The current study shows significant growth delay in anaplastic Dunning prostate R3327-AT1 tumors by administering CTX on a continuous low-dose (M-CTX) schedule, alone or in combination with Tha. Significantly, the data show physiological changes detectable noninvasively by MRI prior to changes in tumor volume. DCE MRI revealed a significant decrease in signal enhancement (AUC) starting on day 3 of treatment through day 7, and a good correlation between very low AUC (<0.1) on day 7 and tumor growth delay. In good agreement, histology confirmed that M-CTX alone or M-CTX + Tha treatment caused a huge central necrotic core, leaving just a thin peripheral region alive.
There are increasing applications of noninvasive imaging for in vivo
tumor diagnosis and prognosis [18,25–27
]. Given the noted tumor heterogeneity, and hence observed heterogeneous responses to treatment, it is vital to be able to assess early therapeutic response and in turn be able to individualize treatment regimens. Functional MRI permits noninvasive evaluation of tumor physiology; thus, MRI may be a useful tool to detect early treatment-induced changes prior to overt changes in tumor size. The detection of early changes following antiangiogenic or vascular targeting treatment may be particularly valuable.
There is increasing interest in using DCE MRI to assess tumor vascularization in both diagnostic and prognostic aspects. Strong signal enhancement on T1
-weighted images is reported to correlate with well-vascularized and highly permeable vessels [16,17
]. Indeed, some studies have indicated that DCE MRI is capable of detecting early changes in tumor vascular perfusion and permeability after treatment with antiangiogenic or vascular targeting agents [16,17
]. In the present study, we demonstrate that DCE MRI provides early indication related to the efficacy of M-CTX therapy (). Our results showed a significant decrease in AUC (tumor perfusion and permeability), especially in central regions, as early as 3 days after initiating combined M-CTX + Tha therapy. Furthermore, tumor size on day 18 is significantly associated with weakly enhancing fraction (AUC < 0.1) on day 7. This is in line with our histologic findings of increased avascular fractions due to central necrosis formation. These results also parallel a study by Klement et al. [2
], who found that a combination of continuous low-dose vinblastine and DC101 caused a 65% decrease in tumor perfusion within 14 days as assessed by histologic analysis of fluorescent perfusion marker.
Qualitative analysis of DCE MRI provides estimates of tumor perfusion and permeability based on the initial slope of the time-signal intensity curve or time-concentration product curve (IAUC) [17,28,29
]. In the present study, we applied a spin-echo multiple slice (SEMS) pulse sequence with 59 seconds acquisition time and integrated signal enhancement during the first 3 minutes for AUC analysis, which gave a lower temporal resolution than some published studies [17,29
]. It is possible that the longer acquisition time and extended AUC missed information regarding the “first pass” of the bolus contrast. However, comparison of AUC estimated over the first 60, 120, or 180 seconds showed essentially equivalent results. We have previously studied DCE MRI in the AT1 tumors by using echo planar imaging with fast acquisition time (4 seconds), which showed similar heterogeneity between tumor center and periphery [30
Diverse MRI techniques have previously been used to study early physiological changes after CTX administered at the higher conventional dose [28,31,32
]. Zhao et al. [31
] found significant changes in apparent diffusion coefficient (ADC) of water in RIF-1 tumors in mice 2 days after a single dose of 150 or 300 mg/kg CTX. Poptani et al. [28
] reported an increase in perfusion of RIF-1 tumors in mice 24 hours after a single dose CTX (300 mg/kg). For the present group of AT1 tumors, we routinely found significantly decreased perfusion in central tumor regions within 3 days and in the periphery by day 7 (; ), whereas control tumors showed no changes in the periphery, but did show change in the tumor center over 7 days (). It is critical to note that the changes are much less apparent when central and peripheral regions were combined for analysis ().
BOLD MRI or susceptibility-weighted R2
* measurement exploits the intrinsic paramagnetic properties of deoxy-hemoglobin. Recently, BOLD MRI has been used to assess tumor response to antiangiogenesis, vascular targeting, and photodynamic therapy agents [17,18,33
]. An increase in R2
* may relate to increased paramagnetic deoxyhemoglobin in tumor tissue, which could be caused by a reduction in tissue perfusion. Some of our treated tumors showed increased R2
* on days 1 and 3, but with return to baseline or even lower levels in our final MRI measurements on day 7. The transient change in R2
* may have resulted from decreased vasculature and increased thrombosis. However, tumor R2
* is also related to several other factors (e.g., necrotic tissue development) [19
]. Overall, R2
* neither correlated with growth in the control group nor with M-CTX or M-CTX + Tha therapy.
Significant increase in tumor apoptosis and evidence of apoptotic vascular endothelia in this study ( and ; ) indicated the antiangiogenic effect of M-CTX alone or M-CTX + Tha. These data coincide with other studies using antiangiogenic agents [34
] or M-CTX [35,36
]. There is increasing evidence that several conventional chemotherapeutic agents including CTX, doxorubicin, vinblastine, and paclitaxel have potential antiangiogenic activity against experimental and clinical cancers, especially administered at continuous low doses [5,37,38
]. Furthermore, recent studies have shown that M-CTX induced thrombospondin 1 expression, which can further enhance antiproliferative and proapoptotic effects [35,36
]. Tha has been recognized as an antiangiogenic agent, which inhibits neovascularization by suppressing TNF-α
, bFGF, and VEGF production [9,11
]. In addition, clinical studies have shown that Tha, alone or combined with chemotherapeutic agents, caused vascular thromboses [39
]. In the current study, we found increased thromboses in microvessels of treated tumors, compared with control tumors.
We observed a significant increase in VEGF expression in the tumors treated with M-CTX or M-CTX + Tha, as also reported in studies of several other antiangiogenic agents (e.g., TNP-470 and SU6668) [16,34
]. Colocalization of VEGF overexpression with hypoxia (pimonidazole staining; ) supports the hypothesis that tumor hypoxia may have mediated a feedback or compensatory increase of VEGF. Poptani et al. [28
] recently reported that RIF-1 fibrosarcoma treated with a single dose of CTX, 300 mg/kg, i.p., showed a significant decrease in tissue pO2
24 hours after treatment. The decrease in glycolytic rates and increase in oxidative metabolism observed in their study suggest that hypoxia resulted from more oxygen consumption by surviving cells rather than shortage of oxygen supply [32
]. In our current study, MVD evaluation by the “hot spot” technique revealed no significant change in vasculature of the tumor periphery between the treated and pretreated tumors or size-matched controls (). However, the overall fraction of vasculature decreased significantly and resulted in a huge avascular central necrotic area, evidenced by both histology and MRI, as also reported for other antiangiogenic studies [16
]. Important to this observation is the fact that AT1 tumors do not normally develop a central necrosis [21–23,40
]. In terms of tumor growth delay, M-CTX, alone or combined with Tha, significantly increased tumor volume doubling time (). Compared with the M-CTX alone, the combination with Tha in this study had an additional inhibitory effect on tumor growth, although the effect of Tha appears to be, at best, additional rather than synergistic. However, a recent phase II study conducted by NCI [41
] concluded that adding Tha to docetaxel resulted in an encouraging PSA decline rate and overall median survival rate in patients with metastatic AIPC. Another phase II trial [42
] also suggests that low-dose Tha (100 mg/kg, daily) may be an option for patients with AIPC. Others reported that this continuous low-dose regimen, if combined with other therapeutic approaches (e.g., antiangiogenesis or immunotherapy), could produce a synergistic effect in a variety of experimental tumors [3,4
]. The mode of action of Tha is complicated and its antiangiogenic effect is believed to be at least partly independent of VEGF. This might explain that the level of VEGF overexpression, accompanied with M-CTX treatment-induced hypoxia, was not reduced in the M-CTX + Tha group. Metronomic therapy has also been shown to be effective at overcoming drug resistance [6
]. In the future, it would be interesting to develop sublines of the Dunning prostate R3327 tumor resistant to traditional CTX doses and to investigate whether the metronomic approach would still be effective. Our higher dose CTX (150 mg/kg) schedule, which is equivalent to the clinical MTD dose, induced significant tumor growth delay during the first 12 days of treatment, but all six animals died after three doses. A similar observation has been reported by Man et al. [3
] that mice with MDA-MB-231 breast tumor xenografts died after three doses of 150 mg/kg CTX. Reduced toxicity of continuous low-dose therapy could be an important advantage and, recently, the combination of metronomic CTX with methotrexate has been reported to have a significant response in patients with advanced or recurrent breast carcinoma [5
In summary, continuous low-dose CTX, alone or combined with Tha, significantly inhibited tumor growth of the syngeneic rat prostate R3327-AT1 tumor with reduced toxicity compared with multiple conventional doses. Our results demonstrate that physiological changes in the early stage of this low-dose regimen can be detected by MRI. MRI revealed intratumoral heterogeneity in situ and in vivo, and changes in temporo-spatial dynamics, which may give an early indication of treatment response and ultimately allow scheduling, dosage, and drug combination to be optimized and individualized.