## 1 Introduction  
Colorectal cancer is the second leading cause of cancer death in the world [1]. For the treatment of this disease, surgery with adjuvant chemotherapy is a standard treatment in clinical practice; however, small nodules that are not easily visible with the naked eye are reportedly a significant poor prognostic factor [2]. Therefore, detection and treatment of such small nodules are important for improving the survival of colorectal cancer patients [2]. Small submillimeter tumors are useful tools for developing novel medicines and methods for detecting or treating small cancer nodules [3]. However, the establishment of useful in vivo tumor xenograft models that are applicable to study submillimeter tumors is challenging [4]. In conventional studies, in vivo xenograft mouse models of subcutaneous tumors approximately 5 mm in size (65 mm\({}^{3}\) in volume), made by subcutaneous injection of colorectal cancer cell suspension, have been generally used. Such models serve as powerful tools to investigate treatment efficacy and drug delivery to tumors because it is easy to observe tumor growth and dynamics in vivo using imaging technology [5; 6]. Previous studies using these models have provided many drugs for treating primary solid tumors [5; 6]. However, a conventional subcutaneous injection of cancer cell suspension makes it difficult to construct submillimeter tumors because cancer cells cannot localize in very small regions [7]. On the other hand, some in vivo mouse models, such as intraperitoneal disseminated models and metastatic models, have been used to study submillimeter tumors of colorectal cancer [8; 9; 10]. These models are constructed by intraperitoneal or intravenous injection of cancer cell suspension and are important for understanding behavior of submillimeter tumors in organs such as, the peritoneal cavity, liver, lung, etc. [8; 9; 10]. However, in these models, in vivo observation of the growth of each tumor, especially of submillimeter tumors, is difficult, and tumor growth regulation is challenging.  
Thus far, various in vitro 3D culture systems have been reported as practical tools to generate uniform multicellular cancer spheroids [11; 12; 13; 14; 15]. Here, we hypothesized that transplantation of a single spheroid cultured using the in vitro 3D culture system would become a core to create a subcutaneous submillimeter tumor xenograft in vivo. For this purpose, we adopted our newly developed radiation-crosslinked gelatin hydrogel microwell (rGHM) arrays [16; 17] which can be used as scaffolds for both spheroid culture and transplantation. The rGHM arrays are produced by applying a radiation-molding technique, which forms micropatterned radiation-crosslinked gelatin hydrogels [16; 17]. In rGHM, gelatin is used as the base material because it is a hydrolyzed form of collagen, the extracellular matrix's main constituent. The radiation-molding technique requires no additives or crosslinkers and, thus, does not alter the biocompatibility and biodegradability of gelatin. Although the sol-gel transition of native gelatin occurs around the mammalian body temperature, the rGHM arrays made of crosslinked gelatin hydrogel do not melt even at 50 \({}^{\circ}\)C and can be stably used for cell culture. Another advantage of rGHM arrays that distinguishes them from other similar technologies is their applicability as a transplantation scaffold. The rGHM arrays allow one to produce single spheroids in the respective rGMMs and transplant them together.  
This study aimed to establish an in vivo xenograft mouse model of a subcutaneous submillimeter tumor of human colorectal cancer HT-29 cells expressing red fluorescent protein (RFP) (HT-29-RFP), using subcutaneous transplantation of a single spheroid made of cancer cells together with rGHM as a scaffold.