Contributions of cell kill and posttreatment tumor growth rates to the repopulation of intracerebral 9L tumors after chemotherapy: an MRI study

Abstract

The drought of progress in clinical brain tumor therapy provides an impetus for developing new treatments as well as methods for testing therapeutics in animal models. The inability of traditional assays to simultaneously measure tumor size, location, growth kinetics, and cell kill achieved by a treatment complicates the interpretation of therapy experiments in animal models. To address these issues, tumor volume measurements obtained from serial magnetic resonance images were used to noninvasively estimate cell kill values in individual rats with intracerebral 9L tumors after treatment with 0.5, 1, or 2 x LD10 doses of 1,3-bis(2-chloroethyl)-1-nitrosourea. The calculated cell kill values were consistently lower than those reported using traditional assays. A dose-dependent increase in 9L tumor doubling time after treatment was observed that significantly contributed to the time required for surviving cells to repopulate the tumor mass. This study reveals that increases in animal survival are not exclusively attributable to the fraction of tumor cells killed but rather are a function of the cell kill and repopulation kinetics, both of which vary with treatment dose.

Figure 1

Demonstration of the mathematical analysis of tumor growth and treatment. Data points are actual values obtained for a 9L tumor treated with a 2 × LD₁₀ BCNU dose. The line marked A is the least-squares fit of the pretreatment growth points to Eq. 1. The line marked C is the fit of the posttreatment growth points. Line C is extrapolated back to the time of treatment to give V_post. Log cell kill is the log of the ratio V_pre/V_post (Eq. 2). The line marked B is the theoretical tumor regrowth curve assuming Td_post = Td_pre. The relative contributions of cell kill and tumor growth rates are related to the calculated repopulation intervals, Tra_pre and Tra_post, as described by Eqs. 3 and 4. Tx, point of BCNU administration; Tra_pre, repopulation interval given equivalent pretreatment and posttreatment growth rates; Tra_post, repopulation interval given the fitted posttreatment growth rate; V_pre, tumor volume at Tx; V_post, theoretical “viable” tumor volume immediately after treatment determined by extrapolation of C.

Figure 2

A series of coronal T₂-weighted MR images of a rat harboring a 9L tumor in the right hemisphere (a) within 2 h after BCNU treatment (2 × LD₁₀) and (b–d) at 263, 433, and 775 h posttreatment, respectively. Each displayed image is from approximately the same region of the brain.

Figure 3

Tumor volumes obtained form serial MR images of four individual rats are displayed versus time after 9L cell implantation. In each case, the individual volume measurements are shown along with the line corresponding to the least-squares fit. (A) A representative plot of an untreated intracerebral 9L tumor that reveals the exponential growth characteristic (Td = 67, r² = 0.997) of this tumor over the life span of the animal. For BCNU-treated animals, the individual volume measurements are shown along with the pretreatment (solid) and posttreatment (dashed) exponential growth fits after treatment with (B) 0.5, (C) 1, and (D) 2 × LD₁₀ BCNU. Pre- and post-BCNU doubling times (Td) were (B) 53 and 70 h, (C) 55 and 86 h, and (D) 46 and 99 h, respectively, revealing a decrease in growth rate during posttreatment exponential tumor regrowth. Calculated log cell kill values were (B) 0.4, (C) 0.9, and (D) 1.6. In D, the arrows denoted by a, b, c, and d correspond with the rat brain MR images shown in Fig. 2 obtained at 330, 593, 763, and 1105 h, respectively.

Figure 4

Mean intracerebral 9L tumor doubling times (± SE) before BCNU administration (Td_pre; empty bars) and during the exponential regrowth phase (Td_post; filled bars) after BCNU treatment. Statistically significant differences found before and after BCNU treatment (P < 0.03) using a two-tailed paired Student’s t test are indicated (∗). In addition, differences between Td_post for each posttreatment group were found to be statistically significant (P < 0.05).

Figure 5

Mean in vivo log cell kill values (± SE) for intracerebral 9L tumors determined from serial volumetric MR tumor images as a function of BCNU dose. The line represents a least-squares fit to the data (y = 0.99x − 0.25, r² = 0.99).