Non-invasive imaging of engineered human tumors in the living chicken embryo

Abstract

The growing interest in engineered tumor models prompted us to devise a method for the non-invasive assessment of such models. Here, we report on bioluminescence imaging (BLI) for the assessment of engineered tumor models in the fertilized chicken egg, i.e, chick chorioallantoic membrane (CAM) assay. One prostate cancer (PC-3) and two osteosarcoma (MG63 and HOS) cell lines were modified with luciferase reporter genes. To create engineered tumors, these cell lines were seeded either onto basement membrane extract (BME) or gelfoam scaffolds, and subsequently grafted in vivo onto the CAM. BLI enabled non-invasive, specific detection of the engineered tumors on the CAM in the living chicken embryo. Further, BLI permitted daily, quantitative monitoring of the engineered tumors over the course of up to 7 days. Data showed that an extracellular matrix (ECM) composed of BME supported growth of reporter gene marked PC-3 tumors but did not support MG63 or HOS tumor growth. However, MG63 tumors engineered on the collagen-based gelfoam ECM showed a temporal proliferation burst in MG63 tumors. Together, the data demonstrated imaging of engineered human cancer models in living chicken embryos. The combination of CAM assay and BLI holds significant potential for the examination of a broad range of engineered tumor models.

Evidence for immortality and autonomy in animal cancer models is often not provided, which causes confusion on key issues of cancer biology

“…The tests had been conducted, ever since 1901 [374,375], mainly with culture of surgically removed tumor tissues or with transplantation of the tissues to animals, the two modern techniques aforetime. Actually, a technique involving both transplantation and culture was done by inoculating tumor cells into a fertile egg and then hatching it [376][377][378][379][380][381][382][383][384][385][386], which is the parentage of some modern chick embryo assays for cancer research [387][388][389][390][391][392][393][394][395][396][397][398][399] such as the chick heart invasion assay [400][401][402][403][404][405]. The rationale for using tissue culture is that neoplastic cells are immortal and can self-renew to forever maintain themselves as a "new organism" by incessant cell division.…”

Multi-Modal PET and MR Imaging in the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Model for Initial In Vivo Testing of Target-Specific Radioligands

“…The method we used was similar to already published HET-CAM models [17,83,[91][92][93][94]. Fertilized chicken eggs (LSL Rhein-Main Geflügelvermehrungsbetriebe GmbH & Co.KG, Dieburg, Germany) were defined upon start of incubation on embryo development day 0 (EDD0).…”

High resolution, label‐free photoacoustic imaging of live chicken embryo developing in bioengineered eggshell

“…Experimental techniques for studying chicken embryo are well established and various methods have been reported for visualizing the embryo. Imaging the embryo through a window in the eggshell is a common method , but the small window size limits the view. This is overcome by transferring the embryo into a petri dish or explanting the embryo before imaging, which implies that the embryo needs to be sacrificed post‐imaging.…”

“…Limited imaging depth of confocal microscopy and OCT makes it difficult to view the embryo at later stages of development. Other imaging techniques like bioluminescence imaging (BLI) , intravital imaging , laser speckle imaging (LSI) , and high‐frequency ultrasound imaging are not widely used for vasculature imaging of chicken embryo as they either need external contrast agent to distinguish the embryonic tissues and organs, have poor imaging resolution or need complicated image processing algorithms to enhance the image quality. Providing the advantages of high ultrasound resolution and good intrinsic optical contrast, photoacoustic imaging (PAI) seems like a promising choice for chicken embryo imaging.…”