Establishment of an In Vivo Xenograft Mouse Model of a Subcutaneous Submillimeter HT-29 Tumor Formed from a Single Spheroid Transplanted Using Radiation-Crosslinked Gelatin Hydrogel Microwell

Colorectal cancer is a frequent cause of death worldwide. The detection and treatment of small nodules are crucial for improving survival of colorectal cancer patients. Submillimeter tumors are useful tools for developing novel methods to approach this issue. However, there are no suitable in vivo models that allow easy monitoring of the growth of these tumors. This study established a xenograft mouse model of subcutaneous submillimeter tumors with human colorectal cancer HT-29 cells. We transplanted a single spheroid formed by HT-29 cells expressing red fluorescent protein (RFP) (HT-29-RFP). Additionally, we adopted our newly developed radiation-crosslinked gelatin hydrogel microwells (rGHMs), which can be used as a culture base to form spheroids and as a transplantation scaffold with biocompatibility and biodegradability. Spheroids approximately 700 μm in size were uniformly created in seven days in the respective rGHMs. Every single spheroid was extracted either with or without rGHM and transplanted into the subcutis of severe combined immunodeficiency (SCID) mice (<i>n</i> = 4). After 21 days, the spheroids inoculated together with rGHM successfully formed uniform subcutaneous submillimeter tumor xenografts that were observable in vivo in a stereoscopic fluorescence microscope in all transplanted mice. In contrast, spheroids transplanted without rGHM also developed small tumors in all mice but showed higher variability in size than those transplanted with rGHM. During transplantation, the rGHM ensured easy handling and stabilization of the position of a single spheroid. Inoculation of spheroids with rGHM in the nude mice was similarly examined (<i>n</i> = 4), showing that only one out of four mice formed tumors. In conclusion, rGHM effectively formed spheroids and created uniformed xenografted submillimeter tumors of HT-29-RFP in SCID mice. Our model could provide a useful platform to develop medicines and methods for detection and treatment of small nodules of colorectal cancer.