FIBRONECTIN DISTRIBUTION IN HUMAN BONE-MARROW STROMA - MATRIX ASSEMBLY AND TUMOR-CELL ADHESION VIA ALPHA-5-BETA-1 INTEGRIN

Tumor cell interactions with fibronectin (FN) are important for the de velopment of secondary tumors inside the bone marrow stroma. We studie d and compared the in situ distribution of FN in paraffin-embedded hum an bone marrow sections and investigated the in vitro regulation of FN assemblage by bone marrow stromal cells (BMSC). Finally, the role of FN in the interaction of BMSC with tumor cells was studied. Fine elong ated FN-positive cell extensions, probably of stromal cell origin, wer e observed as well as a limited amount of extracellular FN deposits in connective tissues around capillaries and sinusoids. In vitro studies , using the confocal laser scanning microscope, showed that BMSC produ ced a high amount of FN with a characteristic extracellular matrix for mation in an extensive network. FN matrix formation was predominantly detected at contact sites between cultured BM:SC. In in vitro cultures with low cell concentrations and in vivo with a limited number of str omal cell contacts only limited matrix was found. From previous studie s it is known that the alpha 5 beta 1 integrin is involved in the regu lation of FN assembly. Here the role of the alpha 5-subunit of this in tegrin was investigated. By using two different monoclonal antibodies (mAb) against the alpha 5-subunit (2H6 and mAb16) the assembly of endo genous FN was completely blocked, indicating that these antibodies are directed against the active epitope. Another mAb (mAb11) against the alpha 5-subunit did not affect the FN assemblage. Codistribution analy sis of alpha 5-subunits, alpha v-subunits, actin, and FN demonstrated that the alpha 5 beta 1 integrin is associated with FN and not with in tracellular actin. Integrins alpha v beta 1, alpha v beta 3, and alpha v beta 5, also ligands of FN, did not colocalize with FN. Codistribut ion of alpha v with the terminal ends of actin and not with FN indicat es that alpha v-subunits are mainly directed to vitronectin rather tha n to FN. The dominant role of alpha 5 beta 1 in FN interaction is unde rlined by effective blocking of tumor cell adhesion with BMSC using an ti-alpha 5, anti-beta 1, and anti-FN antibodies. These results emphasi ze the important role of alpha 5 integrin subunit in FN matrix assembl y in human BMSC and an exclusive role of alpha 5 beta 1 in the anchora ge and regulation of FN-mediated adhesion processes in the bone marrow . (C) 1997 Academic Press