Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
Department of Surgery, YanBian University Hospital, Yanji, Jilin, China.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
Department of Pathology, Seoul National University Hospital, Seoul, Republic of Korea.
Department of Pathology, College of Medicine, Seoul National University, Seoul, Republic of Korea.
Department of Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea.
Division of Surgery, Thyroid Center, Seoul National University Cancer Hospital, Seoul, Republic of Korea.
Department of Molecular Medicine and Biopharmaceutical Sciences Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Republic of Korea.
Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States.
Abstract
INTRODUCTION Securing a well-established mouse model is important in identifying and validating new therapeutic targets for immuno-oncology. The C57BL/6 mouse is one of the most fully characterised immune system of any animal and provides powerful platform for immuno-oncology discovery. An orthotopic tumor model has been established using TBP3743 (murine anaplastic thyroid cancer [ATC]) cells in B6129SF1 hybrid mice, this model has limited data on tumor immunology than C57BL/6 inbred mice. This study aimed to establish a novel orthotopic ATC model in C57BL/6 mice and characterize the tumor microenvironment focusing immunity in the model.
METHODS Adapted TBP3743 cells were generated via in vivo serial passaging in C57BL/6 mice. Subsequently, the following orthotopic tumor models were established via intrathyroidal injection: B6129SF1 mice injected with original TBP3743 cells (original/129), B6129SF1 mice injected with adapted cells (adapted/129), and C57BL/6 mice injected with adapted cells (adapted/B6).
RESULTS The adapted TBP3743 cells de-differentiated but exhibited cell morphology, viability, and migration/invasion potential comparable with those of original cells in vitro. The adapted/129 contained a higher Ki-67+ cell fraction than the original/129. RNA sequencing data of orthotopic tumors revealed enhanced oncogenic properties in the adapted/129 compared with those in the original/129. In contrast, the orthotopic tumors grown in the adapted/B6 were smaller, with a lower Ki-67+ cell fraction than those in the adapted/129. However, the oncogenic properties of the tumors within the adapted/B6 and adapted/129 were similar. Immune-related pathways were enriched in the adapted/B6 compared with those in the adapted/129. Flow cytometric analysis of the orthotopic tumors revealed higher cytotoxic CD8+ T cell and monocytic-myeloid-derived suppressor cell fractions in the adapted/B6 compared with the adapted/129. The estimated CD8+ and CD4+ cell fractions in the adapted/B6 were similar to those in human ATCs but negligible in the original/B6.
CONCLUSION A novel orthotopic tumor model of ATC was established in C57BL/6 mice. Compared with the original B6129SF1 murine model, the novel model exhibited more aggressive tumor cell behaviours and strong immune responses. We expect that this novel model contributes to the understanding tumor microenvironment and provides the platform for drug development.
Figure 1In vivo passaging of TBP3743 cells. TBP3743 cells were subcutaneously injected into C57BL/6 mice and tumor growth were observed for 21 days in 1st and 2nd round, and 10-15 days in 3rd, 4th 5th, and 6th round of in vivo passaging. At each round, the two largest tumors were harvested, primary single cell culture were performed, and established cells were used for the next round of in vivo passaging. (A) Scheme for TBP3743 adaptation into C57BL/6 mice. (B) Tumor growth curves of the original, 3rd and 6th round from day 5 to 11. (C) The morphology of TBP3743 original or subclones (scale bars: 200 µm). (D) Thyroid differentiation genes were analyzed by real-time RT-PCR. To characterize TBP3743-original or -adapted cells, (E) Cell proliferation were measured by the CCK-8 assay at the indicated time, (F) Cell migration assay were performed using Transwell system (scale bars: 50 µm), (G) Cell invasion ability were measured by spheroid culture system. SC, subclone.Figure 2Establishment of orthotopic tumor model using TBP3743-B6 cells. B6129SF1 mice were implanted with 105 cells of each cell line (TBP3743-original and -adapted cells) and C57BL/6 mice were implanted with 105 cells of TBP3743-adapted. After 14 days post-implantation, tumors were harvested. (A) Representative image of tumors and, quantification of tumor volume and weight. (B) Representative image of H&E staining and Ki67 immunohistochemistry staining on each tumors (H&E upper panel scale bars: 50 µm; lower panel scale bars: 10 µm; Ki67 panel scale bar: 50 µm; lower panel scale bars: 10 µm). (C) Quantification of Ki67+ area in tumors. (D) FACS analysis of MHC class 1 on TBP3743-original, -adapted, and SC6 tumors. 129, B6129SF1; B6, C57BL/6. All data are expressed as mean ± SD.Figure 3Molecular characteristics of orthotopic tumors from the adapted TBP3743 cells. B6129SF1 mice were implanted with 105 cells of each cell line (TBP3743-original and -adapted cells) and C57BL/6 mice were implanted with 105 cells of TBP3743-adapted. After 14 days post-implantation, tumors were harvested. Total RNA was harvested from whole tumor lysates and sequenced. (A) Volcano plots showing changes of gene expression. (B) Heatmap of changes in oncogenic signalling pathways and (C, D) each of oncogenic pathways. (E) Proliferation of epithelial and mesenchymal stem cells and (F) cell migration analysis. 129, B6129SF1; B6, C57BL/6. All data are expressed as mean ± SD.Figure 4Molecular characteristics of the immune microenvironment in adapted TBP3743 cell-derived orthotopic tumors. B6129SF1 mice were implanted with 105 cells of each cell line (TBP3743-original and -adapted cells) and C57BL/6 mice were implanted with 105 cells of TBP3743-adapted. After 14 days post-implantation, tumors were harvested. Total RNA was harvested from whole tumor lysates and sequenced. (A) Classification of gene set enrichment analysis and (B) the networking of enriched pathways of immune category. (C) Heatmap of changes in immune response signalling pathways and (D) change of immune response to tumor and change of immune inhibitory signal. (E) Alteration of immune stimulatory related marker and immune inhibitory related marker. (F) B6129SF1 mice were implanted with 105 cells of TBP3743-original cells. After 10 days post-implantation, tumors were harvested. Anti-PD-L1 (0.2 mg/kg) was intraperitoneally injected from 5 days after tumor cell injection. Quantification of tumor volume and weight. (G) C57BL/6 mice were implanted with 105 cells of TBP3743-adapted cells. After 10 days post-implantation, tumors were harvested. Anti-PD-L1 (0.2 mg/kg) was intraperitoneally injected from 5 days after tumor cell injection. Quantification of tumor volume and weight. 129, B6129SF1; B6, C57BL/6. All data are expressed as mean ± SD.Figure 5Immune cell profiling of the orthotopic tumor microenvironment. B6129SF1 mice were implanted with 105 cells of each cell line (TBP3743-original and -adapted cells) and C57BL/6 mice were implanted with 105 cells of TBP3743-adapted. After 14 days post-implantation, tumors were harvested. (A-D) Immune cell fractions were analysed by flow cytometry analysis using whole tumor lysates. (A) Representative plots and quantified graphs of CD45+ cells in each tumors. Subpopulation of (B) T-lymphocytes (CD3+ pan T; CD8+ cytotoxic T; CD4+ helper T; and CD25+FoxP3+ regulatory T), (C) macrophages (CD11b+F4/80+ pan macrophage; CD11b+F4/80+CD80+CD206- M1-macrophage; and CD11b+F4/80+CD80-CD206+ M2-macrophage), and (D) myeloid cells (CD11b+ pan myeloid; CD11b+Ly6C+Ly6G- M-MDSC; and CD11b+Ly6C-Ly6G+ PMC-MDSC). (E) RNA sequencing data of 8 human ATC samples were collected from (8) and deconvolution analysis were performed for comparing immune cell fractions between mice tumors from each group and human ATCs. M-MDSC, mononuclear myeloid-derived suppressor cells; PMN-MDSC, polymorphonuclear myeloid-derived suppressor cells; 129, B6129SF1; B6, C57BL/6. All data are expressed as mean ± SD.Figure 6Effects of BRAFV600E inhibitor on TBP3743 cells. (A) Effect of PLX-4032 on TBP3743-original and -adapted cells were measured by cell viability assay. (B-D) TBP3743-adapted cells (1 × 105/10µL PBS) were injected into the murine thyroid via orthotopic injection, and mice were daily treated with 10 mg/kg of PLX-4032. At 2 weeks after injection, tumors were harvested. (B) bioluminescence imaging and (C) representative image of tumors, and quantification of tumor weight. (D) Representative images of IHC for T cell (CD3), helper T cell (CD4), cytotoxic T cell (CD8), macrophage (F4/80), and M2-macrophage (CD163) in each group tumors. scale bars: 50 µm. All data are expressed as mean ± SD.
그림 출처: PubMed Central 공개 논문 (PMC10403231)
Conflict of interest statement
SC is a co-founder of Cellus, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
