Circadian Influences on Chemotherapy Efficacy in a Mouse Model of Brain Metastases of Breast Cancer

Abstract

Chemotherapy is more effective in the treatment of peripheral tumors than brain metastases, likely reflecting the reduced ability of chemotherapy to cross the blood-brain barrier (BBB) and blood-tumor barrier at efficacious concentrations. Recent studies demonstrate circadian regulation of the BBB. Thus, we predicted that optimally timed chemotherapy would increase anti-tumor efficacy in a model of brain metastases of breast cancer (BMBC). First, we characterized novel daily alterations in BBB permeability to a commonly used chemotherapeutic, ¹⁴C-paclitaxel, within BMBC following injections given at four time points across the day. Peak and trough ¹⁴C-paclitaxel concentrations within BMBC occurred during the mid-dark phase and at the beginning of the light phase, respectively. Notably, chemotherapy injections during the dark phase increased cell death within BMBC and delayed onset of neurological symptoms relative to injections during the light phase. These data provide strong evidence for the beneficial effects of chrono-chemotherapy for the treatment of BMBC.

Keywords: blood-brain barrier; brain metastases; breast cancer; chronotherapeutic; circadian rhythm.

Figure 1

Daily alterations in ¹⁴C-paclitaxel within JIMT-1BR3 BMBC. (A) When examining all tumors independently, mice receiving paclitaxel injections at ZT14 or ZT17 demonstrated significantly increased ¹⁴C-paclitaxel concentrations within BMBC relative to mice receiving paclitaxel injections at ZT0 or ZT5. (B) When examining animal averages, planned comparisons demonstrate an increase in ¹⁴C-paclitaxel concentrations within BMBC at ZT17 relative to ZT0 or ZT 5. (C) Additionally, when combining groups and assessing animal averages based on activity, mice that received paclitaxel injections during their active phase displayed significantly increased ¹⁴C-paclitaxel concentrations within BMBC. (D) Representative cresyl violet and autoradiography images for ZT0 and ZT17. [(A) n= 122, 269, 185, 142 respectively; (B) n= 9, 9, 9, 8 respectively; (C) n= 18, 17 respectively]. The data are presented as mean +SEM. Graph bars that do not share a letter are statistically significantly different at p<0.05. ^#Main effect of time of injection; (A) nonparametric Kruskal-Wallis test; Dunn’s multiple comparisons test (B) one-way ANOVA; Fisher’s LSD multiple comparisons test (C) unpaired t-test.

Figure 2

Optimal timing of chemotherapy delays onset of neurological symptoms and increases cell death within JIMT-1BR3 BMBC. (A) Mice harboring BMBC and receiving paclitaxel injections at ZT17 (ie TC-ZT17, n=15) demonstrated prolonged onset of neurological symptoms relative to mice harboring BMBC and receiving paclitaxel injections at ZT0 (i.e., TC-ZT0, n=16). Mice receiving paclitaxel injections alone did not display neurological deficits irrespective of administration time (i.e., C-ZT0, n=14 and C-ZT17, n=15). Additionally, mice harboring BMBC and receiving paclitaxel injections at ZT17 demonstrated significantly prolonged (B) mean and (C) median number of days until the onset of neurological symptoms and significantly increased (D) cell death with BMBC relative to ZT0. (E, F) Representative images of cell death within BMBC at ZT0 and ZT17. For negative controls see Supplemental Figures 1 and 2 . Green- cytokeratin 5/8/GFP, Red- cleaved caspase-3. (B. n= 16,15 respectively; (C) n= 16,15 respectively; (D) n= 11, 11 respectively). The data are presented as mean +SEM for (B, D) and median +95% CI for (C). Graph bars that do not share a letter are statistically significantly different at p<0.05. * Log-rank p<0.05. (A) Log-rank test (B) unpaired t-test (C) Mann Whitney test (D) unpaired t-test.

Figure 3

Optimal timing of chemotherapy delays onset of neurological symptoms in 4T1-BR5 murine model of BMBC. (A) Administration of paclitaxel at ZT17 (i.e., TC-ZT17, n=13) prolonged the onset of neurological symptoms relative to mice harboring BMBC and receiving paclitaxel injections at ZT0 (i.e., TC-ZT0, n=13). Additionally, mice harboring BMBC and receiving paclitaxel injections at ZT17 demonstrated significantly prolonged (B) mean and (C) median number of days until the onset of neurological symptoms relative to ZT0. The data are presented as mean +SEM for (B) and median +95% CI for (C). Graph bars that do not share a letter are statistically significantly different at p<0.05. * Log-rank p<0.05. (A) Log-rank test (B) unpaired t-test (C) Mann Whitney test.

Figure 4

Timing of Paclitaxel Administration Does Not Alter Sleep/Wake States in Mice Harboring JIMT-1BR3 BMBC. Sleep/Wake states did not significantly differ between (A) baseline, (B) three days following the first paclitaxel injection, or (C) three days following the second paclitaxel injection. There was an unexpected power outage following the first paclitaxel injection, day 1 ZT4-8. Additionally, whereas in both groups there was an increase in (D) total sleep, (E) sleep during the daytime, and (F) sleep during the nighttime in the three days following the second paclitaxel injection relative to baseline sleep, these effects were not specific to one group. Further, there was no change in the (G) diurnal ratio between groups. (A–C). n= 8,7 respectively; (D–F). n= 8,6 respectively; (G) n=7,5 respectively). The data are presented as mean +SEM. (A, B) Two-way repeated measures ANOVA; Fisher’s LSD multiple comparisons test. (C) Mixed effect analysis; Fisher’s LSD multiple comparisons test. (D–G) unpaired t-test.