Membrane, Chorioallantoic

The chorioallantoic membrane (CAM) assay represents a suitable and reliable alternative xenograft system to assess the in vivo growth and aggressiveness of tumour cells.33, 34 To analyse micro‐tumours formed by the HCSC enriched HepG2 subclones in comparison to those of their parental cell line, HepG2, clone 3 and clone 5 cells were grown on the CAM of fertilized chicken eggs. For this, specific pathogen‐free (SPF) eggs (VALO BioMedia, Osterholz‐Scharmbeck, Germany) were bred in an incubator at 37°C at a relative humidity of ~80%. On day 8 of chicken embryo development a window (Ø ~1‐1.5 cm) was cut into the more rounded pole of the eggs and the egg shell membrane was removed. The windows were re‐sealed with silk tape (Durapore^TM, 3M) and the eggs were incubated for another day. Then 1 × 10⁶ cells were embedded in Matrigel (Corning^® Matrigel^® Basement Membrane Matrix, 356237; 1:1 mixture with medium; total volume 40 μL per pellet) and the resulting plugs were placed onto the CAM of the developing embryos (day 9 of incubation). Micro‐tumours with their surrounding CAM were harvested 5 days after engraftment (day 14 of embryonic development), imaged ex ovo and the tumour volume was determined as follows by assuming an ellipsoid shape: V_Tumour = length × width × height × 0.52.35 Finally, the CAM micro‐tumours were fixed in 4% phosphate buffered formalin for 24 hours, dehydrated and embedded in paraffin.

Freshly fertilized eggs of domestic chicks (Gallus gallus), of the Ross 308 (Aviagen) strain, were obtained from a local commercial hatchery (Agricola Berica, Montegalda (VI), Italy), placed in a cold room at 4 °C and maintained in a vertical position for 24–72 h. The eggs were then placed in the dark and incubated at 37.5 °C and 60% relative humidity, with rocking. The first day of incubation was considered embryonic day 0 (E0). Fertilized eggs were then selected by a light test on E14 before injection. Chick embryo injection was performed according to previous reports^{30 (link)}. Briefly, a small hole was made on the egg shell above the air sac, and 35 μmoles of VPA (Sodium Valproate, Sigma Aldrich) were administered to each fertilized egg, in a volume of 200 μl, by dropping the solution onto the chorioallantoic membrane. Age-matched control eggs were injected using the same procedure with 200 μL of vehicle (double distilled injectable water). After sealing the hole with paper tape, eggs were placed in a rocking incubator (FIEM srl, Italy) until E18, when eggs were placed in a hatching incubator (FIEM srl, Italy). Hatching took place at a temperature of 37.7 °C, with 60% humidity, as previously described^{16 (link)}. The day of hatching was considered post-hatching day 0 (P0). All subsequent procedures were performed in complete darkness, so that the chicks remained visually inexperienced until the moment of test. Two independent samples of chicks were used for the two experiments described.

The chick embryo tumor growth assay was performed as previously described [38 (link)] with slight modifications. According to the French legislation, no ethical approval is needed for scientific experimentations using oviparous embryos (decree n° 2013-118, February 1, 2013; art. R-214-88). Animal work was done under animal experimentation permit N° 381029 to jean Viallet and animal experimentation permit N° B3851610001 to Institut Albert Bonniot. Briefly, Fertilized White Leghorn eggs (Société Française de Production Agricole, St.-Brieuc, France), were incubated at 38°C with 60% relative humidity for 10 days. At stage E10, the chorioallantoic membrane (CAM) was dropped by drilling a small hole through the eggshell into the air sac and a 1 cm² window was cut in the eggshell above the CAM. Cultured MDA-MB-231-GFP were detached by trypsinization, washed with complete medium and suspended in serum free DMEM. A 50 µl inoculum of 1 X 10⁶ MDA-MB-231-GFP cells was added onto the CAM of each egg (eggs were randomized in 4 groups of 15). One day later, tumors that began to be detectable were treated every second day at E11, E13, E15 and E17 by dropping 100 µl of either OME (300 µg/mL or 450 µg/mL), colchicine (2µM) or 0.02% ethanol (vehicle) in PBS onto the tumor. At E19 the upper portion of the CAM was removed, transferred in PBS and the tumors were then carefully cut away from normal CAM tissue and weighted. In parallel, a 1cm² portion of the lower CAM was collected to evaluate the number of nodules, containing GFP-expressing cells. The fluorescent nodule were visualized in situ using whole mounts of tissue fixed in 4% formaldehyde in PBS and flattened between a hollow glass slide and a thick coverslip. In order to number the nodule, a thorough and complete visual scan of the piece of the lower CAM was done using fluorescent microscope. Chick embryos were sacrificed by decapitation.

Fertilized eggs were hatched at 37℃ for 7 days, on the eighth day, alive chicken embryos were checked. Then the egg sheet was gently peeled to expose the air chamber. The allantoic membrane was removed and placed with a silicone ring. Next, eggs were treated with control IgG or IgG mut-B2 and cultured at 37℃. After three days the blood vessels of embryos were captured by microscope and digital camera. The vascular and chorioallantoic membrane regions were assessed using the IMAGE J software. The angiogenic area was calculated as a percentage using the following formula: vascular area/CAM area ×100%. The experiments were repeated in triplicate.

We measured the serum concentration of circulating T₄ at several developmental stages in chicks to examine the effects of MMI injection. Blood was sampled by puncturing the medial metatarsal vein in the lower leg 1, 12, or 24 h after hatching (PBS, n = 10; MMI, n = 9). At E20, blood was sampled by cutting the blood vessels of the chorioallantoic membrane 12 h before hatching when embryos were externally pipped (PBS: n = 7; MMI: n = 6). Blood serum was isolated by centrifuging whole blood at 5,000 × g for 25 min at room temperature (approximately 24°C) and was stored at −30°C until T₄ analysis. Serum T₄ levels were measured using an immunoassay kit (Abor Assays, Ann Arbor, MI, United States; Cat. No. K050-H1) following the manufacturer’s protocol (Mishra et al., 2017 (link); Prabhat et al., 2020 (link)). Briefly, 5 µL of serum samples were incubated with dissociation reagent at a 1:1 dilution for 60 min at room temperature (approximately 25°C). Next, assay buffer was added to obtain a sample volume of 850 μL at a final dilution of 1:170. Next, we pipetted 100 µL of each standard and diluted samples to designated wells, as well as 125 μL and 100 µL of assay buffer to the non-specific binding and maximum binding wells, respectively. Then, 25 µL each of T₄ conjugate and antibody was added (except for the non-specific binding wells), and the plate was incubated at room temperature (approximately 25°C) on a plate shaker at 700 rpm for 1 h. After washing the plate four times with a wash buffer, 100 µL of TMB (3,3′,5,5′-tetramethylbenzidine) substrate was added to each well, and the plate was incubated for another 30 min without shaking at room temperature (approximately 25°C). After adding 50 µL of stop solution to each well, the plate was read at 450 nm using an iMark microplate reader (Bio-Rad Laboratories, Hercules, United States).