Chick Embryo Chorioallantoic Membrane as a Platform for Assessing the In Vivo Efficacy of Chimeric Antigen Receptor T-cell Therapy in Solid Tumors

Abstract

The fertilized chicken egg chorioallantoic membrane (CAM), a highly vascularized membrane nourishing the developing embryo, also supports rapid growth of three-dimensional vascularized tumors from engrafted cells and tumor explants. Because murine xenograft models suffer limitations of time, cost, and scalability, we propose CAM tumors as a rapid, efficient screening tool for assessing anti-tumor efficacy of chimeric Ag receptor (CAR) T cells against solid tumors. We tested the efficacy of human epidermal growth factor receptor 2 (HER2)-specific CAR T cells against luminescent, HER2-expressing (FaDu, SCC-47) or HER2-negative (MDA-MB-468) CAM-engrafted tumors. Three days after tumor engraftment, HER2-specific CAR T cells were applied to tumors grown on the CAM. Four days post-CAR T cell treatment, HER2-expressing FaDu and SCC-47 tumors treated with CAR T showed reduced viable cancer cells as assessed by luciferase activity. This reduction in viable tumor cells was confirmed by histology, with lower Ki-67 staining observed in CAR T cell-treated tumors relative to T cell-treated controls. Persistence of CAR T in CAM and tumor tissue 4 days post-treatment was confirmed by CD3 staining. Altogether, our findings support further development of the chick CAM as an in vivo system for rapid, scalable screening of CAR T cell efficacy against human solid tumors.

FIGURE 1.

Preparation and CAR T cell treatment of the chicken egg chorioallantoic membrane. (A) Chicken egg and CAM anatomy. (B) Preparation methodology for tumor cell line engraftment on the CAM. (C) Experimental timeline.

FIGURE 2.

Establishment of tumor models on the CAM. Representative imaging of CAM-grown tumors harvested 7 days after engraftment of tumor cells. (A–F) Characteristics of tumors demonstrated in gross image (A), in vivo luminescence imaging (B), and representative tumor histology demonstrating tumor growth (C and D), HER2 expression (E), and location of vasculature (F) with H&E (C and F) and cytokeratin (D) staining. Scale bar, 100 microns.

FIGURE 3.

CAR T cell infiltration of CAM tissue. (A) Pan-cytokeratin staining of CAM tumors (PanCK). (B) CAR T cell detection in CAM tissue (CD3). The issues were collected on day 7. Scale bar, 100 microns.

FIGURE 4.

Quantification of tumor growth on the CAM following CAR T cell treatment. (A and B) Time course of luciferase activity of CAM tumors on experimental days 3 and 7 captured by IVIS imaging in T cell (A) or CAR T cell–treated (B) tumors. (C) Linear regression of T cell and CAR T cell–treated tumors, with statistical comparison of slopes. ***p = 0.0005, **p = 0.004. (D) Day 7 luciferase activity by IVIS imaging. ****p < 0.0001. (E) Representative regions of interest of pan-cytokeratin (PanCK) and Ki-67 staining on day 7 in T cell or CAR T cell–treated eggs. Scale bar, 200 microns. (F) Quantification of Ki-67 staining of CAM tumors by H-Score using Qupath. Three regions of interest selected per slide. *p < 0.05.

FIGURE 5.

Visual abstract. Overview of chick CAM modeling of solid tumor treatment with CAR T. Engrafted tumor cells are treated three days following engraftment, and harvested seven days after engraftment to characterize T cell infiltration. The visual abstract was created using BioRender.com.