|Home | About | Journals | Submit | Contact Us | Français|
Doxycycline (Dox) controlled Tet systems provide a powerful and commonly used method for functional studies on the consequences of gene overexpression/downregulation. However, whereas Dox delivery in tissue culture in vitro is relatively simple, the situation in vivo is more complex. Several methods of Dox delivery in vivo have been described—e.g., in drinking water containing alcohol, in drinking water containing various concentrations of sucrose, and in feed. Unfortunately there are no reports directly comparing the advantages and disadvantages of these diverse methods, and there is no generally accepted standard. We therefore compared four non-invasive methods of Dox delivery in vivo—in drinking water, by gavage, as a jelly, and in standard feed. To assess the delivery of Dox by these methods, we used a subcutaneous xenograft model based on colorectal carcinoma cells engineered for Dox-inducible expression of an activated mutant of c-Src and the luciferase reporter gene. Our results indicate that feed represents the most favorable method of Dox administration.
Human cancer cells grown as subcutaneous xenografts in immunodeficient mice represent one of the most frequently used in-vivo models for drug-target validation and preclinical drug testing in translational cancer research.1 These cells are often engineered for inducible expression/suppression of a given gene of interest to enable a more precise assessment of its function(s).2–6 Inducible gene expression/suppression based on the use of doxycycline (Dox) controlled Tet systems provides a powerful and commonly used method for functional studies on the consequences of gene overexpression/downregulation.7,8 However, whereas Dox delivery in tissue culture in vitro is relatively simple, the situation in vivo is more complex. Several methods of Dox delivery in vivo have been described—e.g., in drinking water containing alcohol,9 in drinking water containing diverse concentrations of sucrose,2,3,6,10,11 and in feed.4,5,12,13 Unfortunately, there are no reports directly comparing the advantages and disadvantages of these diverse methods, and there is no generally accepted standard.
We therefore compared four non-invasive methods of Dox delivery in vivo—in drinking water containing sucrose, by gavage, as a jelly, and in standard feed (see Table 1). To assess the delivery of Dox by these methods, we used a subcutaneous xenograft model based on the recently described HCT116 Luc-Src527F HighX45 colon carcinoma cells. These cells were engineered for simultaneous, Dox-inducible expression of an activated mutant of c-Src (c-SrcY527F) and a firefly-luciferase reporter gene.5 Xenografts were grown by subcutaneous injection of 5 × 106 cells in 0.1 mL of medium into the mid-dorsal region of the back of 6- to 8-wk-old female Balb/c-NUDE nude mice (Paterson Institute for Cancer Research, Manchester, UK). Mice were housed in individually vented caging systems on a 12 h light/12 h dark environment and maintained at uniform temperature and humidity. They were fed a standard diet of irradiated feed (Cat. No. 2018, Harlan-Teklad, Madison, WI) and allowed water ad libitum. When tumors reached 200 mm3 they were treated with Dox by each method for the periods shown in Table 1. As Dox has limited stability in water, the supply was changed every other day.14
At various times after the start of treatment, mice were killed by cervical dislocation and tumors immediately excised and frozen at −80°C. They were then ground in liquid nitrogen and ~50mg added to 200μL of lysis buffer (Cat. No. E153A; Promega, Madison, WI, for luciferase assay and Cat. No. 9803; Cell Signalling, Danvers, MA, for SDS-PAGE). Samples were sonicated at 10 μ for 3 sec on ice and clarified by centrifugation for 30 min at 20,000g and 4°C. Supernatant was assayed for protein content using a Bradford assay (Cat. No. 500-0006, Bio-rad, Hamel Hempstead, UK) and samples were adjusted to 2 μg/μL protein content with the respective lysis buffer. Luciferase assays and SDS-PAGE were carried out as described previously.15
Initially, Dox in water + 1% sucrose (the latter added to increase palatability) was administered for 48 h. While this gave good induction of c-SrcY527F and luciferase (Figure 1A and B), the mice became noticeably dehydrated, which was evident by a change in skin elasticity, a cyanotic appearance, apparent “thinning” of the skin, and weight loss. To assess whether the observed weight loss was associated with the presence of the tumor, non-tumor-bearing mice were given 1% sucrose water + 2mg/mL Dox. After 72 h, they also became severely dehydrated (>20% loss of body weight) and the experiment had to be curtailed due to ethical guidance restraint (Figure 1C). These observations indicate that dehydration and weight loss were associated with the route of Dox administration rather than the presence of the tumor. Importantly, a similar dehydration effect associated with Dox delivery in drinking water has been reported by others in a different mouse strain, indicating that it may represent a more general phenomenon rather than a strain-specific artifact.6 Dox solutions of varying concentrations administered by gavage over 48 h caused induction of c-SrcY527F similar to that seen with Dox administered via drinking water, but did not result in dehydration. Luciferase induction levels for Dox administration via either method were not significantly different (Figure 1A, B, and Table 1). Over 7 d, all gavage, jelly, and feed regimens were able to elicit induction of c-SrcY527F and luciferase in xenograft tumors (Figure 2A and B). Luciferase induction levels for feed and jelly regimens were not significantly different from those seen for gavage, with the exception of Dox delivered in feed at 200 mg/kg, which was significantly lower. Mice did not become dehydrated under any gavage, jelly, or feed regimen (Figures 1, ,2,2, and Table 1).
Our results indicate that Dox delivery to xenograft tumors is achievable by a variety of methods. Dox-regulated xenograft systems for inducible gene expression are widely reported in the literature and often use drinking water as the route of Dox administration, sometimes for prolonged periods of time.2,3,10,11 We demonstrated that although this administration route is effective, it may lead to animal dehydration. This may result in a significant increase in animal suffering (unacceptable by many ethical guidelines) and compromise the experimental results. A primary use of inducible xenograft models in pharmacological research is the assessment of the effects of gene induction/suppression on tumor growth and drug responses. Dehydration and weight loss subsequent to administration of Dox via drinking water represent a significant confounding factor, as the tumor weight relative to the weight of the animal will alter, in addition to the absorption, distribution and excretion pattern of any drug under investigation.
Dox administered to xenograft-bearing mice by gavage, as a jelly, or in feed ensures good induction without visible dehydration. The latter has been successfully used over longer time courses than studied here,4,5 and is easiest to handle logistically. We therefore conclude that feed represents the most convenient method of Dox delivery for inducible gene expression studies in the subcutaneous xenograft model.
All procedures were carried out in accordance with UKCCCR guidelines 1999 by approved protocols (Home Office Project licenses 40/2746 and 40/2328). This study was supported by program grants from Cancer Research UK (C147) and the Medical Research Council (G0500366) to C.D. and I.J.S., respectively. The authors have no competing interests.