN samples with and without IELC from patients with ovarian and/or peritoneal endometriosis. RESULTS: Gene expression was analyzed in EL (n=13), IELC-positive PSLN (PSLN+, n=11), and IELC-negative PSLN (PSLN-, n=8). Gene expression differences between PSLN+ and PSLN- were analyzed and evaluated in relation to their expression levels in EL. Genes expressed at high levels in EL but not in PSLN- and known to be expressed in IELC (such as ESR1, PGR) served as controls and the expected gene dilution effect was clearly observed. Expression of a set of genes (CXCR4, CD68, MKI67, and CD44) was found to be higher in PSLN+ vs. PSLN-, while lowest in EL, indicating upregulation in IELC. In contrast, EPCAM and E-cadherin, which were strongly expressed in EL, were not found to be expressed in PSLN+, and thus likely absent from IELC. IHC confirmed the expression of CXCR4, CD44s, and CD44v6 in IELC, as well as the absence of E-cadherin from IELC. CONCLUSION: Our data indicate that spread of endometriosis to PSLN is accompanied by differential expression of several genes, including EPCAM, CDH1 (E-cadherin), CXCR4, and CD44, suggesting an involvement of CD44 splice variants as well as CXCR4 signalling in this process.