Noninvasive monitoring of tumor therapy response helps in developing personalized treatment strategies. Here, we performed sequential PET and diffusion-weighted MRI to evaluate changes induced by a ...FOLFOX-like combination chemotherapy in colorectal cancer xenografts, to identify the cellular and molecular determinants of these imaging biomarkers.
Tumor-bearing CD1 nude mice, engrafted with FOLFOX-sensitive Colo205 colorectal cancer xenografts, were treated with FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) weekly. On days 1, 2, 6, 9, and 13 of therapy, tumors were assessed by in vivo imaging and ex vivo analyses. In addition, HCT116 xenografts, which did not respond to the FOLFOX treatment, were imaged on day 1 of therapy.
In Colo205 xenografts, FOLFOX induced a profound increase in uptake of the proliferation PET tracer 3'-deoxy-3'-
F-fluorothymidine (
F-FLT) accompanied by increases in markers for proliferation (Ki-67, thymidine kinase 1) and for activated DNA damage response (γH2AX), whereas the effect on cell death was minimal. Because tracer uptake was unaltered in the HCT116 model, these changes appear to be specific for tumor response.
We demonstrated that
F-FLT PET can noninvasively monitor cancer treatment-induced molecular alterations, including thymidine metabolism and DNA damage response. The cellular or imaging changes may not, however, be directly related to therapy response as assessed by volumetric measurements.
Ketone bodies have both metabolic and epigenetic roles in cancer. In several studies, they showed an anti-cancer effect via inhibition of histone deacetylases; however, other studies observed faster ...tumour growth. The related molecule butyrate also inhibits growth of some cancer cells and accelerates it in others. This "butyrate paradox" is thought to be due to butyrate mediating histone acetylation and thus inhibiting cell proliferation in cancers that preferentially utilise glucose (the Warburg effect); whereas in cells that oxidise butyrate as a fuel, it fails to reach inhibitory concentrations and can stimulate growth.
We treated transgenic mice bearing spontaneous MMTV-NEU-NT mammary tumours with the ketone body β-hydroxybutyrate (β-OHB) and monitored tumour growth, metabolite concentrations and histone acetylation. In a cell line derived from these tumours, we also measured uptake of β-OHB and glucose, and lactate production, in the absence and presence of β-OHB.
β-OHB administration accelerated growth of MMTV-NEU-NT tumours, and their metabolic profile showed significant increases in ATP, glutamine, serine and choline-related metabolites. The β-OHB concentration within the treated tumours, 0.46 ± 0.05 μmol/g, had no effect on histone acetylation as shown by western blots. Cultured tumour cells incubated with 0.5 mM β-OHB showed β-OHB uptake that would be equivalent to 54% of glycolytic ATP phosphorylation and no significant change in glucose consumption or lactate production.
These results suggest that a β-OHB paradox may occur in these mammary tumours in a manner analogous to the butyrate paradox. At low β-OHB concentrations (<1 mM, as observed in our tumour model post-treatment), and in the absence of a Warburg effect, β-OHB is consumed and thus acts as an oxidative energy source and not as an epigenetic factor. This would explain the increase in tumour growth after treatment, the metabolic profiles and the absence of an effect on histone H3 acetylation.
Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We ...evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.
Purpose
The aim of the study was to investigate the potential of diffusion-weighted magnetic resonance imaging (DW-MRI) and 3′-dexoy-3′-
18
Ffluorothymidine (
18
FFLT) positron emission tomography ...(PET) as early biomarkers of treatment response of 5-fluorouracil (5-FU) in a syngeneic rat model of colorectal cancer liver metastases.
Procedures
Wag/Rij rats with intrahepatic syngeneic CC531 tumors were treated with 5-FU (15, 30, or 60 mg/kg in weekly intervals). Before treatment and at days 1, 3, 7, and 14 after treatment rats underwent DW-MRI and
18
FFLT PET. Tumors were analyzed immunohistochemically for Ki67, TK1, and ENT1 expression.
Results
5-FU inhibited the growth of CC531 tumors in a dose-dependent manner. Immunohistochemical analysis did not show significant changes in Ki67, TK1, and ENT1 expression. However,
18
FFLT SUV
mean
and SUV
max
were significantly increased at days 4 and 7 after treatment with 5-FU (60 mg/kg) and returned to baseline at day 14 (SUV
max
at days −1, 4, 7, and 14 was 1.1 ± 0.1, 2.3 ± 0.5, 2.3 ± 0.6, and 1.5 ± 0.4, respectively). No changes in
18
FFLT uptake were observed in the nontreated animals. Furthermore, the apparent diffusion coefficient (ADC
mean
) did not change in 5-FU-treated rats compared to untreated rats.
Conclusion
This study suggests that 5-FU treatment induces a flare in
18
FFLT uptake of responsive CC531 tumors in the liver, while the ADC
mean
did not change significantly. Future studies in larger groups are warranted to further investigate whether
18
FFLT PET can discriminate between disease progression and treatment response.
Purpose
We recently reported that high thymidine phosphorylase (TP) expression is accompanied by low tumor thymidine concentration and high 3′-deoxy-3′-
18
Ffluorothymidine (
18
FFLT) uptake in four ...untreated lung cancer xenografts. Here, we investigated whether this relationship also holds true for a broader range of tumor models.
Procedures
Lysates from
n
= 15 different tumor models originating from
n
= 6 institutions were tested for TP and thymidylate synthase (TS) expression using western blots. Results were correlated to
18
FFLT accumulation in the tumors as determined by positron emission tomography (PET) measurements in the different institutions and to previously published thymidine concentrations.
Results
Expression of TP correlated positively with
18
FFLT SUV
max
(
ρ
= 0.549,
P
< 0.05). Furthermore, tumors with high TP levels possessed lower levels of thymidine (
ρ
= − 0.939,
P
< 0.001).
Conclusions
In a broad range of tumors,
18
FFLT uptake as measured by PET is substantially influenced by TP expression and tumor thymidine concentrations. These data strengthen the role of TP as factor confounding
18
FFLT uptake.
The antivascular actions of disodium combretastatin A-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 carcinosarcoma after single doses of 10 or 30 mg x kg(-1). Pharmacokinetic data showed ...that 10 mg x kg(-1) in the rat gave a plasma exposure similar to that achieved in the clinic. Blood flow rate to the tumor and normal tissues was measured using the uptake of radiolabelled iodoantipyrine (IAP). Quantitative autoradiography was used to determine changes in spatial distribution of tumor blood flow. Both doses caused an increase in mean arterial blood pressure (MABP) and a reduction in heart rate 1 h after treatment. Blood flow rate to the tumor decreased to below 15% of control for both doses at 1 h, whereas the normal tissues were much less affected. A further reduction (to 2% of control at 6 h) was found for 30 mg x kg(-1). Recovery was essentially complete by 24 h for both doses. Vascular resistance increased 80-fold in tumor at 6 h after 30 mg x kg(-1), compared with a maximum 5-fold increase in normal tissues. Analysis of the spatial distribution of tumor blood flow illustrated an overall reduction in all areas of the tumor at 1 h after 10 mg x kg(-1), with a tendency for blood flow in the peripheral regions of the tumor to recover more quickly than in central regions. Tumor blood flow reduction was related to vascular damage including vessel distension, coagulation and haemorrhage, and tumor cell damage culminating in necrosis. No pathology was evident in any of the normal tissues following treatment. The data provide an insight into the mechanisms underlying tissue blood flow changes occurring after clinically relevant doses of CA-4-P. It is currently being used to aid interpretation of pharmacodynamic data obtained from phase I/II clinical trials of CA-4-P and is relevant for future drug development in this area.
Seddon, B. M., Honess, D. J., Vojnovic, B., Tozer, G. M. and Workman, P. Measurement of Tumor Oxygenation: In Vivo Comparison of a Luminescence Fiber-optic Sensor and a Polarographic Electrode in the ...P22 Tumor. Radiat. Res. 155, 835–844 (2001). Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO2 readings lay in a range over twice that of sensor pO2(5min) values (–3.2 to 80 mmHg and –0.1 to 34.8 mmHg, respectively). However, there was no significant difference between the means ± 2 SE of the median pO2 values recorded by each instrument (11.0 ± 3.3 and 8.1 ± 1.9 mmHg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means ± 2 SE of the median pO2 values reported by the electrode and sensor (1.7 ± 0.9 and 2.9 ± 0.7 mmHg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO2 values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO2 values of 0.6 to 28.1 mmHg.
Purpose: Tumor hypoxia is associated with poor prognosis and a more malignant tumor phenotype. SR-4554, a fluorinated 2-nitroimidazole,
is selectively bioreduced and bound in hypoxic cells. We ...present validation studies of SR-4554 as a noninvasive hypoxia marker
detected by fluorine-19 magnetic resonance spectroscopy ( 19 F MRS) in the P22 carcinosarcoma, a tumor with clinically relevant hypoxia levels.
Experimental Design: Tumor-bearing female severe combined immunodeficient mice received SR-4554 at 180 mg/kg. Pharmacokinetic studies of parent
SR-4554 in plasma and tumors were performed using high-performance liquid chromatography-UV. Total SR-4554 (parent SR-4554
and bioreduction products) was monitored in tumor by 19 F MRS using a 4.7 T spectrometer, with continuous acquisition for up to 5 h. A parameter of total SR-4554 retention, the 3-h
19 F retention index ( 19 FRI) was determined. Tumor pO 2 , assessed polarographically, was decreased (5 mg/kg hydralazine or 100 mg/kg combretastatin A-4 phosphate) or increased 1
l/min carbogen (5% CO 2 , 95% O 2 ) plus 500 mg/kg nicotinamide, and the corresponding 19 FRI was measured.
Results: Comparative HPLC-UV- and MRS-derived assessments of parent and total SR-4554, respectively, indicated that concentrations
of total SR-4554 consistently exceeded parent SR-4554, the differential increasing with time. This indicates formation and
retention of SR-4554 bioreduction products in tumor, confirming the presence of hypoxia. The 19 FRI was higher in hydralazine- and combretastatin-treated animals compared with unmodulated animals ( P = 0.004 and 0.15, respectively) and animals receiving carbogen and nicotinamide ( P = 0.0001 and 0.005, respectively). Significant correlations were demonstrated between mean 19 FRI and polarographic pO 2 parameters ( P < 0.002).
Conclusions: Retention of hypoxia-related SR-4554 bioreduction products can be detected in the clinically relevant P22 tumor by 19 F MRS, and the 19 FRI correlates with polarographically measured pO 2 . These findings support the use of SR 4554 as a noninvasive hypoxia marker.
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