(W1030-04-23) Promising Mouse Models for Premetastatic Niche

Purpose: The aim of this study was to examine the changes in the lungs of mice bearing subcutaneous B16F10 (mouse melanoma) tumors and Balb-neu-T mice bearing neu-driven mammary tumors, with the objective of utilizing these as pre-clinical models for therapies targeting the premetastatic niche (PMN).


Methods: B16F10 cell line, an aggressive mouse melanoma cell line, was cultured in vitro and then injected subcutaneously into C57BL/6 albino mice. Different groups of mice were sacrificed once primary tumor volumes reached ~100, 400, 800, and 1500 mm³. A part of the lung was digested to prepare a single-cell suspension for flow cytometric analysis. The remainder of the lung was formalin-fixed and then processed for immunohistochemistry (IHC) analysis. Female neu-T mice were monitored for the development and growth of mammary tumors from 4 weeks of age. Neu-T mice were sacrificed at 20 weeks, and endpoint (~36 weeks, when all 10 mammary tumors had developed). Wild-type mice with no tumors were used as controls. Lungs were processed as previously described. For flow cytometry, monocytic MDSCs (M-MDSCs) were identified as CD45⁺CD11b⁺Gr1⁺Ly6C⁺ while polymorphonuclear MDSCs (P-MDSCs) were identified as CD45⁺CD11b⁺Gr1⁺Ly6G⁺. Lungs from healthy mice without the primary tumor were used as controls. Additionally, MDSCs were also identified with anti-Gr-1 and anti-Ly6G antibodies by IHC.

Results: With B16F10 tumor-bearing C57BL/6 mice, a significant increase (p < 0.01) in P-MDSC counts was observed when the primary tumor was ~800 mm³ or greater (Figure 1b), while the number of M-MDSCs became significantly higher (p < 0.01) than the control when the primary tumors reached 1500 mm³ (Figure 1a). MDSC numbers did not increase significantly beyond 800 mm³. Both M-MDSCs and P-MDSCs showed a significant increase (p < 0.05) as a proportion of CD45⁺ immune cells (Figure 1c and 1d). IHC analysis (Figure 2) of the lung sections for total MDSCs (Gr1⁺) and P-MDSCs (Ly6G⁺) confirmed the increased frequency of MDSCs in the lungs of mice with 800 mm³ or larger primary tumors. With neu-T mice, P-MDSC counts increased with an increase in age and mammary tumors, while higher M-MDSC counts at 20 weeks of age were reduced at 36 weeks of age (Figure 3). IHC analysis of the lungs revealed a higher frequency of Gr-1 and Ly6G⁺ stained cells in tumor-bearing neu-T mice, confirming the flow cytometry results.

Conclusion: Significantly increased lung MDSC counts were observed when the primary tumor size reached 800 mm³ or higher in C57BL/6 mice or when neu-T mice developed all ten mammary tumors. These results suggest that the B16F10 tumor model and the transgenic neu-T model could be useful models to evaluate therapies targeting PMN. Our studies also point to the key role of MDSCs in the formation of the PMN and MDSCs as a potential therapeutic target for PMN disruption and anti-metastasis therapy.

References: [1] Wang Y, Ding Y, Guo N, Wang S. MDSCs: Key Criminals of Tumor Pre-metastatic Niche Formation. Front Immunol. 2019 Feb 7;10:172. doi: 10.3389/fimmu.2019.00172. PMID: 30792719; PMCID: PMC6374299.
[2] Hao, Z., Li, R., Wang, Y. et al. Landscape of Myeloid-derived Suppressor Cell in Tumor Immunotherapy. Biomark Res 9, 77 (2021). https://doi.org/10.1186/s40364-021-00333-5

Figure 1. Increase in lung MDSCs with the increase in primary tumor volumes. (a) lung M-MDSC count (b) lung P-MDSC count (c) lung M-MDSC proportion of CD45+ immune cells (d) lung P-MDSC proportion of CD45+ immune cells. Data represented as mean ± SD. Each symbol represents value from one mouse.

Figure 2. Lung MDSC results with increased primary tumor volume by Immunohistochemistry-paraffin. (a) lung total MDSCs (b) lung P-MDSCs. Data represented as mean ± SD. Each symbol represents data from one mouse.

Figure 3. Increase in lung MDSCs with the increasing age in neu-T mice. (a) lung M-MDSC count (b) lung P-MDSC count (c) lung M-MDSC proportion of CD45+ immune cells (d) lung P-MDSC proportion of CD45+ immune cells. Data represented as mean ± SD, n=3.