Characterization of a B16-F10 melanoma model locally implanted into the ear pinnae of C57BL/6 mice

Abstract

The common experimental use of B16-F10 melanoma cells focuses on exploring their metastatic potential following intravenous injection into mice. In this study, B16-F10 cells are used to develop a primary tumor model by implanting them directly into the ears of C57BL/6J mice. The model represents a reproducible and easily traceable tool for local tumor growth and for making additional in vivo observations, due to the localization of the tumors. This model is relatively simple and involves (i) surgical opening of the ear skin, (ii) removal of a square-piece of cartilage followed by (iii) the implantation of tumor cells with fibrin gel. The remodeling of the fibrin gel within the cartilage chamber, accompanying tumor proliferation, results in the formation of blood vessels, lymphatics and tissue matrix that can be readily distinguished from the pre-existing skin structures. Moreover, this method avoids the injection-enforced artificial spread of cells into the pre-existing lymphatic vessels. The tumors have a highly reproducible exponential growth pattern with a tumor doubling time of around 1.8 days, reaching an average volume of 85mm3 16 days after implantation. The melanomas are densely cellular with proliferative indices of between 60 and 80%. The induced angiogenesis and lymphangiogenesis resulted in the development of well-vascularized tumors. Different populations of immunologically active cells were also present in the tumor; the population of macrophages decreases with time while the population of T cells remained quasi constant. The B16-F10 tumors in the ear frequently metastasized to the cervical lymph nodes, reaching an incidence of 75% by day 16. This newly introduced B16-F10 melanoma model in the ear is a powerful tool that provides a new opportunity to study the local tumor growth and metastasis, the associated angiogenesis, lymphangiogenesis and tumor immune responses. It could potentially be used to test different treatment strategies.

Fig 1. Tumor inoculation method.

(A-B) Ventral view of an ear during surgery (A: dotted square = area from ventral skin flap reflected; small square = outline of the region of full thickness cartilage to be removed (4 mm²). Clot containing tumor cells (B: brown nodule) placed on the region from which cartilage removed. (C) A large black tumor growing on both sides of the ear (day 13). (D) Dorsal view of an ear showing a non-established tumor (greyish) (day 8). (E) Dorsal view of an ear established black tumor (day 11). (F) Time related changes in the mean percentage of “established tumors” as evaluated by color. The mean percentage of black tumors detected between day 8 and 11 based on the 5 different studies are represented by the error bars. Results for each individual study are represented by colored symbols. (G) Correlation plot between the percentage incidence of black tumors and their size as a function of time after trans-plantation. r: correlation coefficient. Data shown as mean ± SEM. *: p<0.05, ***: p<0.001, ****: p<0.0001.

Fig 2. Tumor growth and reproducibility.

(A) Changes in tumor volume as a function of time after implantation. Tumor growth curves are shown for the 5 different studies (each individual experiment is represented by a colored symbol). Data shown as mean ± SEM. (B) Tumor doubling times and the number of tumors (N) available for measurements, per repeat study, at day 8 (the highest number) the lowest number day 16.

Fig 3. Hemic tumor vascularization: Vascular density, vascular area and perfusion.

(A) Time related changes in the number of blood vessels per field of view on days 10, 13 and 16 after implantation. (B) Time related changes in the blood vessel surface area (in μm²) at 10, 13 and 16 days after implantation. (C) Time related changes in the percentages of perfused blood vessels. Each symbol represents the value for a single tumor, the horizontal lines mean percentage. (D) Concentration of VEGF-α produced in tumors at 10, 13 and 16 days after implantation. (E, F, G) Representative merged mosaics of CD31 and DAPI staining showing the blood vessels in tumors at D10 (E), D13 (F) and D16 (G) after implantation. White squares: Areas enlarged in Fig 3G, 3H and 3I. (H, I, J) Zoom in of pictures D (H), E (I) and F (J), represented by the white square representing the vascular density at higher magnification. (K, L) MicroangioCT of a melanoma on D13. (K) 3D reconstruction of the whole mouse ear and its vasculature including the implanted tumor (encircled). The main feeding vessels are coming from the base of the ear (blue arrows). (L) Virtual section through the tumor at higher magnification (= 3D-microangioCT dataset). A feeding vessel (blue arrow) is entering the tumor mass. Areas with dense network of smaller vessels (asterisks) and many bigger vessels (black arrows). Data shown as mean ± SEM. **: p<0.01. Scale bars: 1 mm (E, F, G) and 200 μm (H, I, J).

Fig 4. Lymphatic tumor vascularization: Density and area.

(A) Time related changes in the number of lymphatic vessels per field of view. (B) Time related changes in lymphatic vessel surface area per field of view (in μm²). (C, D, E) Representative merged mosaics of Lyve-1 (red) and DAPI (blue) staining showing lymphatic vessels in tumors on D10 (C), D13 (D) and D16 (E) after implantation. (F, G, H) Zoom in of white square in pictures D (F), E (G) and F (H) represented tumor lymphatics at higher magnification. Data shown as mean ± SEM. **: p<0.01. Scale bars: 1 mm (D, E, F) and 100 μm (G, H, I).

Fig 5. Quantification of tumor cell characteristics.

(A) Time related changes in the cell density obtained by the number of nuclei in an area of 10000 μm² of hematoxylin-eosin stained sections. (B) Time related changes in the percentage of KI67 positive cells among 100 Dapi-positive nuclei. (C, D, E) Representative merged images of KI67 and DAPI staining in frozen sections showing proliferating cells (Ki 67, red) among side blue Dapi-positive nuclei on D10 (C), D13 (D) and D16 (E) after implantation. (F, G, H) Zoom in of the white square in pictures A (F), B (G) and C (H) represented the cell density at higher magnification. Data shown as mean ± SEM. *: p<0.05, **: p<0.01. Scale bars: 100 μm (C, D, E) and 50 μm (F, G, H).

Fig 6. Characterization of macrophages and T cells.

(A, B, C) Representative plots for the identification of the macrophage population (A) and of their two subtypes, M1-like (B) and M2-like (C) macrophages versus days after implantation. (D, E) Percentage of macrophages (D) and macrophage subtypes (E) versus days after implantation. (F, G) T cells (CD3) identification strategy (F) for the CD4+ and CD8+ subpopulations (G). (H, I) Percentage of T cells among viable cells (H) and T cells subpopulations (I) among all T cells, both versus days after implantation. (J, K, L) Representative plots for identifying T regulatory cells (CD4+CD127-, J, CD25+, K) among T cells, versus days after implantation. (L). Data shown as mean ± SEM. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Fig 7. Metastases.

(A, B, C) Percentage mice with melanotic cervical lymph node metastases on D10 (A), D13 (B) and D16 (C). (D) Localization of sampled lymph nodes (red). (E, F, G, H) Representative pictures of cervical lymph nodes harboring different metastatic volumes. Each node illustrates the amount of metastatic black tissue that characterizes the semi-quantitative scores of 1 to 4. (I) Estimation of the metastatic volume based on the score in E, F, G and H pictures versus days after implantation. Data shown as mean score ± SEM. *: p<0.05.