## 4 Discussion  
We established a xenograft mouse model of a subcutaneous submillimeter tumor of HT-29-RFP cells by transplanting a single spheroid cultured in biocompatible and biodegradable rGHM in a SCID mouse. By using a stereoscopic fluorescence microscope, submillimeter subcutaneous tumors were easily observed, and tumor growth could be monitored in vivo using this model. Considering that the model established in this study showed uniformity in the tumor shape and timing of the formation of submillimeter xenografts, this model would be a useful tool to develop novel methods and screen drugs for diagnosis and treatment of submillimeter tumors of colorectal cancer. Our subcutaneous submillimeter tumor xenografts were uniformly developed in SCID mice, but not in nude  
Figure 5: Observation of mice transplanted with HT-29-RFP spheroids cultured on rGHM in nude mice. (**A**) Representative images of a mouse transplanted with HT-29-RFP spheroids with rGHM, which showed no tumor formation on day 0, 14, and 21, respectively (nude mouse 1). This pattern was observed in three out of four mice (75%). (**B**) Representative images of a mouse transplanted with HT-29-RFP spheroids with rGHM, which showed tumor formation on day 0, 14, 21, 28, 30, and 35, respectively (nude mouse 2). This pattern was observed in one out of four mice (25%). Bright-field and red fluorescence channels are shown in left and right rows, respectively. mice. The success rate of transplantation of human tumor cells in these mice has been reported to be higher than that in the nude mice due to the mutations present in SCID mice [18; 19]; therefore, our findings are in agreement with the expectations. This study used a well-characterized human colorectal cancer cell line HT-29 [20], which would be an advantage to use HT-29 in this model. To date, in vivo subcutaneous xenograft mouse models made by subcutaneous injection of cancer cell suspension have been widely used in many studies on colorectal cancer because of the convenience of such model creation and an easy observation in vivo [5; 6]. However, the traditional subcutaneous injection of cancer cell suspension is not possible in forming submillimeter tumors in mouse models. In this study, we found that the goal of forming uniform submillimeter tumors in vivo can be successfully achieved by the transplantation of a single spheroid together with rGHM into SCID mice. In this method, the subcutaneous transplantation of a single spheroid was easily performed using rGHM as a scaffold for both spheroid culture and transplantation. We observed that the gel under the skin disappeared a few days after transplantation, suggesting that rGHM underwent enzyme-mediated degradation in vivo, which facilitated nutrition and blood supply from the mouse body to the transplanted spheroids and activated tumor formation. Given these advantages, rGHMs would be useful not only for generating multicellular cancer spheroids but also for developing mouse models with uniform subcutaneous submillimeter tumors. Although rGHM transplantation is more invasive than the subcutaneous injection of cancer cell suspension, the model established in this study has the advantage of producing submillimeter tumors. As in other conventional models, mouse models constructed by intraperitoneal or intravenous injection of cancer cell suspension have been widely used to study disseminated metastatic small nodules of colorectal cancer [8; 9; 10]. These dissemination models are useful tools because they can accurately imitate the disease condition when small tumors are disseminated to organs. However, in vivo identification and observation of each tumor lesion are quite difficult when using these models. The model established in this study achieved in making easily observable and uniformly growing submillimeter tumors by subcutaneous transplantation of a single spheroid cultured in rGHM.