Abstract
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 (n = 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 (n = 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.
Highlights
Colorectal cancer is the second leading cause of cancer death in the world [1]
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 radiation-crosslinked gelatin hydrogel microwells (rGHMs) as a scaffold
On day 0, cell aggregation was observed at the bottom of the well after the HT-29-RFP cells were seeded on the rGHM arrays (Figure 2, left column)
Summary
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; small nodules that are not visible with the naked eye are reportedly a significant poor prognostic factor [2]. In vivo xenograft mouse models of subcutaneous tumors approximately 5 mm in size (65 mm 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]. 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. In these models, in vivo observation of the growth of each tumor, especially of submillimeter tumors, is difficult, and tumor growth regulation is challenging
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