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Historically, the late arrival of optical microscopy is something of a puzzle, although it may be a testament to the fact that the development of new technology is driven by the desire for scientific knowledge, and not the other way around. The explorations that began in the 17th century are by no means complete, but who is carrying them forward now?

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Cell line MDA 886Ln was established from a laryngeal lymph node metastasis. When grown as a multicellular tumor spheroid (MTS), it exhibits squamous differentiation. We studied the effects of beta-all-trans retinoic acid (RA) on the growth, differentiation and glycoprotein content of this MTS model for squamous carcinomas of the head and neck. The growth of MTSs was inhibited in a dose-dependent manner by 10(-6) to 10(-10) M RA. Growth inhibition occurred between 3 and 5 days of RA treatment (10(-6)M). Immunohistochemical and electrophoretic analyses revealed that RA suppressed the morphological markers of squamous differentiation (squames), involucrin expression, and keratin expression. Gly-coprotein expression was examined by metabolic labelling using 3H-glucosamine, in situ labelling of polyacrylamide gels with 125I-labelled wheat-germ agglutinin (WGA), localization of fluorescein isothionate-WGA in frozen sections, and determination of sialyltransferase activity. Treatment using 10(-6) M RA altered glycoprotein expression both biochemically and morphologically, and WGA was shown to bind preferentially to sialic acid residues. The sensitivity of this MTS model to RA treatment and its ability to be analyzed through morphological, immunohistochemical and biochemical techniques suggest that it will prove useful in studying the relationships between growth, differentiation and RA-induced alterations in squamous carcinomas.

Retinoic acid (RA) treatment of F-9 embryonal carcinoma cells resulted in cell flattening and increased production of laminin B1 chain, both indicating differentiation to endoderm-like cells. In addition, RA caused a time- and dose-dependent decrease in growth rate in monolayer culture and a dose-dependent decrease in the ability of the cells to form colonies in soft agarose. Differentiation was accompanied by an increase in the fucosylation of specific high-molecular-weight cellular and cell-surface glycoproteins. The fucosylation of glycoproteins of Mr 175,000 (gp175), 250,000 (gp250), and 400,000 (gp400) increased as early as 24 hr after the addition of 5 x 10(-6) M RA to the culture medium. These changes preceded both growth inhibition and the induction of laminin B1 expression, which were detected 48 to 72 hr after addition of RA. The increased fucosylation of these glycoproteins showed a distinct dose-response relationship. Both gp175 and gp250 showed the greatest increase in fucosylation at 10(-5) M, which was also the dose at which RA induced laminin maximally, while the fucosylation of gp400 was greatest at 10(-8) M RA and declined at higher concentrations. The overall synthesis of large fucosylated glycopeptides decreased in RA-treated cells, in spite of the increases in the fucosylation of specific cellular glycoproteins. RA-induced differentiation of F-9 cells was also accompanied by a time- and dose-dependent increase in fucosyltransferase activity. Although the functions of these glycoproteins are not currently known, the early increase in their fucosylation can be considered as a marker of differentiation in this system.

Tuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

Soluble endogenous lactoside-binding lectins, galectins, have been implicated in cell adhesion, growth, differentiation, neoplastic transformation, and metastasis. Two major classes of these lectins, galectin-1 and galectin-3, are developmentally regulated. To explore the mechanisms by which the expression of the galectins is regulated and to examine their association with the differentiation processes induced by all-trans retinoic acid (RA), dibutyryl cyclic AMP (Bt2cAMP) and their combination, we used the murine embryonal carcinoma (EC) cell line F9 and its RA-resistant mutant, RA-3-10. RA induced endodermal differentiation and a concurrent induction of galectin-1 and its complementary glycoconjugates (laminin and lysosomal-associated membrane protein, LAMP) in the F9 wild-type (wt) line, but failed to induce differentiation and had no effects on or even reduced the expression of galectin-1, laminin, and LAMP in the RA-3-10 line. On the other hand, RA inhibited expression of galectin-3 in the wild-type line but had no effect on the RA-3-10 line. The galectin-1 gene is at least partially regulated at the transcriptional level. These results demonstrate a parallel association between differentiation and induction of galectin-1, and inhibition of galectin-3 in F9 cells by RA. The study suggests that a regulated expression of galectins and their complementary glycoconjugates is involved in the differentiation pathway induced by RA in F9 cells.