Fabrication of flexible thin polyurethane membrane for tissue engineering applications.

Thin and flexible polymeric membranes play a critical role in tissue engineering applications for example organs-on-a-chip. These flexible membranes can enable mechanical stretch of the engineered tissue to mimic organ-specific biophysical features, such as breathing. In this work, we report the fabrication of thin (<20 µm), stretchable, and biocompatible polyurethane (PU) membranes. The membranes were fabricated using spin coating technique on silicon substrates and were mounted on a frame for ease of device integration and handling. The membranes were characterized for their optical and elastic properties and compatibility with cell/tissue culture. It was possible to apply up to 10 kilopascal (kPa) pressure to perform cyclic stretch on 4 mm-diameter membranes for a period of 2 weeks at 0.2 hertz (Hz) frequency without mechanical failure. Adenocarcinomic human alveolar basal epithelial (A549) cells were cultured on the apical side of the PU membrane. The morphology and viability of the cells were comparable to those of cells cultured on standard tissue culture plates. Our experiments suggest that the stretchable PU membrane will be broadly useful for various tissue engineering applications in vitro.

Osteopontin expression in a group of lymph node negative breast cancer patients.

The aim of this study was to examine the cellular distribution of osteopontin (OPN) protein [by immunohistochemical (IHC) analysis] and mRNA [by in situ hybridization (ISH)] in the primary tumors of lymph node negative (LNN) breast cancer patients and to determine whether the level of immunodetectable OPN may be associated with tumor aggressiveness. We examined OPN levels in tumors from 154 patients with LNN breast cancer who were followed for a median of 7 years (range 1.7-16.3 years). IHC staining for OPN was seen in tumor infiltrating macrophages and lymphocytes in 70% of these tumors, and in the carcinoma cells themselves in 26%. ISH was performed to determine cellular distribution of OPN mRNA expression in sections from selected tumors. OPN mRNA was detected in groups of tumor cells, individual tumor cells and tumor infiltrating macrophages and lymphocytes. Matched sections showed that some tumor cells with IHC staining for OPN protein were also positive for OPN mRNA by ISH, in contrast with previous studies which have shown OPN mRNA expression only in tumor infiltrating inflammatory cells. Our results thus indicate that OPN protein can be produced by breast cancer cells in vivo and suggest that it may also be taken up from the environment (i.e., secreted by inflammatory cells or other tumor cells). Tumor cell IHC staining intensity was then assessed using a semiquantitative scoring system. Univariate analysis showed tumor cell OPN positivity above an optimized cutpoint to be significantly associated with decreased disease-free survival (DFS) and overall survival (OS). The results of this pilot study thus suggest that the ability of breast cancer cells to either synthesize OPN or to bind and sequester OPN from the microenvironment may be associated with tumor aggressiveness and poor prognosis.

Coordinate expression of OPN and associated receptors during monocyte/macrophage differentiation of HL-60 cells.

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.

Osteopontin and p53 expression are associated with tumor progression in a case of synchronous, bilateral, invasive mammary carcinomas.

OBJECTIVE: To examine the association between expression of osteopontin (OPN), p53, other molecular markers (Ki-67, c-erb B2, and estrogen receptor protein) and tumor progression in a case of synchronous, bilateral, invasive mammary carcinomas of the same histology. DESIGN: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections. Plasma OPN level was determined by a quantitative antigen capture assay. SETTING: The patient was seen, treated, and followed up for a period of 5 years at the London Regional Cancer Centre, Ontario, Canada. PATIENT: A 60-year-old woman presented with bilateral infiltrating mammary carcinomas of the same histologic type and grade. Bilateral mastectomy and axillary node dissection showed involvement of 3 of 12 right axillary and 0 of 11 left axillary lymph nodes. She later developed a right chest wall recurrence, followed by widespread metastatic disease to the skull, liver, and left femur. RESULTS: The primary tumor of the right breast was OPN- and p53-positive, whereas the tumor of the left breast was negative for both markers. The development of right axillary lymph node metastases, chest wall recurrence, and distant metastases was associated in all instances with an immunohistochemical profile of high level expression of OPN and p53. Plasma assay for OPN at the time of last admission showed a markedly elevated OPN level. CONCLUSIONS: Increased p53 expression was found to be associated with increased tumor aggressiveness. The association of increased OPN expression with increased malignancy in breast cancer is a novel finding and raises the possibility of a role for OPN in tumor progression, as well as the potential for this marker in predicting clinical aggressiveness.

Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival.

Osteopontin (OPN) is a secreted, integrin-binding phosphoprotein that has been implicated in both normal and pathological processes; qualitative increases in OPN blood levels have been reported in a small number of patients with metastatic tumors of various kinds. We measured plasma OPN levels in 70 women with known metastatic breast carcinoma, 44 patient controls who were on follow-up after completion of adjuvant treatment for early breast cancer, and 35 normal volunteers. The median plasma OPN of patients with metastatic disease was 142 microgram/liter (range, 38-1312 microgram/liter) and was significantly different (P < 0.0001, Mann Whitney U test) from both control groups (medians, 60 and 47 microgram/liter; ranges, 15-117 and 22-122 microgram/liter). Furthermore, we found that increasing plasma OPN is associated with shorter survival (P < 0.001) when patients were grouped in terciles for plasma OPN. This was also demonstrated when using a Cox proportional hazards model. Median plasma OPN levels were significantly increased for three or more sites of involvement (median, 232 microgram/liter; n = 13) versus 1 or 2 metastatic sites (medians, 129 and 130 microgram/liter; n = 29 and 28, respectively). Plasma OPN levels were correlated with other biochemical markers related to the extent of disease, such as serum alkaline phosphatase, aspartate succinate aminotransaminase, and albumin (r = 0.81, 0.62, and -0.56, respectively; all P < 0.001). This study demonstrates a statistically significant elevation in plasma OPN in the majority ( approximately 70%) of a large series of patients with metastatic breast cancer when compared (95th percentile) to healthy women or patients who had completed adjuvant treatment for early-stage breast cancer. Furthermore, this is the first study to demonstrate that higher OPN levels in patients with metastatic breast cancer may be associated with an increased number of involved sites and decreased survival.

Osteopontin expression in lung cancer.

Osteopontin (OPN), an integrin-binding, transformation-associated protein, is secreted by tumor cell lines in culture and is associated with increased malignancy in some experimental tumor systems. Little is known, however, about the significance of OPN expression in human cancers. The aims of this study were to determine if OPN was expressed in a series of surgically resected lung cancers, and if there was a relationship between OPN expression and clinico-pathologic findings or outcome. Twenty-five patients who underwent curative pulmonary resection were studied prospectively. RNA was extracted from primary tumor and distant normal lung tissue for each patient. OPN RNA levels were evaluated by northern blotting. Immunohistochemistry on paraffin-embedded tissue, using an anti-OPN monoclonal antibody, was performed to assess tissue distribution of OPN protein. OPN RNA and protein were over-expressed in the majority of tumors, relative to paired normal tissue. There was variation in the cells of the tumor that were OPN-immunopositive. In some cases OPN was present in tumor cells, while in the majority of cases OPN was detected primarily in tumor-infiltrating macrophages and necrotic areas. Over-expression of OPN RNA or protein generally was not related to clinico-pathological findings. However, there was a statistically significant association between OPN-immunopositivity in the tumor and patient survival. These findings suggest that OPN levels in lung tumors have the potential to provide clinically important predictive information on patient outcome, and that OPN may play a role in the biology of lung cancer.

Low-molecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones.

Osteopontin (OPN) is a multifunctional glycosylated phosphoprotein found in body fluids, including urine, and has been implicated in urinary stone formation. We tested the hypothesis that OPN levels in urine of patients with kidney stones differed from normal individuals. To quantify OPN levels in the urine, we developed an ELISA using a combination of a mouse monoclonal and rabbit polyclonal antibodies raised against a recombinant glutathione-S-transferase-human OPN fusion protein. In a group of 34 patients diagnosed with kidney stones compared with a control group of 23 normal individuals, we found that OPN levels in urine of the patient and control groups ranged from 0.01 to 2.7 micrograms/ml, with no significant difference in their medians (P > 0.8, Mann-Whitney test). OPN in urine was qualitatively assessed by Western blotting using a biotinylated monoclonal antibody to detect various molecular forms. The urine of most individuals contained OPN species within in the 55- to 66-kDa electrophoretic mobility range. However, a significantly higher proportion of individuals in the patient group (13 of 34) was found to have aberrant urine OPN species (< or = 40 kDa) compared to 2 of 23 for the control group (P < 0.03, chi 2 test). Mixing experiments indicated that urine samples with aberrant OPN contain proteases inhibitable with phenylmethylsulfonyl fluoride. Such proteases could break down normal urine OPN in vitro. Therefore, urine from a high frequency of kidney stone patients contains serine proteases that contribute to proteolytic cleavage of OPN.

Quantification of osteopontin in human plasma with an ELISA: basal levels in pre- and postmenopausal women.

OBJECTIVES: To develop an immunoassay for osteopontin (OPN), a secreted phosphoglycoprotein that is implicated in a number of human diseases, and establish basal plasma OPN levels in healthy women. DESIGN AND METHODS: An antigen-capture ELISA was developed to quantity OPN in plasma using a combination of mouse monoclonal and rabbit polyclonal antibodies. Basal OPN levels were determined in blood plasma of 21 pre- and 14 postmenopausal women obtained at 7-day intervals over a 4-week period. RESULTS: A group of 35 healthy women had a median OPN level of 31 micrograms/L (range = 14-64 micrograms/L). Comparison between pre- and postmenopausal women showed that their 4-week average OPN levels did not differ significantly (p > 0.16, Mann-Whitney test), and that levels in each premenopausal individual remained constant during the menstrual cycle, unaffected by cyclical levels of leuteinizing hormone and progesterone. CONCLUSION: Systematic quantification of plasma OPN can now be done by ELISA, which was used to establish basal plasma OPN levels in a group of healthy women. Levels in pre- and postmenopausal women appeared relatively stable over a 4-week period.

Inhibition of Arg-Gly-Asp (RGD)-mediated cell adhesion to osteopontin by a monoclonal antibody against osteopontin.

Osteopontin (OPN), a secreted phosphoprotein, has been implicated in various biological phenomena (e.g. bone development, sepsis, tumor progression, and metastasis). Its role in any context is poorly understood. OPN contains a conserved Gly-Arg-Gly-Asp-Ser (GRGDS) sequence, and binds to cells via integrin-mediated mechanisms. Using recombinant human osteopontin-glutathione S-transferase fusion protein and our improved hybridoma fusion partner (Sp2/mIL6), we raised murine monoclonal antibodies against osteopontin. We characterized two antibodies that recognize not only recombinant but also native human osteopontin. These antibodies do not cross-react with mouse osteopontin (recombinant protein or that secreted by ras-transformed NIH 3T3 cells), or bovine bone osteopontin, suggesting that they recognize epitopes unique to human OPN. One antibody specifically inhibited adhesion of MDA-MB-435 human breast cancer cells and ras-transformed NIH 3T3 cells to human osteopontin. This antibody failed to recognize osteopontin cleaved by thrombin, which cleaves adjacent to the cell binding domain. We previously showed that thrombin cleavage reduces osteopontin cell binding activity. Thus we postulate that this monoclonal antibody recognizes and interferes with the function of the RGD/thrombin cleavage region of human OPN.

Effect of vitrectomy and cytopreparatory techniques on cell survival and preservation.

Obtaining vitreous fluid by means of vitrectomy frequently results in a specimen that is difficult to assess cytologically. We devised an experimental model to examine the effect of the vitrector on human leukemic cancer (HL60) cells in suspension and to evaluate the cytopreparatory techniques of membrane filtration and cytocentrifugation. Eighteen 3-mL specimens of cells at concentrations ranging from 1 to 9 x 10(5)/mL were vitrectomized, and eighteen 3-mL control samples matched for cell concentration were obtained atraumatically. No significant difference in cell loss, as determined by means of staining with nigrosin vital dye, was found at any cell concentration between the vitrectomized and control specimens. The specimens were then processed cytologically. On cytologic assessment it was not possible to distinguish the vitrectomized and control specimens. A higher degree of cell preservation was noted at higher cell concentrations regardless of the cytopreparatory technique, but at lower concentrations membrane filtration resulted in a higher proportion of cytologically assessable specimens than did cytocentrifugation (42% vs. 22%). The results suggest that the vitrector causes minimal cellular damage and that to obtain optimal results both cytopreparatory techniques should be used with all vitrectomy specimens.

Increased frequency of both total and specific monoclonal antibody producing hybridomas using a fusion partner that constitutively expresses recombinant IL-6.

The addition of auxiliary feeder cells or conditioned medium has been shown to augment the yield of mouse hybridomas obtained following the cell-cell fusion of myeloma and B lymphocytes. The addition of one of these factors, interleukin-6 (IL-6) has been found to increase the proportion of hybridomas secreting monoclonal antibodies of desired specificity. As an alternative genetic approach, we have examined the efficacy of a retroviral infectant of Sp2/0 cells that constitutively expresses recombinant murine IL-6 (Sp2/mIL-6) as fusion partner. The results demonstrated that the yields of both viable Ig-secreting hybridomas, and antigen-specific monoclonal antibodies were increased 3-15-fold and 5-9-fold, respectively, with the Sp2/mIL-6 relative to Sp2/0 or Sp2/neo cells as fusion partner. Sp2/mIL-6 cells generated hybridomas with comparable growth rates, stability, and Ig production. The results of staining nascent hybridoma colonies immunohistochemically for Ig production suggest that Sp2/mIL-6 cells as a fusion partner increased the viability and/or stability of nascent hybrid cells that are producing Ig. Thus the Sp2/mIL-6 cells are an improved myeloma parent for the generation of large numbers of antibody-producing hybridomas against specific antigens.

Radioimmunoassay of metallothionein in rabbit, rat, mouse, Chinese hamster, and human cells.

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.

Cefmenoxime and bilirubin: competition for albumin binding.

Certain drugs are known to compete with bilirubin for albumin binding; therefore, all drugs administered to neonates should be tested to determine the degree of competition. The effect of cefmenoxime on bilirubin-albumin binding was determined by comparing the oxidation rate of free bilirubin in the presence and absence of drug. The reserve albumin concentration (RAC) of pooled cord serum was also measured using the MADDS dialysis rate method. We show that cefmenoxime competes with bilirubin for albumin binding with a displacement constant, of 3.1 x 10(3) l/mol. The maximal displacement factor (MDF) is used to determine the clinical effect of the drug at usual serum concentrations. The MDF for cefmenoxime is 1.10, representing approximately a 10% increase in free bilirubin concentration. In comparison, the MDF for a known bilirubin displacing drug, sulfisoxazole, is 2.43. The MADDS method showed an estimated 28% decrease in the RAC at 150 mumol/l, the mean peak serum concentration (MPSC) of cefmenoxime. These results show while cefmenoxime affects bilirubin-albumin binding, the degree of the effect is relatively small. However, cefmenoxime may pose a hazard to very sick, premature infants, especially if the infant is jaundiced.

Modulation of clonal progression in B16F1 melanoma cells.

We have examined the effects of the microenvironment on the frequency and generation of metastatic variant cells in both parental B16F1 melanoma cells and nascent clones. The metastatic abilities of cultured B16F1 cells were tested after a period of growth in the presence or absence of a second cell population separated from each other by a transwell membrane (0.45 micron pore size). The first population is defined as the 'responder' cells and the second as the 'stimulator' cells. We found that the presence of 10(5) B16F1 stimulator cells during the growth of responder B16F1 cells from approximately 10(4) to approximately 10(6) cells resulted in cells with an increased metastatic phenotype (greater than 8-fold increase in median number of lung tumors relative to untreated B16F1 parental cells). The presence of stimulator cells also increased the metastatic phenotype of nascent clones, which were grown to a population size of less than 10(6) cells, suggesting that the rate of generation of metastatic variants of the responder B16F1 clones was affected by the stimulator cells. Other cell lines, including highly metastatic B16F10 and BL6 melanoma cells, and KHT35-L1 fibrosarcoma cells, were effective stimulator cells when as few as 10(4) cells were added to transwells. In addition, normal immortalized NIH 3T3 cells were effective stimulator cells only at 10(5) cells/transwell. The cell density at which untreated parental B16F1 cells were harvested (3 x 10(3)-3 x 10(5) cells/cm2) did not affect the median number of lung tumors significantly. These results suggest that factors released from both tumor and immortalized normal cells can modulate epigenetic changes in the metastatic phenotype of B16F1 melanoma cells.

Spontaneous and radiation-induced genetic instability of heteromyeloma hybridoma cells.

We have examined the genetic stability of heteromyeloma cells both spontaneously and following ionizing radiation. Clones of E10 cells (SHM-D33 heteromyeloma X human lymphoblastoid) were examined for the stability of human immunoglobulin (Ig) production (mu, lambda), relative human and mouse DNA, and total DNA content. The stability of recloned E10 cells was improved more than fourfold relative to the stability of the nascent E10 cells. The spontaneous loss of human Ig production in the established E10 cells was approximately 1.5 x 10(-3) events/cell per generation, which is comparable to mouse hybridomas. In contrast to the relative stability of antibody production, the relative human DNA content of antibody producing clones of E10.26 cells showed considerable variation (median, 15%; range, 4 to 23% for 30 clones) although the total DNA content of the clones was relatively constant (1.2(+/- 0.1) x 10(-12)g/cell). The frequency of Ig(mu-) antibody loss variants was increased in three subclones of E10 cells following irradiation (P less than 0.05, 20 to 90 Ig(mu-) variants/10(5) cells per Gray. In addition, the human DNA content per cell was significantly reduced (P less than 0.001) in a sample of irradiated E10 clones, while the total DNA content per cell was constant. We conclude that, although the antibody production is relatively stable in heteromyeloma cells, the relative human DNA content is constantly drifting by small amounts while maintaining a constant DNA content.

Sulfated glycoconjugate determinants recognized by monoclonal antibody, SG-1, correlate with the experimental metastatic ability of KHT fibrosarcoma cells.

We have examined the binding and functional activity of monoclonal antibody (MAb) SG-1 that was raised by immunization against embryonal carcinoma cells and screened using KHT fibrosarcoma cells. Quantitative absorption, binding and in situ immunochemical staining assays indicate that the MAb SG-1-defined epitopes are expressed preferentially by the highly metastatic KHT35-L1 cells relative to the weakly metastatic, parental KHTp cells. Furthermore, there was a significant correlation (p less than 0.05) between the expression of MAb SG-1-defined antigen on the cells, following trypsin treatment, and their metastatic ability. Binding of MAb SG-1 to antigen was inhibited by specific sulfated polysaccharides including cerebroside sulfate (brain sulfatide), fucoidan, and dextran sulfate (500 kD) but not by heparan, chondroitin, keratan or dextran (5 kD) sulfates. Initial characterization of antigen from KHT cells indicates that the epitope of MAb SG-1 is defined by sulfated glycoconjugates containing galactose and sulfate but not N-acetylglucosamine. In the total lipid extracts of KHT35-L1 cells the antigen was detected in the delipidated protein fraction as well as in the chloroform/methanol fraction. These results suggest that the sulfated glycoconjugate determinants identified by MAb SG-1 may be relevant to the metastatic process of KHT fibrosarcoma cells.

Some ras-transformed cells have increased radiosensitivity and decreased repair of sublethal radiation damage.

We examined the effect of 60Co irradiation on the clonogenic survival of rat NRK cells, NRK cells carrying a temperature-sensitive viral K-ras oncogene (tsK-NRK), mouse NIH 3T3 cells, and NIH 3T3 cells transformed with the human bladder cancer (T24) H-ras oncogene (PAP2). We tested the hypothesis that ras oncogene expression renders cells more resistant to radiation, but found in both systems that ras-transformed cells were more, not less, sensitive to radiation. We also found indications of altered repair of sublethal radiation damage. PAP2 cells were more sensitive to radiation than NIH 3T3 cells. Increased sensitivity was reflected in a decreased shoulder region of the survival curve with little effect on its slope (D0). TsK-NRK cells were also slightly more sensitive to radiation than NRK and exhibited decreased repair of sublethal damage at both the permissive and nonpermissive temperatures. Thus, we found that expression of ras oncogenes is not always associated with increased radiation resistance. In summary, our results suggest that (1) ras oncogene expression in some cells may be associated with increased, rather than decreased, radiation sensitivity, and (2) ras oncogene expression may alter the shoulder region of the dose response curve, suggesting changes in the repair of sublethal radiation damage.

Dynamic heterogeneity: metastatic variants to liver are generated spontaneously in mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells derived from F9 cells form predominantly liver tumors following the intravenous injection (i.e. experimental metastasis assay) of EC cells into syngeneic 129/J male mice. In this study, EC cells (OTF9) expressing stage-specific embryonic antigen-1 (SSEA-1) are compared with cells (SOTF9) lacking SSEA-1 antigen in the experimental liver metastasis assay. When parallel clones of EC cells were grown to a measured cell number and tested in the experimental metastasis assay, it was observed that the frequency of experimental liver metastases increases with the population size. When the clonal population size is less than the critical number of cells (approximately 2 x 10(5) cells), the frequency of liver tumors is reduced relative to that of the parent EC population. The metastatic ability of clones derived from individual liver metastases did not differ from that of the parental cells. An analysis of the recessive biochemical and immunochemical markers of parental cells and of independent liver metastases suggests that somatic hybridization to host cells by the EC cells is not involved. These results are consistent with predictions from our dynamic heterogeneity model that was formulated by examining the experimental lung metastasis of KHT fibrosarcoma and B16 melanoma cells. Mathematical analysis of the results indicates that the effective rate of generation of the liver metastasizing variant cells is (7 +/- 3) x 10(-6) per cell per generation for both OTF9 and SOTF9 cells.

Rates of generation of methotrexate-resistant variants in cells temperature-sensitive for malignant transformation.

We used tsLA23-NRK cells, in which malignant transformation is under the control of a temperature-sensitive (ts) src oncogene, to examine the relationship between genetic instability and malignant transformation. Cells were maintained in vitro as either transformed cells (at 36 degrees C) or as normal cells (at 39 degrees C). Genetic instability was assessed in these two cell populations by determining (1) the frequencies of methotrexate (MTX)-resistant variants present in these populations, and (2) the rates of generation of MTX-resistant variants, as determined from Luria-Delbrück fluctuation analyses. We observed no significant differences, in either of these parameters of genetic instability, that could be attributed to transformation status. We conclude that in this defined cellular system there is no obligatory relationship between malignant transformation and increased genetic instability, as assessed by either frequencies or rates of generation of variants resistant to MTX.