In vivo Wnt pathway inhibition of human squamous cell carcinoma growth and metastasis in the chick chorioallantoic model

Abstract

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with poor overall survival. New therapeutic strategies that target specific molecular lesions driving advanced disease are needed. Herein we demonstrate the utility of the chicken chorioallantoic membrane (CAM) assay for in vivo human HNSCC tumor growth and metastasis and the tumor suppressive effects of a new chemotherapeutic agent.

Methods: We tested anti-metastatic effects of a WNT pathway inhibitor, WNT974 (also known as LGK974), which targets porcupine (PORCN) the palmityl-transferase that is essential for secretion of Wnt proteins. CAM assays were performed with 8 HNSCC cell lines: UM-SCC-1, UM-SCC-10A, UM-SCC-10B, UM-SCC-11A, UM-SCC-14A UM-SCC-17A, UM-SCC-17B, UM-SCC-25, and UM-SCC-34.

Results: UM-SCC-1 (University of Michigan Squamous Cell Carcinoma cell line) CAM xenografts contain CD44+ and ALDH+ cancer stem cell (CSC) proportions similar to UM-SCC-1 mouse xenografts supporting the applicability of the CAM assay for study of CSCs. Inhibition of WNT signaling by the PORCN inhibitor WNT974 reduced metastatic spread of UM-SCC cells, especially in UM-SCCs with Notch1 deficiency.

Conclusions: Our data demonstrate decreased tumor growth and metastases in tumors from cell lines that showed in vitro responses to WNT974, providing evidence that this agent may have a role in future HNSCC therapy.

Keywords: Cell lines; Chorioallantoic membrane; Human squamous cell carcinoma; In vivo cancer model; NOTCH1 mutation; UM-SCC; WNT pathway inhibition; WNT974.

Fig. 1

CAM xenografted UM-SCC cell lines metastasize to the liver. This figure compares the relative content human DNA in the tissue as determined by amplified human Alu DNA sequences. The values were calculated by comparing the Alu qPCR cycle threshold (Ct) of a given liver specimen to the organs of non-xenografted control animals. Each qPCR reaction was completed in quadruplicate. Error bars are standard error of the mean

Fig. 2

Analysis of ALDH positive cells in UM-SCC-1 CAM xenografts. a Flow cytometry of unstained sample of UM-SCC-1 primary tumor cells grown in the CAM assay. b Flow cytometry of UM-SCC-1 CAM xenograft tumor cells stained with Aldefluor substrate and DEAB inhibitor. The Aldefluor substrate only reacts with the mammalian ALDH enzyme, so inclusion of the substrate will allow the human cells to shift forward while the chick cells will show no shift from the unstained sample. c Removal of the DEAB inhibitor results in a right-shift of the ALDH+ population. 1.55 % of the total cell population is ALDH+, but only 18.97 % of the total cell population is human cells. Therefore, 8.17 % of the UM-SCC-1 cells in the CAM xenograft were analyzed to be ALDH+

Fig. 3

Analysis of CD44 positive cells in UM-SCC-1 CAM xenografts. a Flow cytometry results of unstained sample of UM-SCC-1 primary tumor cells grown in the CAM assay. b Flow cytometry results of UM-SCC-1 primary tumor cells stained with CD44-APC antibody. 15.44 % of the total cell population is CD44+, but only 18.97 % of the total cell population is human cells. Therefore 81.39 % of the UM-SCC-1 cells in the CAM xenograft are CD44+

Fig. 4

WNT974 blocks UM-SCC-1, -11A and -25, but not -14A, CAM xenograft growth. UM-SCC cell lines were implanted on CAM models and treated with 1 μM WNT974 or vehicle control (DMSO) every other day for eight days. At the conclusion of the experiment, tumors were harvested from the CAM and weighed. We assessed 22, 33, 24 and 27 viable animals UM-SCC-1, -11A, -25 and -14A, respectively, with half receiving vehicle control and the rest WNT974. Averages of all groups are shown along with standard error. ** P < 0.05

Fig. 5

WNT974 blocks liver metastasis of UM-SCC-11A and UM-SCC-25 CAM xenografts. Livers from UM-SCC-11A and -25 CAM xenografts (from Fig. 4) were harvested at the conclusion of the experiment and assessed for human ALU DNA sequences by qPCR. WNT974 was administered every other day to a final concentration of 1 μM. Livers from non-xenografted animals were used as a negative control and Ct difference from the median centered average of normal livers is shown. All qPCR assays were run in quadruplicate. ** P < 0.05