Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo

Abstract

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

Keywords: cancer spheroids; cell culture; grafting; skin; tissue engineering; tissue-engineered substitutes; tumor.

Figure 1

Schematic Representation of the Two Tumorigenic Assays Tested. (A) Schematic representation of the subcutaneous injection and (B) healthy tissue-engineered skin substitute (TES) and cancerous tissue-engineered skin substitute (cTES) method.

Figure 2

Evaluation of Mass Development After Injection In Vivo. (A–C) Evaluation of mass growth over time following subcutaneous injection of 10 million HeLa cells (A), healthy keratinocytes (B) or healthy fibroblasts (C). (D,E) Representative pictures of the tumor formed in HeLa cell-injected mice (D) or residual mass formed by keratinocyte-injected mice (E) after 3 months. (F) Representative injection site without any visible mass in fibroblast-injected mice after 3 months. (G,H) Hematoxylin and eosin staining of the mass formed in HeLa- and keratinocyte-injected mice.

Figure 3

HeLa Spheroid Size Analysis. (A) HeLa spheroids’ appearance and diameter before seeding on dermal sheets (day 0); (B) macroscopic appearance of cTES cultured 14 days at the air–liquid interface and spheroid diameter before grafting; (C) macroscopic appearance of TES produced with healthy cells.

Figure 4

Appearance of TES or cTES and Tumor Weight After Grafting. (A) Macroscopic appearance of healthy TES at day 7, day 14 and day 21 after grafting; (B) macroscopic appearance of cTES at day 7, day 14, day 21 and day 28 after grafting; (C) tumor weight of cTES (in grams) at sacrifice for the four grafted mice (day 28 or day 43 after grafting).

Figure 5

Histological Analysis Before and After Grafting. (A) Hematoxylin and eosin (H&E) and Masson trichrome (MT) staining of cTES before grafting. Hematoxylin and eosin staining (H&E) and immunofluorescence staining against HLA-ABC (green) of the cTES graft site 28 days after grafting. Arrows point to the epidermal remnants in cTES. Cell nuclei were stained with Hoechst (blue). (B) Hematoxylin and eosin staining (H&E) of healthy TES before and after grafting. E: epidermis; D: dermis; T: tumor.

Figure 6

Histology for Metastasis Analysis of Mice Injected with HeLa Cells or Grafted with cTES. (A) Hematoxylin and eosin staining of organs (lung, lymph node, brain, kidney, spleen and liver) from mice grafted with cTES or injected with HeLa cells. (B) HLA-ABC (red) immunofluorescent staining of organs from mice grafted with cTES or injected with HeLa cells. Nuclei were stained with Hoechst (blue). The primary tumor of mice grafted with cTES was used as a positive control for HLA-ABC staining.