Abstract Collagen sponge-gel histoculture was developed by Leighton (In: Encyclopedia of Life Sciences. John Wiley and Sons, Ltd.: Chichester, 2010, Published Online. DOI: 10.1002/9780470015902.a0002573.pub2). In the 1950s, Leighton showed that placing cells in histoculture enables them to form 3-dimensional structures. Because of its architectural resemblance to native tissue, sponge gel histoculture represents a unique in vivo-like model to study cancer-cell behavior (Brenner's Encyclopedia of Genetics, 2nd Ed., Vol. 7, pp. 73-76. Elsevier, 2013). For example, Leighton observed that when C3HBA mouse mammary adenocarcinoma cells were grown on sponge-matrix histoculture, the cells aggregated similar to the original in vitro tumor. Distinct structures were formed within the tumors such as lumina and stromal elements, with some of the glandular structures similar to the original tumor (above-reference). We have further developed sponge gel histoculture using Gelfoam to grow tumors, nerves, hair follicles, skin with growing hair (above-reference). In the present report, we use fluorescence ubiquitination cell cycle indicator (FUCCI) imaging and Gelfoam® collagen sponge gel histoculture to demonstrate that the cell cycle phase distribution of cancer cells in Gelfoam® and in vivo tumors is similar whereby only the surface cells proliferate and interior cells are quiescent in G1/G0. This is markedly contrary to 2D culture where most cancer cells cycle. This observation explains, at least in part, the resistance of solid tumors to chemotherapy which targets only cycling cells. Citation Format: Shuya Yano, Shinji Miwa, Sumiyuki Mii, Yukihiko Hiroshima, Fuminari Uehara, Hiroyuki Kishimoto, Hiroshi Tazawa, Ming Zhao, Toshiyoshi Fujiwara, Robert M. Hoffman. Three-dimensional Gelfoam® histoculture enables cancer cells to mimic in vivo cancer cell cycling as visualized with FUCCI imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1979. doi:10.1158/1538-7445.AM2014-1979