br Metastatic Tumors Develop in the Abdominal
Metastatic Tumors Develop in the Abdominal Wall of NSG Mice With Intraperitoneal Ovarian Cancer Following Puncture of the Abdominal Wall
Based on our findings in wild type C57BL/6 mice, we sought to assess if metastasis could be established in immune compromised mice as well. This has relevance because immune modulated mice can be used to elucidate the role of certain immune cell groups on the establishment and growth of metastasis. NSG mice were inoculated with ID8-Luc Z-Guggulsterone as previously described and sacrificed 3–4 weeks following abdominal wall puncture. The biopsy specimens were processed and
H + E slides were reviewed with pathology [Figure 4A]. Similar to the immunocompetent C57BL/6 mice, NSG mice had intra-abdominal wall tumor metastases specific to the puncture site. In total, tumor was
Translational Oncology Vol. 12, No. 1, 2019 Port site metastasisWilkinson-Ryan et al. 65
Figure 2. Puncture sites and control sites can be easily processed for histologic evaluation. Once mice were sacrificed and the abdominal hair was removed the puncture site was identified by either a scar or palpable tumor nodule at the injection site (just medial to the nipple). If no scar or tumor nodule was identified, the area medial to the nipple was marked (white arrow, A). The abdominal wall was then excised
(B) and a Keyes punch biopsy was used to take a biopsy of a control site remote from the right or left lower quadrant (red arrow) and a biopsy of the puncture site (blue arrow). The black marker was visible transperitoneal if the abdominal wall specimen was placed skin side down on a white surface and this was used to guide the biopsy of the puncture site.
identified in 12/18 (70% ± 10.0) NSG puncture site biopsies and 0/18 (0%) of control site biopsies (P b .002, CI 42.24–97.76) [Figure 4B]. Thus, we determined that puncture site metastasis can be established and replicated in immune compromised mice. This now enables the study of the role of various immune components in establishment of port site tumor metastasis by comparing frequencies and rates of metastasis in mice with various immune deficiencies and the contributions of specific subsets of immune cells.
Metastatic Intra-Abdominal Wall Tumors are Infiltrated With Immune Cells
In order to expand our understanding of how puncture site metastasis can be used for immunologic studies, including potential immunotherapies, we performed immune staining to detect the presence of immune cell populations within the metastasis. Abdominal wall specimens with evidence of invasive disease as assessed by H + E were stained with anti-murine CD3 and CD11b antibodies in order to detect T cells and leukocytes, predominantly macrophages, respectively. Robust populations of CD3+ cells and CD11b + cells were detected within the stroma surrounding metastatic intra-abdominal wall tumor [Figure 5A]. Evidence of CD3+ and CD11b+ cells were identified in 4/4 samples tested (100%) [Figure 5B]. Based on these findings we determined that T-
cells and leukocytes infiltrate the abdominal wall puncture site metastases. The presence of immune cells within the stroma of puncture site metastases confirms the potential utility of this model for studying the immune related mechanism of tumor cell infiltration and implantation that promotes and facilitates metastases.
Advancements in the study of EOC require development of clinically relevant animal models. Here we describe the establishment and reproducibility of a port site metastasis model of EOC based on the clinical pathophysiology of port site metastasis. The advantage of the model described in this study is that it can be used in an immunocompetent mouse and therefore can be used to study the role of immune infiltrates in the development of port site metastasis as well as the efficacy of new immunotherapies. To develop this model we leveraged ID8 cells which are derived from C57BL/6 mice and are therefore syngeneic to mice on a BL/6 background. Moreover, ID8 tumors have been widely used in syngeneic murine ovarian cancer models . Based on our findings, syngeneic puncture site metastasis can be used to study the role of immune cells in metastasis formation and the effectiveness of immunotherapies .