Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids

Abstract

The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogenous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.

DC electrical field-induced c-fos expression and growth stimulation in multicellular prostate cancer spheroids

“…The effects of radiation, chemotherapy or immunotherapy have been tested on MCSs (Soranzo and Ingrosso, 1988;Carlsson et al, 1989;Stuschke et al, 1993; Wartenberg and Acker, 1996). In comparison with single tumour cells grown in suspension or as monolayers, MCSs exhibit a higher complexity and therefore can be better compared with the situation in a threedimensional tissue (Carlsson et al, 1983).…”

Coupled cellular trafficking and diffusional limitations in delivery of immunotoxins to multicell tumor spheroids

“…Heterogeneous antigen expression has been reported in other small spheroids (e.g., Carlsson et al, 1989), and we postulated that our system may also be subject to receptor expression heterogeneity. TfR expression is related to cell iron and growth requirements, with more actively proliferating cells expressing more receptors (Gatter et al, 1983).…”

Penetration of Tumor Therapy Interesting Substances in Non-Vascularized Metastases: Review of Studies in Multicellular Spheroids

“…The method has previously been described in detail [18,21,22,23]. Briefly, after incubation with the radioactive labeled therapy substance, the spheroids were placed on cover slips, quickly frozen in liquid propane cooled by liquid nitrogen.…”

“…However, not much attention has been given to studies performed using the so-called "freeze drying method" [18][19][20][21][22], which has been extensively used at our laboratories since more than 25 years. This method allows spatial analysis of non-bound radiolabeled substances and, in combination with conventional histology and autoradiography techniques, gives possibilities to evaluate penetration in regions without using binding as a criterion for penetration.…”