A Babsky, Ju Shenghong - Apparent diffusion coefficient of waterin evaluation of treatment response in animal body tumors - страница 2

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Chinnayan et al. monitored the human breast MCF-7 cancer xenografts therapy with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [65]. TRAIL is a ligand molecule which induces caspase-8-dependent apoptosis. It is a type II transmembrane protein with homologous to other members of the tumor necrosis factor family that can binds to the death receptors, DR4 and DR5. The authors have found an increase in waterADC from 0.7x10-3mm2/s to 1.7x10-3mm2/s in combined TRAIL- and radiation-treated tumors seven days post-treatment but not in tumors treated with these therapies separately. Histological analysis confirmed a decrease in cellularity and activation of apoptotic activity in tumor cells after the TRAIL and radiation therapy.

Morse et al. treated the human breast MCF-7 and MDA-mb-231 cancer xenografts with the chemotherapeutic agents docetaxel (15 and 30 mg/kg). The main mode of therapeutic action of docetaxel is the suppression of microtubule dynamic assembly and disassembly, rather than microtubule bundling leading to apoptosis, or the blocking of Bcl-2 [66] Docetaxel has greater cytotoxicity than paclitaxel, possibly due to its more rapid intracellular uptake. MCF-7 cells are partially deficient for apoptosis, and MDA-mb-231 cells are not. The authors examined whether MRI-measured ADC is altered in response to therapies that induce cell death via non-apoptotic mechanisms and corre­lates ADC changes with cell death modalities regionally within the tumor. There was a post-treatment increase in tumor ADC that was generally associated with the central region of the tumor, which had low cellularity as determined by histology. The water ADC values from multiple slices in both MCF-7 and MDA-mb-231 tumors show an in­crease from about 0.5x10-3 mm2/s to values approaching 1.0 - 1.5x10-3 mm2/s two to four days post-treatment. The authors observed a general trend towards increasing waterADC with increasing dose. Both tumors showed a decrease in the number of pixels after treatment relative to carrier-treated controls, implying tumor shrinkage. These re­sults indicated that early and significant changes in ADC can be related to mitotic catast­rophe and lytic necrosis in the absence of apoptosis. The authors proposed to use changes in ADC as a generalized measure of cytotoxic reaction to chemotherapy.

Lemaire et al. examined the effect of 5FU (100 mg/kg, ip) on the waterADC in a rat mammary tumor induced by N-methyl-N-nitrosurea [44]. They showed that the 5FU-treated tumors were not distinguishable in terms of tumor volume change up to day 5,

apparent diffusion coefficient of water in evaluation of treatment response in animal body tumors

whereas the waterADC over the same period of time did distinguish the sensitive and non-sensitive groups. They found that mammary tumors with low initial ADC values re­sponded to a single 5FU bolus therapy by a 30% increase in ADC at day 7 after treat­ment, whereas tumors with high initial ADC showed a 30% decrease and tumors with intermediate values showed no significant change in ADC.

5. Subcutaneous Brain Tumor

Post-treatment changes in waterADC in rat intracranial glioma have been well descri­bed [62, 67-70]. In some studies 9L glioma was inoculated subcutaneously and thus it can be classified as a body gliosarcoma. The effects of alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on sc model of 9L glioma have been studied [38, 39, 41, 71]. BCNU or carmustine is a mustard gas-related a-chloro-nitrosourea compound used as an alkyla­ting agent that attacks the 0-6 group of guanine in DNA. It is a lipid-soluble drug that can penetrate into the central nervous system. BCNU is used in the treatment of several types of brain cancer (including glioma, glioblastoma multiforme, medulloblastoma, and astrocy­toma), multiple myeloma and lymphoma (Hodgkin's and non-Hodgkin).

Babsky et al. have shown that in sc-implanted 9L glioma, waterADC increased with growth in sham-treated control as well as non-responsive tumors (NRBCNU) after BCNU treatment [41]. The waterADC did not change significantly after effective treatment with BCNU (RBCNU) when the tumor growth was arrested (Fig. 3). The increase in waterADC with tumor growth may be due to an increase in ECS that may result from inefficient cell packing and aberrant and leaky vasculature with tumor growth. However, a previous study showed that the relative extracellular spaces, which were measured by destruc­tive chemical analysis, in untreated control and BCNU-treated tumors are identical [71]. Several researchers have shown decreases in tumor ATP/Pi and PCr/Pi ratios with un­treated growth [71-73]. This decrease in ATP level should decrease the activity of the Na+/K+-ATPase. On the other hand, Na+/H+ exchanger activity may increase because of increased glycolytic rates and intracellular acid production in poorly perfused and hypoxic tumor tissue. These two effects should increase [Na+]i with untreated growth. The de-

Fig. 3. Effect of 1,3-bis(2-chloroethyl)-1-nitro-sourea (BCNU) therapy on water ap­parent diffusion coefficient (ADC) in subcutaneously (sc) implanted 9L glio­ma. Control - non-treated animals, NRBCNU - BCNU non-responsive, RBCNU - BCNU responsive. Signifi­cance: p < 0.05 *(vs. before treatment), 1.6




1          1 1

Control "   ■ NRbcnu D Rbcnu


















1  ****   1           1           1           1           1 1

"(vs. Control), ****(Rbcnu vs. NRbcnu

1 3 Days after Therapy




Біологічні Студії / Studia Biologica • 2009 • Том 3/№1 С 3-24

cline in cellular energetics and the increase in [Na+]i could also result in altered water-macromolecular interactions and changes in cytoplasmic viscosity due to cell swelling. These changes could alter water ADC in the intracellular compartment as well. Another reason for an increase in waterADC in untreated sc glioma could be increased necrosis and cyst [74]. Lemaire et al. [44] showed that the waterADC of necrotic tissue is be­tween two- and three-fold higher than for non-necrotic tumor tissue. The observed in­creases in waterADC with growth of sc-implanted 9L glioma and interruption of these changes with BCNU therapy are in contrast to previous results reported for intracranial 9L glioma [52] and sc-implanted tumors (presented in Table), which show increases in waterADC aftertherapy. However, Dzik-Jurasz et al. [75] reported a decrease in water ADC for some patients with rectal adenocarcinoma after neoadjuvant chemoradiation, and Mardor et al. [76] showed that changes in waterADC varied from patient to patient after chemotherapy of human glioma. Moffat et al. [35] have analyzed the sensitivity/ resistance of human brain tumors to treatment by evaluating the changes or lack there­of in waterADC. The authors associated an increase in waterADC after treatment with cell lysis, apoptosis, necrosis involving cell shrinkage, and blebbing followed by phago­cytosis. A decrease in tumor waterADC they explained by 1) cell enlargement associ­ated with mitotic catastrophe, 2) a reduction in tumor blood flow resulting in focal ische-mia/hypoxia, and 3) by drainage of tumor water (Fig. 1). The authors concluded that a significant increase or decrease in tumorADC values occurring after treatment indi­cates likelihood some response to therapy, but a lack of change in tumorADC values indicates a therapeutically unresponsive tumor. However, these observations were made on the basis of clinical observations during weeks and months after different treat­ments. All these data show that changes in waterADC following therapy can be different in different tumors, or even in the same tumor cell line at different locations, e.g., intracra­nial versus sc-implanted glioma. A proper characterization and understanding of the changes in waterADC and 23Na MRI SI in the same tumors at different locations is very important for application of these techniques for monitoring therapy in orthotopic tumors.

6. Liver, Prostate, and Other Tumors

The effects of chemotherapy in animal liver tumors have been studied using ortho­topic tumors. Transcatheter hepatic arterial chemoembolization is a classic interven­tional therapy for hepatocellular carcinoma and secondary liver cancers. In chemoem­bolization, the blood supply to the tumor is blocked surgically or mechanically and anti­cancer drugs are administered directly into the tumor. This permits a higher concentra­tion of drug to be in contact with the tumor for a longer period time. Geschwind et al. used transcatheter hepatic arterial chemoembolization with carboplatin to treat VX2 tu­mor in the rabbit liver [13]. Carboplatin is a platinum-based chemotherapy drug used to treat various types of cancers, including sarcomas, lymphomas, and germ cell tumors. Platinum complexes are formed in cells, which bind and cause cross-linking of DNA-ultimately triggering apoptosis. The waterADC in the rabbit VX2 liver carcinomas tumor was evaluated after sacrificing of the animals to prevent motion artifacts. DWI deli­neated regions of tumor cell death as zones of lower signal intensity (SI) in both control and treated groups. WaterADC was significantly greater in the area of tumor necrosis compared to the area of viable tumor cells. Histological analysis showed a significantly lower amount of viable cells after treatment (< 1%) compared to the untreated control group (55%). Bsl-2 was expressed in the liver tumor after chemoembolization, sugges­ting an apoptotic pathway of cell death.

Yuan et al. used another type of chemoembolization of the rabbit VX2 tumor in the liver when iodized oil (0.3 mL/kg) and pharmorubicin (2 mg/kg) were infused into the he­patic artery [77, 78]. Similar to doxorubicin pharmorubicin is an anthracycline antibiotic that interacts with DNA by intercalation and inhibition of macromolecular biosynthesis [79]. This inhibits the progression of the enzyme topoisomerase II, which unwinds DNA for transcription. The authors anesthetized rabbits with 3% soluble pentobarbitone into auri-border vein to slow and stabilize animal breathing. Using b-values of 100 s/mm2 and 300 s/mm2, waterADC in surrounding liver tissue (2.71x10-3mm2/s and 2.30x10-3mm2/s, respectively) tissue was higher than in the tumor center (1.77x10-3 mm2/s and 1.55x10-3mm2/s, respectively). They have found that under both b-values 100 s/mm2 and 300 s/mm2 the waterADC decreased in the tumor periphery 6 and 16 hr post-treatment while at 32 and 48 hr post-treatment it started to increase. A similar trend was found for waterADC changes in normal liver parenchyma area around the tumor. The authors con­cluded that necrotic liver tumors manifests low signal and high ADC value, while viable tumor manifests high 1H signal and low ADC value after chemoembolization.

An orthotopic pancreatic tumor model was used to study the early therapeutic effi­cacy of monometric monoclonal anti-death receptor 5 antibody (TRA-8) combined with gemcitabine using DW 1H MRI [80]. An anti-death receptor 5 is predominantly expressed in most cancer cells but not in normal cells. TRA-8 was developed specifically to target this receptor. As with fluorouracil and other analogues of pyrimidines, gemcitabine re­places cytidine of nucleic acids, during DNA replication, arresting tumor growth, as new nucleosides cannot be attached to the „faulty" nucleoside, resulting in apoptosis. To prevent the transfer of the respiratory motion in the chest and abdominal area, an or­thogonally bent plastic board was used. At day 1 after the beginning of the combined TRA-8 and gemcitabine therapy waterADC in tumor regions was 27% higher than in untreated controls or those treated with gemcitabine only. At that time point no statistical difference in tumor volume was found. The mean waterADC values gradually increased over three days, which were concurrent with tumor volume regression. An increase in ADC was correlated with an increase in apoptotic cells density of tumors and with mean survival times of animals treated with the same drugs.

Jennings et al. studied the effect of docetaxel (10, 30, and 60 mg/kg) on waterADC in prostate cancer xenografts sc-implanted into the flanks of SCID mice, which are se­verely deficient in T and B cells and fail to reject allogenic grafts or produce antibodies to common antigens [81]. They showed that tumor volumes and secreted prostate-specific antigen both vary inversely with docetaxel dose, and that the waterADC in­creased significantly by day 2 and day 4 post-treatment with all drug doses. Authors concluded that DW MRI can be used for early detection of prostate carcinoma xenograft response to docetaxel chemotherapy, and waterADC changes may be used to optimize timing of fractioned chemotherapy such that effective doses may be applied when the tumor shows its highest ADC related to the high tumor vulnerability.

Vogel-Claussen et al. used the vascular-targeting agent ZD6126 to treat sc DU-145 human prostate cancer xenografts in SCID mice [82]. ZD6126 is a prodrug for N-acety-cholinol (NAC), a tubulin-binding agent that inhibits tubulin polymerization and leads to microtubule destabilization selectively in the immature endothelial cells ofthe tumor neo-vasculature [83]. After iv administration ZD6126 rapidly converts to NAC. In non-resistant to the drug tumors, the treatment caused progressive cell necrosis in the central tumor region within 24 hr post-treatment that was associated with a trend towards restricted dif­fusion. The authors explained this effect as a result of acute tumor ischemia, followed by

cell swelling and a relative decrease in extracellular space. These data are consistent with the results of Thoeny et al. for angiogenic therapy in rhabdomyosarcomas pre­sented above [43]. A significant linear correlation was observed between the area of ADC values > 0.9x10-3 mm2/s and the amount of necrosis in tumors pretreatment at 48 and 72 hr after ZD6126 treatment. The authors mentioned that in cystic malignancies with high pre-treatment ADC values, DW MRI may not adequately assess early tumor response to antivascular therapy.

Dev et al. used a new type of cancer treatment that combines pulsed electric fields (PEF) with the anticancer drug bleomycin. PEF may create transient pores in the mem­branes which allow entry of drugs into the cells [84]. Bleomycin is a glycopeptide antibi­otic that acts by induction of DNA strand breaks and/or inhibition ofthe incorporation of thymidine into DNA strands. DNA cleavage by bleomycin depends on the presence of oxygen and metal ions, at least in vitro. It is believed that bleomycin chelates metal ions (primarily iron) producing a pseudoenzyme that reacts with oxygen to produce super­oxide and hydroxide free radicals that cleave DNA. Intratumor injection of bleomycin with PEF significantly increased waterADC 24 to 48 hrs post-treatment of sc laryngeal tumor from 0.73x10-3 mm2/s to 0.83x10-3 mm2/s. During this interval, spin-lattice T1 relaxation time was unchanged while spin-spin T2 relaxation time significantly increased in the treated group. The longer T2 values may reflect early apoptosis and tumor death reflec­ting less density ofthe tumor cells and higher water ADC may indicate loose structural organization and necrosis after treatment.

The studies presented above allow one to conclude that in animal models, with a few exceptions, effective tumortherapy is associated with an increase in waterADC.

7. Does Initial Level of ADC Predict Chemotherapeutic Efficiency?

Predicting treatment response on the basis ofthe pre-treatment ADC value could have considerable clinical benefit as it might indicate the eventual outcome of therapy. As described above, Lemeire et al. found that mammary tumors with low initial ADC values (< 0.95x10-3 mm2/s) responded to a single 5FU bolus therapy by a 30% increase in ADC at day 7 after treatment, whereas tumors with high initial ADC (> 1.2x10-3 mm2/s) showed a 30% decrease; tumors with intermediate values showed no significant change in ADC [44]. The high and low initial ADC groups both showed a significant decrease in tumor volume after the treatment. High and low ADC values in tumors were correlated with high and low necrosis, respectively. The authors explained these results by higher absolute concentration of 5FU in tumors with low necrosis. Kamm et al. [85] previously have shown that the absolute concentration of 5FU and metabolites was lower in the necrotic regions of tumors compared to viable regions, an effect attributed to better vascularization ofthe viable regions.

Studies in human rectal carcinoma [16], [17], and colorectal hepatic metastases [86] have shown that cellular tumors with low baseline pre-treatment ADC values re­spond better to chemotherapy or radiation treatment than tumors that exhibit high pre­treatment ADC values. One possible explanation of these results is that tumors with high pre-treatment ADC values are likely to be more necrotic than those with low values. Necrotic tumors frequently are hypoxic, acidotic, and poorly perfused, leading to dimi­nished sensitivity to chemotherapy and to radiation therapy.

However, it is still unclear if the initial ADC values can predict the clinically relevant tumor chemosensitivity in all types of tumors. Babsky et al. have found that the initial le­vels of water ADC as well as 23Na SI were higher in BCNU-responsive sc-implanted 9L

gliomas compared to the BCNU non-responsive group [41]. These data suggested that a higher initial ADC level (1.1 - 1.7x10-3mm2/s) was a promising sign for effective BCNU treatment, and in contrast, tumors with a lower initial ADC value (0.6 - 0.9x10-3 mm2/s) were most likely to be resistant to BCNU treatment. The higher pretreatment ADC levels in BCNU-responsive tumors may be related to an overall weakened condition ofthe cells making them vulnerable to toxic therapy. Furthermore, Seierstad et al. [46] did not find correlations between pretreatment ADC values and changes in colon adenocarcinoma HT29 xenograft volumes after chemoradiation, whereas early changes in mean ADC quantitatively correlated with treatment outcome. They conclude that „it is a prerequisite for comparing different chemotherapy regimens in animal models that initial xenografts be similar so that observed effects are therapy-induced effects and not effects originating from the initial composition of necrosis, fibrosis, and viable cells in tumors".

8. Tumor Tissue Water ADC and 23Na MRI Signal Intensity Reflects Structural Post-treatment Changes

The increase in extracellular space following therapy can cause not only an in­crease in waterADC, but also an increase in total tumor tissue (Na{), as [Na+]e is 10-15 times higher than [Na+]i. Because of its concentration, ubiquity, and short T1,23Na is the second most sensitive MR nucleus in tissue, with only 1H being more sensitive. It has been shown that, on average, both 23Na SI and water ADC increased throughout the tumor after Cp (Fig. 2) or 5FU (Fig. 4) treatment of RIF-1 [40, 42]. The increase in 23Na SI after chemotherapy could be because of an increase in concentration of total tumor tissue sodium ([Na+]t) or a change in 23Na relaxation times. The authors showed that Cp treatment or untreated growth of RIF-1 tumors did not significantly change the T1, T2s, and T2f values or the relative contributions of T2sand T2f. These results suggest that the observed increase in 23Na MRI SI after Cp treatment was due to increased [Na+]t caused by Cp treatment. The inductively coupled plasma-mass spectroscopy (ICP-MS) data confirmed that in Cp-treated tumors, [Na+]t is significantly increased three days after treatment (45 + 7 mM control, 58 + 10 mM Cp-treated). The value of the Cp-induced increase in [Na+]t was comparable for both MRI (36.8%) and ICP-MS (29.4%) methods.

Although the extracellular space increases after therapy, [Na+]e may remain con­stant. [Na+]e can be maintained constantly by transport of Na+ from vascular and/or in­terstitial space ofthe nearby uncompromised tissue even in hypoxic or necrotic regions. Moreover, previous 1H MRI studies show that tumor perfusion is increased after therapy [87]. Thus, transport of Na+ from the vascular space can maintain [Na+]e, and an in­crease in extracellularspace results in increased [Na+]t within the tumoraftertherapy.

There was a good correlation between 23Na SI and waterADC in the Cp (R2 = 0.97) and 5FU (R2 = 0.99) treated tumors (Figs. 2 & 4). One possible reason for this effect may be that [Na+]t increases with increased extracellular space because of cells lost via apoptosis and/or necrosis. Schepkin et al. [38, 39, 52] showed that an increase in 23Na MRI SI occurred 7-9 days following treatment with BCNU, which correlated (R2 = 0.97) to the period of greatest chemotherapy-induced cellular necrosis based on waterADC changes and histopathology. However, in untreated growing sc 9L glioma, [Na+]t also increased during nine days of observation by 20 + 2 mM while the mean ADC remained constant at 1.07 + 0.02x10-3 mm2/s [38].

In general, necrotic tumor regions showed higher water ADC and 23Na MRI SI com­pared to viable regions in most tumors, but this is not true in some cases. In Fig. 5, for example, the histological region of sc 9L glioma (b) clearly corresponds to low waterADC

Day 0 Day 1 Day 2 Day 3 0    12 3

Days after Therapy Days after Therapy

Fig. 4. Correlation of changes in water apparent diffusion coefficient (ADC) (A) and single-quantum (SQ) 23Na signal intensity (SI) (B) after 5-fluorouracil (5FU) therapy in subcutaneously (sc) implanted RIF-1 tumors. a-on the leftside the representative examples of waterADC maps and 23Na MRI for a control and a 5FU-treated tumor before (Day 0) and one, two, and three days after 5FU injection are pre­sented; b - the mean waterADC and SQ 23Na SI from the whole control and treated tumors. A vial filled with a NaCl solution was placed near the tumor as a reference. Significance: p < 0.05 (* - vs. before treatment), p < 0.01 (** - Control vs. 5FU-treated). Data are presented as mean + SEM

Fig. 5. Stained with hematoxylin and eosin (H&E) histological slices (A - C), waterapparent diffusion coeffi­cient (ADC), and 23Na images of subcutaneous 9L glioma. Histological slice from the middle part of the tumor is presented. Arrows point the regions identified as .viable" (A, B) and ..necrotic" (C). These regions are presented as high-resolution (original magnification, x200) histological images

and 23Na MRI SI. However, another region with high cellular density on the left side of the tumor (a) corresponds to an area with high waterADC and low 23Na MRI SI. On the other hand, the region with low cellular density on the right side of the tumor (c) corresponds to high 23Na MRI SI and intermediate waterADC. This discrepancy may reflect the fact that [Na+] and water diffusion in tissue are influenced by different factors. Tissue [Na+] depends mainly on relative ECS and [Na+]i because [Na+]e is largely constant, while waterADC is affected not only by the size of different tissue compartments but also by the biophysical environments in these compartments. For example, a tumor region that is rich in collagen may have low water ADC but high tissue [Na+]. Thus, 23Na MRI may provide additional information about the effects of therapy and untreated growth on tumor microenvironment compared to that which is available from waterADC measurements alone.

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A Babsky, Ju Shenghong - Apparent diffusion coefficient of waterin evaluation of treatment response in animal body tumors