Typical data with this new method in TRAMP mice are shown in . The tumor in had a high lactate signal, high lactate to pyruvate ratio, and no alanine, all of which are typical for TRAMP tumors, as was demonstrated previously (11
). The representative image shown included the liver, which had some of the highest detected [1-13
C]-alanine. The hyperpolarized 13
C MRSI data were collected from throughout the mouse abdomen, and various metabolite amounts and dynamics were observed. The effect and timing of the injection were also observable in the detected pyruvate (). The large pyruvate signal in the neck was from the jugular catheter used for the injection. In the pyruvate curves, a second peak was seen 20 sec after injection that corresponds to the pyruvate in the catheter that is displaced by saline flushed between 12 and 18 sec after injection, creating a second bolus. This was done to clear the catheter line of hyperpolarized pyruvate and also resulted in modulations of the lactate and alanine in some voxels. Representative metabolic dynamics in a normal prostate, early-stage tumor, and late-stage tumor are shown in . In the normal prostate, low amounts of hyperpolarized pyruvate, lactate, and alanine were detected, while high lactate was observed in the tumors. The largest lactate was seen in the late-stage tumor, which is consistent with previous nondynamic studies (11
). The low metabolite signal in the normal prostate makes it difficult to compare the dynamic characteristics between normal and cancerous prostates.
FIG. 2 Typical spectra and dynamic curves. a: The coronal T2-weighted FSE anatomical image shows the majority of the voxel locations. b: A typical single MRSI, acquired 25 sec following injection, demonstrates the spectral characteristics and typical SNR obtained (more ...)
Images of the lactate FWHM and MT, as well as metabolite tSNR, pyruvate FWHM, and pyruvate MT, are shown in a large, late-stage tumor in . The coronal orientation allowed for comparison between the tumor and other organs, such as the liver. While the highest lactate tSNR was within the tumor, there were voxels in the gut with lactate on the level of many tumor voxels. The data analysis showed that the lactate dynamics were distinct within the prostate tumors, with the highest lactate FWHM and lactate MT seen in the tumor, as is shown in . Other tissues with high hyperpolarized lactate levels, such as the gut, did not share these lactate dynamic characteristics. The pyruvate dynamics also provided new information. In particular, the pyruvate MT () showed areas of more rapid uptake in the right, superior portion of the tumor (pyr MT = 23.69 sec), which also had the highest pyruvate tSNR (12.80). This region had the highest lactate tSNR (44.08) and an earlier lactate MT (34.63 sec), suggesting it was a better-perfused and vascularized region of the tumor, in addition to being highly metabolically active. The rest of the tumor had relatively high lactate tSNR (25.03) but a later pyruvate MT (25.72 sec) and lactate MT (36.93 sec), suggesting that perfusion was delayed in these regions. The regional differences between pyruvate and lactate in both their tSNR and MT maps demonstrate the unique heterogeneity information provided by dynamic imaging. Alanine was seen in the liver and gut but not in the tumor.
FIG. 3 Large, late-stage heterogeneous tumor. a: Coronal T2-weighted FSE anatomical image showing a subset of the voxel locations. b: Metabolite curves for pyruvate, lactate, and alanine in each voxel. The green box highlights the liver, while the red highlights (more ...)
shows a comparison of the pyruvate and lactate characteristics between tumor and liver voxels in eight TRAMP mice, as well as the liver voxel characteristics in three wild-type normal mice, showing differences that were strongly statistically significant for many parameters. Only animals with primary prostate tumors and no evidence of metastases were included in these comparisons, and voxels used were required to have a minimum SNR to ensure accurate and consistent dynamic parameterizations. The liver was chosen as the healthy reference tissue because it was more metabolically consistent than the gut, where activity probably varies with digestion and peristalsis, and some portion of the liver was always present and viable in the coronal orientation. The pyruvate and lactate characteristics in normal mice livers (x’s) agree with this hypothesis of the liver as a healthy reference and also show the variance of these parameters resulting from physiologic (cardiac and liver function, flow), experimental (injection, bolus, RF calibration), and/or other sources. In agreement with the visual observations shown in , there were significant statistical differences in the lactate dynamics (FWHM and MT), and both were strictly increasing from liver to tumor within each animal. This resulted in the strongest significance when the paired-sample t test was applied, which controls for interanimal and interexperiment variations. Both the tSNR and fraction of alanine (not shown) were statistically significant, as expected because of the normally high alanine in the liver and little to no alanine in the prostate. The pyruvate to tHC and lactate to tHC ratios were significantly different between tumor and liver, but this was not true for the lactate tSNR or pyruvate tSNR (not shown). The tHC ratio differences are most likely due to alanine, which is included in tHC and detected in the liver. The lack of statistically significant tSNR differences is because the liver often had substantial lactate, especially relative to early-stage tumors. The pyruvate FWHM and MT (not shown) showed no statistically significant differences, suggesting similar perfusion and flow in the tumor and liver. These data suggest the lactate FWHM is the most sensitive dynamic parameter for differentiating tumor and normal tissue.
FIG. 4 Comparison of pyruvate and lactate characteristics between tumor voxels and liver voxels in TRAMP mice (circles) and normal mice (x’s, liver only). The lines connect data points for the same mouse, and each color also represents a different mouse. (more ...)
compares hyperpolarized 13C dynamic data acquired in two TRAMP tumors with different pathologic grades and rates of cellular proliferation based on Ki-67 staining. The TRAMP in had a relatively sparse and ill-defined tumor consisting of 75% moderately differentiated and 25% poorly differentiated tissue. There was no clearly delineated tumor nodule or mass in the FSE image, and Ki-67 staining showed a low density of proliferating tumor cells. On the other hand, the TRAMP in had a well-defined tumor on the FSE image and a high density of proliferating tumor cells with 100% poorly differentiated tissue. The lactate was substantially larger in the more aggressive tumor, with a lactate tSNR of 18.88 as compared to 7.07 for the less aggressive tumor. The pyruvate was similar, with an average pyruvate tSNR of 2.39 and 1.41 for the less and more aggressive tumors, respectively. The dynamic data analysis yielded lactate MTs of 38.37 and 34.43 sec, indicating an earlier arrival and likely better vascularization in the more advanced, aggressive tumor. The lactate FWHM values were 35.00 and 30.62 sec, showing more persistent lactate in the less aggressive tumor, which may be due to poorer vascularization. Heterogeneity was observed across the more aggressive tumor, which demonstrated better pyruvate perfusion and more lactate in the top-left voxels corresponding to the more homogenous region in the FSE image.
FIG. 5 a–c: Low cellular density of proliferating cancer cells. a: The coronal T2-weighted FSE anatomical image suggests an ill-defined tumor. b: Histology of tumor tissue with Ki-67 (cell proliferation) staining, shown in brown at 10x magnification, (more ...)
summarizes the tSNR, lactate MT, and lactate FWHM from 10 hyperpolarized [1-13
C]-pyruvate studies in six of the TRAMP tumors analyzed with histology (N
= 3 had early- to intermediate-stage tumors, N
= 3 had late-stage tumors, as determined by histological analysis). There was a significant difference between the late- and early/intermediate-stage tumors in lactate tSNR, which is consistent with previous results (11
). On average, the dynamic parameters (MT and FWHM) for lactate and pyruvate showed no statistically significant differences between early- to intermediate- and late-stage tumors. One possible explanation is that the majority of lactate and pyruvate detected was from actively proliferating cells, which could have a specific dynamic profile (MT and FWHM), where the amount of lactate corresponds to the density of these cells while the dynamic profile is stable. The pyruvate tSNR was highly variable, indicating a wide range of tissue perfusion. Also, a metabolic difference from normal tissues was that all tumors demonstrated little to no alanine signal, which is high in the normal liver and muscle tissues.
Dynamic imaging data and pathology from the corresponding tissue in a heterogeneous tumor are shown in . Overall, the pyruvate and lactate signal variations were consistent with the variations in cellular density. In particular, the left anterior portion of this tumor (green arrows) had less lactate (lac tSNR = 12.34), which corresponded to a region of reduced cellular density (lighter and pinker hematoxylin and eosin staining) and reduced tissue density (tissue integrity compromised). As shown in the histology, the tissue separated during processing, indicating weakness or lack of structure that was most likely due to the presence of necrosis. There was also lighter staining along the anterior and right sides of the tumor, which had relatively less lactate and pyruvate (lac tSNR = 12.55, pyr tSNR = 4.21). The central posterior region had the greatest tumor cell density and the highest lactate and pyruvate (lac tSNR = 30.30, pyr tSNR = 10.42). The pyruvate and lactate had a similar distribution across the tumor, but the contrast was greater in the lactate signal.
FIG. 6 Large, late-stage heterogeneous tumor. a: Axial T2-weighted FSE anatomical image. b: Hemotoxylin and eosin staining of a tumor cross-section at approximately the same location and orientation as (a). c,d: Pyruvate and lactate curves. No alanine was detected. (more ...)
The perfusion information provided by the dynamic imaging is shown in the lactate and pyruvate MT (). Within the tumor, the shortest MT for both pyruvate and lactate was near the central posterior region (pyr MT = 23.42 sec, lac MT = 36.70 sec), nearest to the major vessels, and the MT increases in the peripheral regions (pyr MT = 24.40 sec, lac MT = 38.64 sec). In the probable-necrosis region (green arrows), the lactate MT is particularly high (39.54 sec), most likely because it has the worst vascularization and perfusion. The lactate FWHM was relatively similar across the entire tumor (average ± standard deviation = 31.29 ± 1.53 sec), as compared to nontumor tissue (data shown in and ).
A lymph node metastasis in this slice (red arrow) was easily identified by its high lactate signal (lac tSNR = 27.41). The lymph node had low pyruvate (pyr tSNR = 5.37) and a later pyruvate MT (24.90), both similar to the peripheral, lower-density regions of the primary tumor. This is related to its naturally slower lymphatic fluid supply and/or relatively poorer vascularization. However, the lactate tSNR, MT (35.31 sec), and FWHM (29.07 sec) were similar to the central primary tumor region, suggesting high active cellular density in the lymph node metastasis.