(31) P and (1) H MRS of DB-1 melanoma xenografts: lonidamine selectively decreases tumor intracellular pH and energy status and sensitizes tumors to melphalan.

In vivo (31) P MRS demonstrates that human melanoma xenografts in immunosuppressed mice treated with lonidamine (LND, 100 mg/kg intraperitoneally) exhibit a decrease in intracellular pH (pH(i) ) from 6.90 ± 0.05 to 6.33 ± 0.10 (p \u3c 0.001), a slight decrease in extracellular pH (pH(e) ) from 7.00 ± 0.04 to 6.80 ± 0.07 (p \u3e 0.05) and a monotonic decline in bioenergetics (nucleoside triphosphate/inorganic phosphate) of 66.8 ± 5.7% (p \u3c 0.001) relative to the baseline level. Both bioenergetics and pH(i) decreases were sustained for at least 3 h following LND treatment. Liver exhibited a transient intracellular acidification by 0.2 ± 0.1 pH units (p \u3e 0.05) at 20 min post-LND, with no significant change in pH(e) and a small transient decrease in bioenergetics (32.9 ± 10.6%, p \u3e 0.05) at 40 min post-LND. No changes in pH(i) or adenosine triphosphate/inorganic phosphate were detected in the brain (pH(i) , bioenergetics; p \u3e 0.1) or skeletal muscle (pH(i) , pH(e) , bioenergetics; p \u3e 0.1) for at least 120 min post-LND. Steady-state tumor lactate monitored by (1) H MRS with a selective multiquantum pulse sequence with Hadamard localization increased approximately three-fold (p = 0.009). Treatment with LND increased the systemic melanoma response to melphalan (LPAM; 7.5 mg/kg intravenously), producing a growth delay of 19.9 ± 2.0 days (tumor doubling time, 6.15 ± 0.31 days; log(10) cell kill, 0.975 ± 0.110; cell kill, 89.4 ± 2.2%) compared with LND alone of 1.1 ± 0.1 days and LPAM alone of 4.0 ± 0.0 days. The study demonstrates that the effects of LND on tumor pH(i) and bioenergetics may sensitize melanoma to pH-dependent therapeutics, such as chemotherapy with alkylating agents or hyperthermia

Multimodality Imaging of Abnormal Vascular Perfusion and Morphology in Preclinical 9L Gliosarcoma Model

This study demonstrates that a dynamic susceptibility contrast-magnetic resonance imaging (DSC-MRI) perfusion parameter may indicate vascular abnormality in a brain tumor model and reflects an effect of dexamethasone treatment. In addition, X-ray computed tomography (CT) measurements of vascular tortuosity and tissue markers of vascular morphology were performed to investigate the underpinnings of tumor response to dexamethasone.One cohort of Fisher 344 rats (N = 13), inoculated intracerebrally with 9L gliosarcoma cells, was treated with dexamethasone (i.p. 3 mg/kg/day) for five consecutive days, and another cohort (N = 11) was treated with equal volume of saline. Longitudinal DSC-MRI studies were performed at the first (baseline), third and fifth day of treatments. Relative cerebral blood volume (rCBV) was significantly reduced on the third day of dexamethasone treatment (0.65 ± .13) as compared to the fifth day during treatment (1.26 ±.19, p < 0.05). In saline treated rats, relative CBV gradually increased during treatment (0.89 ±.13, 1.00 ± .21, 1.13 ± .23) with no significant difference on the third day of treatment (p>0.05). In separate serial studies, microfocal X-ray CT of ex vivo brain specimens (N = 9) and immunohistochemistry for endothelial cell marker anti-CD31 (N = 8) were performed. Vascular morphology of ex vivo rat brains from micro-CT analysis showed hypervascular characteristics in tumors, and both vessel density (41.32 ± 2.34 branches/mm(3), p<0.001) and vessel tortuosity (p<0.05) were significantly reduced in tumors of rats treated with dexamethasone compared to saline (74.29 ± 3.51 branches/mm(3)). The vascular architecture of rat brain tissue was examined with anti-CD31 antibody, and dexamethasone treated tumor regions showed reduced vessel area (16.45 ± 1.36 µm(2)) as compared to saline treated tumor regions (30.83 ± 4.31 µm(2), p<0.001) and non-tumor regions (22.80 ± 1.11 µm(2), p<0.01).Increased vascular density and tortuosity are culprit to abnormal perfusion, which is transiently reduced during dexamethasone treatment

An assessment of vascular normalization in 9L gliosarcoma brain tumor: Using dynamic susceptibility contrast (DSC) imaging and dynamic contrast enhancement (DCE) imaging

Emerging concepts and evidence have purported that certain antiangiogenic drugs can transiently normalize the abnormal morphology and function of tumor vasculature. It is suggested that this transitory period of vascular normalization is suitable for drug delivery to tumor microenvironment, and may improve the efficiency of chemotherapy or radiotherapy. From another perspective, this view seems rather counterintuitive since the normalization of tumor vasculature could potentially augment the growth rate of tumor. Understanding this process has significant implications for improving therapeutic outcomes. For if normalization provides the best time during which treatment should be administered, we want to know when the normalization occurs, and how to optimize timing, dosing and possibly the combinations of drugs administered. If normalization results in advanced tumor growth, rather than a window of opportunity, we want to make sure treatment strategies avoid normalization as an endpoint. We hypothesize that advanced physiologic imaging methods can provide the information necessary to detect and characterize normalization and subsequently optimize treatment strategies. DSC can provide parametric measurements such as relative cerebral blood volume (CBV), cerebral blood flow (CBF) and mean transit time (MTT) which can be use to estimate the vascular morphology (i.e. micro/macro vessels) and vascular function of tumors. Rat brain was implanted with tumor and examined on a 3 Tesla MR system by implementing DSC and DCE techniques. Cerebral perfusion (CBF/MTT) in brain tumor is quite inefficient as demonstrated by DSC. This inefficient perfusion is attributed to the density of irregularly shaped vessels in rat brain tumors as elucidate by micro-computed tomography of intact vessel tree. It is shown that the density of tortuous vessels is the prime suspect of abnormal perfusion. DSC parameters were validated by use of microscopy on brain tissues. Immunohistochemistry, anti-CD31, showed some regression in vessel size after dexamethasone therapy as possible indication of vascular normalization . Dexamethasone treatment also reduced vascular permeability in terms of Ktrans as computed from DCE. Cerebral blood volume measure from DSC is a potential surrogate marker to characterize the normalization of tumor vasculatures during antiangiogenic therapy. And Temozolomide treatment in this window produced a synergistic effect. Therefore, it is suggested here that DSC-MRI measurements could play an important role for assessing vascular normalization of tumor and the efficacy of combining drug treatment

Representative post gadolinium-enhanced T1-weighted MR image of the same rat brain acquired longitudinally on [A] 1^st (baseline) day, [B] 3^rd day, and [C] 5^th day during saline treatment with corresponding parametric relative cerebral blood volume (rCBV) maps on [D] 1^st (baseline) day, [E] 3^rd day, and [F] 5^th day.

<p>Representative post gadolinium-enhanced T1-weighted MR image of the same rat brain acquired longitudinally on [A] 1^st (baseline) day, [B] 3^rd day, and [C] 5^th day during saline treatment with corresponding parametric relative cerebral blood volume (rCBV) maps on [D] 1^st (baseline) day, [E] 3^rd day, and [F] 5^th day.</p

Longitudinal results of [A] quantitative relative cerebral blood volume (rCBV) values of saline treated (N = 11) and dexamethasone treated (N = 13) cohorts <i>in vivo</i>, and [B] tumor growth rate.

<p>Data points are displayed as mean±SEM, and the p-values were evaluated by one-way ANOVA (^nsp-value>0.05, *p-value≤0.05, ** p-value≤0.01, *** p-value≤0.001).</p

Qualitative morphology from micro-CT data of representative <i>ex vivo</i> rat brains.

<p>Top view of vessel tree skeleton in [<b>A</b>] saline treated rat and [<b>B</b>] dexamethasone treated rat. Side view of 3D volume rendered image of [<b>C</b>] saline treated rat and [<b>D</b>] dexamethasone treated rat.</p

Immunohistochemistry of vessels (anti-CD31, brown) and cell nuclei (hematoxylin, blue) from rat 9L gliosarcoma brain tissue with 0.26 mm² field-of-view at 20× magnification.

<p>Tumor regions of [<b>A</b>] saline treated and [<b>B</b>] dexamethasone treated rats.</p

Quantitative morphology from micro-CT data of <i>ex vivo</i> rat brains.

<p><b>[A]</b> Density of vessel branches in tumor volume of interest for saline treated (N = 4) and dexamethasone treated (N = 5) rats. All bar graphs are displayed as mean±SEM, and the p-value was evaluated by unpaired t-test (*p-value ≤0.05, ** p-value ≤0.01, *** p-value ≤0.001). [<b>B</b>] Histogram of tortuosity in saline treated (N = 4) and dexamethasone treated (N = 5) rats. The p-value was evaluated by Mann Whitney U test (p<0.05).</p

Mean vessel area from anti-CD31 photomicrographs of saline treated (N = 4) rats and dexamethasone treated (N = 4) rats.

<p>All bar graphs are displayed as mean±SEM, and the p-values were evaluated by unpaired t-test (*p-value ≤0.05, ** p-value ≤0.01, *** p-value ≤0.001).</p