Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Adaptation of EGF receptor signal transduction to three-dimensional culture conditions: changes in surface receptor expression and protein tyrosine phosphorylation.

A431 cells grown as three-dimensional spheroids show growth stimulation in response to nanomolar concentrations of EGF in contrast to monolayer cultures that show inhibition. In investigating the alterations in EGF signal transduction that underlie this modification of the proliferative response, we have compared the expression of EGF receptors on A431 cells under these conditions and related our findings to tyrosine phosphorylation and the growth response. EGF receptors were measured by 125I-EGF binding to trypsin-dispersed cells. Unexpectedly, dispersion of the monolayers caused an 80% decrease in surface EGF receptor, although, after dispersion, EGF receptor was digested by trypsin with a half-life of 69 +/- 32 min. No evidence for a comparable loss of cellular EGF receptor was seen on trypsin dispersion of spheroids. After allowing for this effect, we found that the receptor density on nondispersed monolayers (5 x 10(6) per cell) was twentyfold greater than that on spheroids (0.25 x 10(6) per cell). EGF-induced tyrosine phosphorylation was confined to the outermost cells of the spheroid, although the presence of surface-expressed EGF binding sites could be demonstrated throughout the structure and the number of EGF receptors/cell on dispersed spheroid cells showed a single distribution peak by flow cytometry, with no evidence for more than one population. Using RCM-lysozyme as a substrate, tyrosine phosphatase activity in spheroids lay within the range observed in monolayer cultures. Autophosphorylation of the EGF receptor following EGF stimulation in monolayer cultures of A431 cells rose rapidly in the first 10 seconds and then slowly increased for at least 3 h. In spheroids, it reached a maximum within 10 seconds and then declined over 3 h. Since the microenvironment within a tumor resembles that in a spheroid, a similar reduction in surface EGF receptor expression may be expected in tumors relative to monolayer cultures, together with corresponding growth stimulation in response to EGF.

Financial and emotional cost of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) in infants with neonatal respiratory disease significantly increases the duration of hospitalization and cost of medical care. Early discharge on home oxygen therapy results in cost savings for third-party payers and the hospital, but adds financial and emotional burdens for the family. The median cost of initial hospitalization for 59 infants was $173,160 each. The median duration of home oxygen therapy was 92 days; the median cost was $5,195, compared with a projected cost of $46,920 for hospitalization for the same period. Two thirds of the 59 families experienced increased financial stress associated with marital status, reduced income, type of health insurance, and/or lack of respite or nursing help. Emotional stress was assessed in 26 (44%) of the families; one half coped well. Parents' perception of adequate insurance and stable income was significantly associated with positive coping. Providing home care for an infant with BPD on oxygen therapy is rewarding in many respects, but success requires appreciating its financial and emotional impact on families and providing them with social and financial support.

Bronchopulmonary dysplasia: a review for the pediatrician.

In this review we have attempted to introduce bronchopulmonary dysplasia as a new chronic lung disease of infancy and childhood. The major risk factors for this illness are preterm birth and the respiratory distress syndrome. The precise etiology of BPD is not understood but trauma from mechanical ventilation and toxicity from exposure to supplemental oxygen are thought to be important. Problems in diagnosis and diagnostic criteria have been discussed as have the details of the unfavorable pulmonary mechanics. We have mentioned some of our own practices in regard to a large and successful home oxygen therapy program. Suggestions have been made for establishing readiness for discharge and for follow-up of these children. Medical management of these patients presently suffers from a lack of prospective and controlled studies. Medical care draws heavily from experience with pediatric asthma. What is known about the long-term outcome of these children has been reviewed with an attempt to highlight controversies between published reports and underscore the need for further investigation. The greatest future success in this area would be the prevention of premature birth. Prior to this, we must await the completion of future controlled and prospective studies.