Studying primary tumor-associated fibroblast involvement in cancer metastasis in mice

Abstract

Stromal cells have been studied extensively in the primary tumor microenvironment. In addition, mesenchymal stromal cells may participate in several steps of the metastatic cascade. Studying this interaction requires methods to distinguish and target stromal cells originating from the primary tumor versus their counterparts in the metastatic site. Here we illustrate a model of human tumor stromal cell-mouse cancer cell coimplantation. This model can be used to selectively deplete human stromal cells (using diphtheria toxin, DT) without affecting mouse cancer cells or host-derived stromal cells. Establishment of novel genetic models (e.g., transgenic expression of the DT receptor in specific cells) may eventually allow analogous models using syngeneic cells. Studying the role of stromal cells in metastasis using the model outlined above may take 8 weeks.

Figure 1

Design of the experiment. (a–d) The use of human tumor (carcinoma)-associated fibroblasts (CAFs) enables selective depletion of stromal cells in a spontaneous metastasis model in mice with DT treatment using the following steps: primary tumor inoculation using metastatic mouse cancer cells (red) and human CAFs (blue) (a); primary tumor resection (b); depletion of human CAFs using diphtheria toxin 1 d after resection (c); and metastases formation in the lungs (d). All animal procedures were performed according to the guidelines of public health service policy on humane care of laboratory animals and in accordance with an approved protocol by the institutional animal care and use committee of MGH.

Figure 2

Isolation of tumor-associated fibroblasts (TAFs) from breast cancer specimens. (a–c) Representative images of immunocytochemical staining for human vimentin (a), cytokeratin AE1-AE3 (b) and α-SMA (c), showing characteristic mesenchymal phenotype. Blue, DAPI nuclear stain (in a–c). Red, vimentin (in a) and α-SMA (in c). Images are 315 µm across.

Figure 3

Spontaneous metastasis of the passenger stromal cells in the coimplantation model. (a–c) Human CAFs coimplanted in the primary tumor site (a) and metastasizing to the lung (b,c). Immunohistochemical markers have been used to identify human cells using Cy3-labeled antihuman vimentin antibody (a,b) and FITC-labeled antihuman nuclear antigen antibody (c). Images are 700 µm across. Reproduced with permission from ref. . All animal procedures were performed according to the guidelines of public health service policy on humane care of laboratory animals, and in accordance with an approved protocol by the institutional animal care and use committee of MGH.

Figure 4

Contribution of circulating stromal cells to spontaneous tumor metastases. (a,b) Selective depletion of stromal cells in the circulating fragments using DT results in prolonged survival (survival experiment in a) and a decrease in the number of lung metastases (time-matched evaluation in b). *P < 0.05. Reproduced with permission from ref. . All animal procedures were performed according to the guidelines of public health service policy on humane care of laboratory animals and in accordance with an approved protocol by the institutional animal care and use committee of MGH.