## Results
### Observation of an In Vivo Model of a Subcutaneous Submillimeter Xenograft Tumor  
#### 3.2.1 Experiment 1  
In this experiment, transplantation of spheroids either with or without rGHM in SCID mice was successfully performed (\(n=4\)). No body weight loss caused by the surgical procedures was observed in any of the examined mice. For the transplantation of a single spheroid, rGHM was helpful for easy handling and position stabilization of the spheroid, which was more difficult to achieve without rGHMs. We observed that the appearance of the gel under the skin completely disappeared by day 7 after transplantation. Figures 3 and 4 show the in vivo observation of tumor growth after transplantation of HT-29-RFP spheroids either with or without rGHM in SCID mice. The in vivo observation was performed using a stereoscopic fluorescence microscope. From the images acquired at day 0 (just after spheroid transplantation) and day 14, no tumor RFP signals were detected at any spheroid transplantation sites either with or without rGHM (Figure 3). From the images acquired at day 21, RFP signals of small tumors from all spheroid transplantation sites were observed in the examined mice; the tumor major axis was 0.55 \(\pm\) 0.20 mm (coefficient of variation (CV) = 0.37) and 1.48 \(\pm\) 1.33 mm (CV = 0.90) and tumor volume was 0.08 \(\pm\) 0.06 mm\({}^{3}\) (CV = 0.70) and 3.94 \(\pm\) 5.80 mm\({}^{3}\) (CV = 1.47), for the spheroids transplanted with or without rGHM, respectively; the spheroids transplanted together with rGHM formed subcutaneous submillimeter xenograft tumors at day 21 that showed lower variability in tumor major axis and volume (with a smaller CV) than those formed from spheroids transplanted without the rGHM (0.41-fold in tumor major axis and 0.48-fold in tumor volume). The tumors were actively grown thereafter, as shown in Figures 3 and 4. There was no significant difference in the tumor growth curve between the groups with or without rGHM (Figure 4). From the measurements of isolated tumor weight at day 42, there was no significant difference in tumor weight, but the CV in the group with rGHM was 0.47-fold smaller than that in the group without rGHM (Figure 4). These results indicated that the use of rGHM in the transplantation of spheroids helped construct uniform subcutaneous submillimeter xenograft tumors growing uniformly in vivo.  
Figure 3: Observation of in vivo xenograft mouse model of a subcutaneous submillimeter HT-29-RFP tumor in SCID mice. (**A,B**) Representative images of a mouse transplanted with HT-29-RFP spheroids either with or without rGHM, respectively, on day 0, 14, 21, 28, 35, and 42. Bright-field and red fluorescence views are shown in left and right rows, respectively.  
Figure 4: Measurement of tumor parameters in in vivo xenograft mouse model of a subcutaneous submillimeter HT-29-RFP tumor in SCID mice. (**A**,**B**) Tumor major axis and volume (left and right) at day 21 with or without rGHM, respectively (\(n=4\)/group). CV = coefficient of variation. There are no significant differences between the two groups in tumor major axis and volume (a 2-tailed \(t\)-test), while the values of CV in the group with rGHM are smaller than those in the group without rGHM (0.41-fold in tumor major axis and 0.48-fold in tumor volume). (**C**) Changes of tumor volume over time. The Y axis is scaled to focus on tumor growth at the time point after day 21. There is no significant difference in the tumor growth curve between the two groups (two-way analysis of variance). Weight measurement in isolated tumors at day 42 after transplantation of HT-29-RFP spheroids in SCID mice. (**D**) Values of the tumor weight in the group with or without rGHM at the transplantation of spheroids. CV = coefficient of variation. There is no significant difference in tumor weight between the two groups (a 2-tailed \(t\)-test), while the CV in the group with rGHM is 0.47-fold smaller than in the group without rGHM.