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Oxygenation and Differentiation in Multicellular Spheroids of Human Colon Carcinoma¹

University of Rochester Cancer Center and Departments of Radiation Biology and Biophysics and of Radiation Oncology, Rochester, New York [R. M. S.]; Swiss Institute for Experimental Cancer Research, 1066 Epalinges, Switzerland [B. S., J. B.]; and Departments of Pathology [H. G.] and of Applied Physiology [B. B., W. M-K.], University of Mainz, D-6500 Mainz, Federal Republic of Germany

Oxygenation and development of necrosis were evaluated in multicellular spheroids of poorly differentiated (HT29) and moderately well-differentiated (Co112) human adenocarcinoma of the colon. Spheroids were grown in vitro under well-controlled oxygen and nutrient conditions in spinner flasks up to sizes of 2800-µm diameter after 5 wk of culture. Morphological studies showed that the Co112 spheroids contained pseudoglandular structures with lumen, very similar to the characteristics of the original tumor specimen from the patient and to the cells when grown as xenograft tumors in nude mice. Microelectrodes were used to measure the oxygen tension (PO₂) profile within individual spheroids at different stages of growth. Histological sections through the centers of spheroids were measured to determine the thickness of the viable rim of cells surrounding spheroid necrotic centers in order to estimate the size of the severely hypoxic zone of cells by comparison with the PO₂ profiles of the same spheroids. The data demonstrate significant differences between these two human colon tumor spheroid systems. Both spheroid types exhibited steep PO₂ gradients at relatively small sizes of <600-µm diameter, but for any given size in this range, the more differentiated Co112 spheroids were more hypoxic. Although severe hypoxia (PO₂, <10 mm of Hg) was present in both spheroid types at larger sizes, there was a significant difference in the central PO₂ values which were between 5 and 10 mm of Hg in large Co112 spheroids but remained at or close to 0 mm of Hg in large HT29 poorly differentiated human colon tumor spheroids. The presence of pseudoglandular structures and lumen in the Co112 spheroids was associated with changes in the shape of PO₂ profiles. Such profiles have not previously been seen in other poorly differentiated human or rodent tumor spheroids. Furthermore, the PO₂ profiles of both of these human tumor spheroid types were often continuously curving with a very shallow gradient in the inner edge of the viable rim of cells surrounding the necrotic center. Regulation of oxygen consumption and/or diffusion in these inner regions of human spheroids could produce these continuously curving PO₂ gradients.

¹ This research was supported by NIH Grants CA 11051, CA 11198, and CA 20329; by Grants MU576/1 and MU576/2-1 from the Deutsche Forschungsgemeinschaft; and by the Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland.

² Supported by the Alexander von Humboldt Foundation by a Senior United States Scientist Award during part of the period during which this research was performed. To whom requests for reprints should be addressed.

Received 7/23/85. Revised 4/22/86. Revised 6/16/86. Accepted 6/20/86.

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