Noninvasive assessment of tumor cell proliferation in animal models

Abstract

Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, and noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, and proliferation of these cells in irradiated severe combined immunodeficiency (SCID) mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1x10(3) cells in the peritoneal cavity, 1x10(4) cells at subcutaneous sites and 1x10(6) circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1x10(6) cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, and this method will accelerate development of novel therapeutic strategies.

Figure 1

Kinetics of tumor cell growth following intraperitoneal injection of tumor cells. HeLa-luc cells were injected into SCID mice and the animals were imaged weekly for 28 days. (A) One representative animal injected with 1x10⁵ HeLa-luc cells is shown. Substrate was administered IP and signals were obtained with 5 minute integration times. Days 0 to 14 are displayed at a 0–3 bit range, days 21 and 28 at the less sensitive 0–7 bit range to optimize anatomical localization. (B) Quantification of tumor signals was obtained for the three cell concentrations tested. Data are plotted as mean photon counts from three animals per group, with respect to time.

Figure 2

Temporal analysis of tumor cell growth at subcutaneous sites. HeLa-luc cells were injected at four discrete sites on the dorsum of each of three SCID mice at concentrations of 1x10⁶ (A) or 1x10⁴ cells per site (B). Data were obtained on the day of injection and serially every 7 days until day 28. Images are presented at a bit range of 0–3. Additionally, images of the mouse injected with 1x10⁶ cells are also displayed at the less sensitive 0–7 bit range for better anatomical localization of the signal source. Quantification of the tumor signals was obtained for each injection site, and the data plotted as mean photon counts, with respect to time (C). Data were obtained with 5 minutes integration times. These data indicated that growth kinetics of the HeLa-luc cells could be monitored at subcutaneous sites over time.

Figure 3

Localization of tumor signal following intravenous injection. Representative animals that received between 1x10⁶ and 1x10⁷ HeLa-luc cells via IV injection are shown. Signals were apparent from the lungs initially, and then at a variety of anatomical sites at later times. Images, displayed here at a bit range of O–3, were obtained at 2 and 7 days after injection of cells.

Figure 4

Probability of survival of SCID mice bearing the HeLa-luc tumor. The survival of 15 SCID mice following IP injection of 1x10⁴ HeLa-luc cells was monitored over time.