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DOI:10.2214/AJR.07.2371
AJR 2007; 189:726-736
© American Roentgen Ray Society

Original Research

Gene Expression Profiles, Histologic Analysis, and Imaging of Squamous Cell Carcinoma Model Treated with Focused Ultrasound Beams

Walter Hundt1,2, Esther L. Yuh1, Mark D. Bednarski1 and Samira Guccione1

1 Department of Radiology, Lucas MRS Research Center, Stanford University School of Medicine, Stanford, CA, 94305.
2 Present address: Department of Clinical Radiology, Clinic of Grosshadern, Ludwig Maximilians University, Marchioninistrasse 15, Munich 81377, Germany.

Received October 4, 2006; accepted after revision April 7, 2007.

 
Partially supported by the Lucas Foundation and the Phil Allen Trust.

Address correspondence to W. Hundt.


Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
OBJECTIVE. The purpose of our study was to evaluate the effect of short-pulse high-intensity focused ultrasound (HIFU) on inducing cell death in a head and neck cancer model (SCCVII [squamous cell carcinoma]) compared with continuous HIFU to get a better understanding of the biologic changes caused by HIFU therapy.

MATERIALS AND METHODS. HIFU was applied to 12 SCCVII tumors in C3H/Km mice using a dual sonography system (imaging, 6 MHz; therapeutic, 1 MHz). A continuous HIFU mode (total time, 20 seconds; intensity, 6,730.6 W/cm2) and a short-pulse HIFU mode (frequency, 0.5Hz; pulse duration, 50 milliseconds; total time, 16.5 minutes; intensity, 134.4 W/cm2) was applied. Three hours later, MR images were obtained on a 1.5-T scanner. After imaging, the treated and untreated control tumor tissue samples were taken out for histology and oligonucleotide microarray analysis.

RESULTS. Prominent changes were observed in the MR images in the continuous HIFU mode, whereas the short-pulse HIFU mode showed no discernible changes. Histology (H and E, TUNEL [terminal deoxynucleotidyl transferase-mediated dUTP {deoxyuridine triphosphate} nick-end labeling], and immunohistochemistry) of the tumors treated with the continuous HIFU mode revealed areas of significant necrosis. In the short-pulse HIFU mode, the H and E staining showed multifocal areas of coagulation necrosis. TUNEL staining showed a high apoptotic index in both modes. Gene expression analysis revealed profound differences. In the continuous HIFU mode, 23 genes were up-regulated (> twofold change) and five genes were down-regulated (< twofold change), and in the short-pulse HIFU mode, 32 different genes were up-regulated and 16 genes were down-regulated.

CONCLUSION. Genomic analysis might be included when investigating tissue changes after interventional therapy because it offers the potential to find molecular targets for imaging and therapeutic applications.

Keywords: ablation • cancer • focused ultrasound • gene expression • histology • MRI • squamous cell carcinoma


Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Procedures using guided external energy deposition such as radiofrequency ablation, cryoablation, and high-intensity focused ultrasound (HIFU) are increasing in clinical use. HIFU technology makes possible the deposition of controlled energy doses deep into soft tissue. Despite the expanding use of these procedures, little is known about the changes in gene expression and the relationship to the doses of energy exposure. Knowledge about the changes in gene expression induced by external energy deposition can be useful in characterizing changes in tissues morphology and design of new therapies. For example, in studies related to defining cellular changes during radiation therapy, it has been shown that immediate and early gene expression is induced after exposure of tissue to various stresses, and these genes can mediate cell proliferation, differentiation, survival, and even cell death [1–3]. These investigations led to new therapy options being evaluated clinically [4].

Currently, only imaging and histologic analysis have been used to characterize tissue during an interventional radiologic procedure, including HIFU therapy. HIFU causes physical changes in tissues through both energy deposition and physical alteration such as cavitation. It has been used clinically for thermal ablation of tumors using continuous-wave HIFU [5, 6] and is being evaluated for drug and gene delivery to tissue using short-pulse wave HIFU [7, 8]. Application of microbubbles can accentuate cavitation, and HIFU has been used to disrupt microbubbles for drug delivery [9]. In thermal ablation therapy, ultrasound beams penetrate soft tissues and can be focused to target sites and cause localized high temperatures (55–90°C) for a short and controlled period of time. As a result, well-defined areas of tissue change, protein denaturation, and coagulation necrosis can be produced in specific regions of tissue whereby damage to the overlying and surrounding tissues can be reduced [10]; however, there is a risk of injury to important structures such as vessels or nerves.


Figure 1
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  		Fig. 1 —Modified Sonablate 200 (Focus Surgery) with ultrasound probe. Photograph shows experimental setup of single-component ultrasound transducer, high-frequency amplifier, diagnostic and therapeutic ultrasound probe, and experimental water bath.

 
Squamous cell carcinoma (SCCVII) is a syngeneic tumor cell line of the C3H mice and has been used extensively as a model for human head and neck cancers. This tumor model exhibits imaging and therapeutic characteristics that are similar to human head and neck carcinomas [11].

The purpose of this study was to determine the differences in genomic expression profiles between tumors receiving the continuous or short-pulse wave modes of HIFU and to correlate these changes with those observed by MRI and immunohistochemistry (IHC). In addition, we evaluated whether functional genomics can be used to provide pertinent information after interventional radiologic procedures such as HIFU that would not be available through imaging or histology.


Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All animal experiments were performed in compliance with institutional animal care committee guidelines and with the approval of the animal care committee.

Tumor Implantation
Twelve C3H/Km mice 10–12 weeks old were anesthetized with intraperitoneal Nembutal (pentobarbital, Abbott Laboratories) (58 mg/kg), and their right and left flanks were shaved and prepared with isopropyl alcohol. An average of 2 x 105 tumor cells (mouse SCCVII) in Hanks' solution were injected intradermally in the right and left flank region of each mouse with a 27-gauge needle. The total volume of injection was 0.2 mL. The sizes of the tumors were measured twice a week to monitor the growth of the tumor. Approximately 3 weeks were required for tumors to grow to 1,300 mm3 in size.

Focused Ultrasound System
A modified Sonablate system (Focus Surgery) (Fig. 1) was used for mechanistic studies. The system contains both imaging and therapy components in a single spherical and concave transducer. The therapy portion of the transducer has a frequency (f0) of 1.0 MHz, aperture diameter of 50 mm, focal length of 40 mm, maximum total acoustic power of 120 W, and maximum focal intensity in water of 8,000 W/cm2. Focal area was 1.5 mm2, and electrical impedance magnitude at f0 was 81 {Omega}. Efficiency was calculated to be 80%. The imaging portion of the transducer has an f0 of 6.0 MHz, bandwidth of 80%, aperture diameter of 8 mm, and focal length of 40 mm.

Two different modes of focused ultrasound beams were applied. To each HIFU mode, six animals were assigned. In the continuous-wave HIFU mode, the transducer was powered continuously at 200 mV for 20 seconds. This sinusoidal wave signal was amplified by a 50-dB radiofrequency power amplifier (Model 2100L, Electronic Navigation Industry) coupled to the transducer device (50-{Omega} impedance). The dose peak intensity at the focal point was approximately 6,730.7 W/cm2. For the short-pulse HIFU mode, a cycle of 50 milliseconds with a frequency of 0.5 Hz was applied. The same power was chosen and the HIFU application lasted for 16.5 minutes. The resulting average intensity at the focal point was 134.6 W/cm2. These parameters led to a total acoustic power of 100.3 W and a pressure of 142.1 kPa. The total energy deposition was nearly the same in both modes (continuous-wave HIFU mode, 134,613.6 J/cm2; short-pulse wave HIFU mode, 133,267.4 J/cm2) (Table 1).


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  		TABLE 1: Applied High-Intensity Focused Ultrasound (HIFU) Parameters

 

Animal and Experimental Setup
For this study, each of the 12 mice was anesthetized to a surgical plane of anesthesia (no response to toe-pinch) with an intraperitoneal injection of ketamine and xylazine (xylazine 5–10 mg/kg plus ketamine 80–100 mg/kg). The anesthetized mouse was placed into a degassed 37°C water bath in a specially designed holder to provide a proper sonographic coupling. Using the imaging portion of the transducer, the mouse was positioned so that the treated tumor was aligned with the center of the therapeutic portion of the transducer, and HIFU treatment commenced. The control tumor located on the opposite side of the flank was not exposed to ultrasound beams.

MRI
A clinical 1.5-T Signa MR scanner (GE Healthcare) was used with a custom-designed quadrature high-pass birdcage coil tuned to 127 MHz for signal reception. The animals were imaged 1 day before and 3 hours after the application of focused ultrasound beams. To obtain accurate and reproducible images, the mouse was placed prone and fixed in wrapped tissue. The body temperature was maintained throughout the MRI studies with a warm blanket. The following scanning protocol was performed in the axial plane with a field of view of 6 cm, 256 x 192 pixels, and slice thickness, 2.0 mm: first, unenhanced T1-weighted spin-echo (TR/TE, 400/15); second, unenhanced T2-weighted fast spin-echo (4,000/85; echo-train length, 12); third, diffusion-weighted simulated acquisition mode (STEAM) sequence (6,000/30; number of excitations, 3; b values, 50, 250, 500, 750, 1,000, 2,000, 3,000, and 4,000 s/mm2; small delta, 9 milliseconds; T2 time determination [6,000/30, 50, 80, 120]); and fourth, contrast-enhanced T1-weighted spin-echo (400/15) at 2 minutes after the injection of 200 µL of 0.5 mol/L solution (1:6 dilution) of gadopentetate dimeglumine (Magnevist, Berlex) administered via tail vein at a rate of 40 µL/s.

After MRI and about 4 hours after the application of HIFU, the animals were euthanized, and the tissue where focused ultrasound beams had been applied was taken out and snap-frozen for histologic and microarray analysis. The MR images were used to locate tissue changes that had taken place due to the application of focused ultrasound beams.

Image Analysis
Postprocessing of the MR images was performed using MRVISION, version 1.5.4b, (MRVision Co.) for analyzing the tissue pixel intensities of the treated and untreated tumors. For semiquantitative analysis, the mean signal intensities of the whole tumor, fat tissue, and oil phantom were measured on the T2-weighted fast spin-echo sequence and the T1-weighted spin-echo sequence before and after contrast administration. They were measured using a monitor-defined circular region of interest (ROI) with 16–150 pixels for the whole tumor, 3–11 pixels for the fat tissue, and 29–64 pixels for the oil phantom. For signal intensity measurement, an ROI was placed on the image that contained the maximal cross-sectional area of each location. For measurement at different times, the size and location of the ROI were kept constant. The signal-to-noise ratios (SNRs) were calculated using the mean signal intensities of the areas and the SD of the background noise. The diffusion coefficient and the T2 time of the tumor tissue before and after heat exposure were determined by using Matlab software, version 2.0 (Mathworks).

Histology and Immunohistochemistry
To assess the histologic changes due to the focused ultrasound beams, we performed H and E staining, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL) staining, and immunohistochemistry for heat shock protein 70 (HSP70). For H and E staining, the tissue samples were preserved in 10% formalin solution for 96 hours. Subsequently, they were embedded in paraffin, sectioned, stained with H and E, and mounted on glass slides. TUNEL assay is used for detection of tumor apoptosis. The tumor TACS Kit (R&D Systems) was used for TUNEL staining procedures. Briefly, tumor samples were first fixed with 3.7% formaldehyde and the cell membranes were permeabilized with Cytonin reagent (protease-free saponin-based buffer, Trevigen). DNA strand breaks were then labeled with biotinylated nucleotides in TUNEL buffer at 37°C for 1 hour. Apoptotic cells were visualized with brown precipitates generated by streptavidin-conjugated horseradish peroxidase (HRP) in the presence of diaminobenzidine (DAB). The samples were then counterstained with 1% methyl green to show viable cells.

For the immunohistochemistry, the air-dry slides were fixed in acetone, dried, and put into a 0.3% hydrogen peroxide solution. Slides were incubated with the HSP70 primary antibody (Stressgen Biotechnologies) and then incubated with a biotinylated goat antimouse immunoglobulin (Jackson Immunoresearch Laboratories) followed by incubation with Streptavidin-HRP (Jackson Immunoresearch Laboratories). In the final steps, the sections were reacted with diaminobenzidine (DAB) chromogen and counterstained in hematoxylin, dehydrated, cleared, and mounted.

Microarray Analysis
The tissue samples (> 500 mg) were flash-frozen at a temperature of –80°C. The total RNA was isolated using TRIzol Reagent (GibcoBRL Life Technologies), and the double-stranded cDNA (complementary DNA) was synthesized by using the SuperScript Choice system (Life Technologies). Next, the cDNA was extracted and precipitated. Biotinilated cRNA (complementary RNA) was synthesized using the Bio Array High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). After incubation, the labeled cRNA was cleaned up according to the RNeasy Mini Kit (Qiagen) protocol. The cRNA was fragmented and hybridized on the Murine Genome U74Av2 Set Array (Affymetrix). The chips were washed and stained with streptavidin phycoerythrin (SAPE, Molecular Probes). To amplify staining, streptavidin phycoerythrin solution was added twice with an antistreptavidin biotinylated antibody (Biotinylated Anti-Streptavidin, Vector Laboratories) step in between. The probe array was scanned on a Hewlett Packard Gene Array Scanner at an excitation wavelength of 488 nm. The amount of light emitted at 570 nm was proportional to the bound target at each location on the probe array.

After hybridization and scanning, the microarray images were analyzed using Microarray Suite 4.0 (Affymetrix) and Gene Spring 4.0 (Silicon Genetics) software. All samples were prepared as described and hybridized onto the Affymetrix Murine Genome U74Av2 Set array, which represents nearly 36,000 full-length murine genes and expressed sequence tag (EST) sequences. Each chip contains 16–20 oligonucleotide probe pairs per gene or cDNA clone. A significant fold change between treated and untreated controls was considered at an average change of ≥ 2.0. The fold change calculations include a series of statistical parameters considering background and noise intensity within each gene chip [12]. The software calculates an average of the two images, defines the probe cells, and computes intensity for each cell.

Western Blot
The HIFU-treated tissue samples and the nontreated tissue samples were placed in lysis buffer (8 mol/L urea/2% 3[3-cholaminopropyl diethylammonio]-1-propane sulfonate [CHAPS] with proteinase inhibitor) and homogenized on ice. The total protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad) with bovine serum albumin as the standard. The total protein (100 µg) of the sample was electrophoresed on 4–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto nitrocellulose membrane. Heat-shocked HeLa (human epithelial cell line) cell extract (LYC-HL101F, StressGen) was run as a positive control. The nitrocellulose membrane was blocked with nonfat dry milk in tris-buffered saline with tween (TBST) buffer overnight and probed with the anti-HSP70 monoclonal antibody (alkaline phosphated conjugated SPA-810 AP, StressGen). Immunodetection of the protein was achieved through use of ECF Reagent (Amersham/Vistra) and imaged on a phospher imager.

Statistical Analysis
To test the SNR changes, the differences of the diffusion coefficients and the T2 times of the tumors exposed to focused ultrasound beams were compared with the untreated controls. The statistical analysis was performed using the Mann-Whitney U test, assuming a significance level of < 5%. The statistical analysis comparing the relative gene expression levels (fold change) of genes of the continuous-wave HIFU- and the short-pulse wave HIFU-treated tumors with the untreated controls was performed using the Student's paired t test, assuming a significance level of < 5%. In addition, the fold change of the continuous-wave HIFU-treated tumors was compared with the short-pulse wave HIFU-treated tumors.


Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MRI
The MR images showed significant differences between the tumors treated with continuous-wave HIFU (Figs. 2D–2F) and those treated with short-pulse wave HIFU (Figs. 2G–2I). In both applied modes, unenhanced T1-weighted images showed homogeneous intensity throughout HIFU-treated tumors and their untreated controls (Figs. 2A–2C). Contrast-enhanced T1-weighted images obtained 2 minutes after injection of gadopentetate dimeglumine showed homogeneous intensity in the tumors treated with the short-pulse wave HIFU mode. However, in the tumors treated with the continuous-wave HIFU mode, total contrast medium uptake was dramatically decreased by about –35.3% ± 3.1% (p < 0.0001) (Figs. 2D–2F). At the border between normal and devascularized tissue, a rim of higher contrast medium uptake was seen. In the T2-weighted images, no changes in the short-pulse wave HIFU mode were seen, whereas in the continuous-wave HIFU mode, a signal intensity increase appeared (Figs. 2D–2F). The increase in SNR values of the T2-weighted images was statistically significant (p = 0.0001). In the short-pulse mode, a slight increase in the diffusion coefficient could be found; however, this increase was not significant. In the continuous-wave mode, there was also a significant increase in the diffusion coefficient (p < 0.0001), and a significant increase in the T2 time (p = 0.011) could be observed (Table 2).


Figure 5
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  		Fig. 2D —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Images after application of continuous-wave HIFU. Unenhanced T1-weighted image is D; no contrast material uptake (arrow) on contrast-enhanced T1-weighted image (E) was seen as sign of devascularization. On T2-weighted image (F), treated tissue shows higher signal intensity (arrow).

 

Figure 7
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  		Fig. 2F —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Images after application of continuous-wave HIFU. Unenhanced T1-weighted image is D; no contrast material uptake (arrow) on contrast-enhanced T1-weighted image (E) was seen as sign of devascularization. On T2-weighted image (F), treated tissue shows higher signal intensity (arrow).

 

Figure 8
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  		Fig. 2G —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Images after pulsed-wave HIFU. No differences in signal intensities were seen between untreated tumors and tumors treated with pulsed-wave HIFU.

 

Figure 10
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  		Fig. 2I —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Images after pulsed-wave HIFU. No differences in signal intensities were seen between untreated tumors and tumors treated with pulsed-wave HIFU.

 

Figure 2
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  		Fig. 2A —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Control images; no application of high-intensity focused ultrasound (HIFU). Unenhanced T1-weighted images. In unenhanced T1-weighted image (A), no contrast uptake was seen as sign of devascularization.

 

Figure 4
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  		Fig. 2C —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Control images; no application of high-intensity focused ultrasound (HIFU). Unenhanced T1-weighted images. In unenhanced T1-weighted image (A), no contrast uptake was seen as sign of devascularization.

 

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  		TABLE 2: MRI Before and After Application of High-Intensity Focused Ultrasound (HIFU)

 

Histology and Immunohistochemistry
In the samples treated with continuous-wave HIFU (Figs. 3D–3F), H and E staining revealed necrosis in the center of the treated area. At the margins, the cells appeared to be very compact and dense. In these areas, TUNEL staining shows apoptotic cells. In the same region where the apoptosis occurred, the immunohistochemistry revealed a high expression of HSP70. In the short-pulse wave HIFU mode (Figs. 3G–3I) of focused ultrasound beams, H and E staining showed multifocal and coalescing areas of acute coagulation necrosis. Cells composing these irregular necrotic areas are characterized by condensation and pyknosis of nuclear chromatin and shrinkage and hypereosinophilia of cell cytoplasm compared with the nontreated tumor. TUNEL staining revealed a number of cells with markedly increased positive staining compared with the nontreated areas of the tumor. In the immunohistochemistry, no significant heat shock protein expression could be detected.


Figure 14
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  		Fig. 3D —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. After application of continuous-wave high-intensity focused ultrasound (HIFU), necrosis in tumor (arrows) was seen. In TUNEL staining (E), apoptotic cells and in immunohistochemistry, positive stain for HSP70 (F) was seen.

 

Figure 16
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  		Fig. 3F —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. After application of continuous-wave high-intensity focused ultrasound (HIFU), necrosis in tumor (arrows) was seen. In TUNEL staining (E), apoptotic cells and in immunohistochemistry, positive stain for HSP70 (F) was seen.

 

Figure 17
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  		Fig. 3G —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. In pulsed-wave mode, note many apoptotic cells (arrow, H) in TUNEL staining (H) but no stain for HSP70 (I) in immunohistochemistry.

 

Figure 19
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  		Fig. 3I —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. In pulsed-wave mode, note many apoptotic cells (arrow, H) in TUNEL staining (H) but no stain for HSP70 (I) in immunohistochemistry.

 
Microarray Analysis
Gene expression analysis revealed profound changes in expression levels of 23 genes being up-regulated and five genes being down-regulated in the continuous-wave HIFU-treated tumors. These genes are related to reaction to ischemia, immune system and inflammation, replication of DNA, transcription and protein synthesis, signal transduction and regulation of gene expression, regulation of cell cycle and cell proliferation, metabolism, and surface proteins and receptors. Three genes encoded for HSP70, HSP40, or related major histocompatibility index (MHC) class III proteins. The highest degree of up-regulation occurred for HSP70 and its related MHC class III gene (fold change = 23.5 ± 18.5, p = 0.001 and fold change = 12.9 ± 4.0, p = 0.001, respectively). The fold change increase of the other genes varied between 10.8 ± 24.6 (p = 0.001) for the cytochrome c oxidase and 2.3 ± 0.8 (p = 0.034) for the gene HSP40. The insulinlike growth factor binding protein 10 (IGFBP10) (fold change = 7.0 ± 3.3, p = 0.001), the aminolevulinic acid synthase (fold change = 9.4 ± 11.5, p = 0.002) and the mouse mRNA (messenger RNA) for profilin (fold change = 10.3 ± 13, p = 0.001) were also highly up-regulated genes. The histamine receptor H2 (fold change = 6.3 ± 3.1, p = 0.021), S-adenosylmethionine decarboxylase 3 (fold change = 6.2 ± 11.4, p = 0.003), Mus musculus adipocyte specific protein adipoQ (adipoQ) mRNA (fold change = 5.3 ± 9.4, p = 0.032), the interferon-inducible guanosine triphosphatase (GTPase) (fold change = 5.3 ± 6.2, p = 0.021), the hemoglobin ß adult minor chain gene (fold change = 6.4 ± 4.3, p = 0.004), and the eukaryotic translation elongation factor 1 alpha 2 (fold change = 5.8 ± 1.4, p = 0.031) were up-regulated, too. Less up-regulated were the intracellular calcium-binding protein MRP8 (myeloid related proteins) (fold change = 4.5 ± 5.7, p = 0.045) and the early growth response 1 (fold change = 4.5 ± 1.9, p = 0.024). The most down-regulated genes were the mast cell protease 2 (fold change = -5.1 ± 8.9, p = 0.003); the lectin, galactose binding, soluble 1 (fold change = -6.4 ± 2.4, p = 0.002); and the mouse 3 mRNA for ß-galactoside–specific lectin (14kDa) (fold change = -6.1 ± 15.2, p = 0.001).

In contrast, the pulsed-mode treated samples showed a completely different pattern of up-regulated and down-regulated genes. Thirty-four genes were up-regulated and 16 genes were down-regulated. None of the genes up-regulated in the pulsed-wave mode were up-regulated in the continuous-wave mode. Three genes up-regulated in the pulsed-wave mode were down-regulated in the continuous-wave mode. Four genes down-regulated in the pulsed-wave mode were up-regulated in the continuous-wave mode. The most up-regulated genes are related to the immune system and inflammation. The highest up-regulated genes were the histocompatibility 2, T region locus 23 (fold change = 9.7 ± 14.7, p = 0.001), and the cytotoxic T lymphocyte-associated protein 2 alpha (fold change = 9.7 ± 14.7, p = 0.001). In the group of regulation of the cell cycle and cell proliferation, the highest up-regulated genes were the mouse histone H2A.1 gene (fold change = 13.2 ± 20.6, p = 0.001), calcyclin (fold change = 11.8 ± 17.8, p = 0.001), and epithelial membrane protein 3 (fold change = 12.1 ± 20.7, p = 0.001). The most down-regulated genes were genes related to metabolic processes: the pyruvate dehydrogenase E1 alpha 1 (fold change = -12.6 ± 20.0, p = 0.001); the glycerol-3-phosphate acyltransferase, mitochondrial (fold change = -11.9 ± 18.5, p = 0.001); and the carnitine acetyltransferase (fold change = -8.8 ± 10.3, p = 0.001) (Table 3).


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  		TABLE 3: Gene Expression Profile for Mouse Tumors Treated with High-Intensity Focused Ultrasound (HIFU)

 

Western Blot
In the tissue lysate of the SCCVII tumors, HSP70 protein was present in the tumors treated with continuous-wave focused ultrasound beams. The detection of the HSP70 protein expression is, however, not immediately possible after focused ultrasound beam exposure (Fig. 4, lane 4 [continuous wave]). Four hours later, corresponding to the high up-regulation of the HSP70 gene, the Western blot analysis revealed the presence of the HSP70 protein (Fig. 4, lane 5 [continuous wave]). After applying the pulsed-wave mode, no HSP70 protein could be found (Fig. 4, lane 6 [pulsed wave]). This correlates with the gene expression analysis results. In the pulsed-wave mode, no up-regulation of the HSP70 gene was found, with the consequence of no increased synthesis of the HSP70 protein.


Figure 20
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  		Fig. 4 —Western blot analysis of heat shock protein 70 (HSP70) expression in SCCVII (squamous cell carcinoma) cells. Lane 1: marker proteins (M), Bio-Rad Protein Assay (Bio-Rad); lane 2: positive heat shock cell lysate (pos C), product # LYC-HL101 (StresssGene); lane 3: SCCVII cell lysate not exposed to high-intensity focused ultrasound (HIFU) treatment (notx); lane 4: continuous-wave HIFU-treated SCCVII tumor cells harvested directly after application (cw); lane 5: continuous-wave HIFU-treated SCCVII tumor cells harvested after 4 hours (cw-4h); lane 6: short-pulse wave HIFU-treated SCCVII tumor cells harvested after 4 hours (pw-4h). Each sample contained 100 µg of proteins from tissue lysates prepared as described.

 

Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thermal therapy, like radiofrequency ablation [13] and cryotherapy [14], is a therapy option for different malignant diseases. Like these therapeutic techniques, HIFU causes changes in tissues through energy deposition. HIFU has been extensively tested for trackless surgery of the brain in animals [15] and humans [16]. It is noninvasive and does not require insertion of a probe. The noninvasive nature of this technique keeps tissue intact for further evaluation after treatment. We compared two models of energy deposition (continuous- and short-pulse wave mode) using MRI, immunohistochemistry, and genomic analysis, to obtain information about the two HIFU modes. In addition, we wanted to correlate MRI, immunohistochemistry, and genomic information for developing targeted therapeutics.


Figure 3
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  		Fig. 2B —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Control images; no application of high-intensity focused ultrasound (HIFU). Unenhanced T1-weighted images. In unenhanced T1-weighted image (A), no contrast uptake was seen as sign of devascularization.

 


Figure 6
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  		Fig. 2E —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Images after application of continuous-wave HIFU. Unenhanced T1-weighted image is D; no contrast material uptake (arrow) on contrast-enhanced T1-weighted image (E) was seen as sign of devascularization. On T2-weighted image (F), treated tissue shows higher signal intensity (arrow).

 


Figure 9
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  		Fig. 2H —MRI of SCCVII (squamous cell carcinoma) tumors. From left to right, images are unenhanced T1-weighted, contrast-enhanced T1-weighted, and T2-weighted. Images after pulsed-wave HIFU. No differences in signal intensities were seen between untreated tumors and tumors treated with pulsed-wave HIFU.

 

The continuous HIFU mode is associated with high energy deposition in a short period of time (20 seconds), whereas the short-pulse HIFU mode delivers the same amount of energy over a much longer time (16.5 minutes). The continuous HIFU mode causes thermal damage in the tissue [17], whereas the short-pulse HIFU mode does not cause direct thermal damage, as reflected by MR images in our study. Postprocedural T1- and T2-weighted imaging revealed signal intensity changes in the treated tissue. It has been shown that the effect could be judged on the basis of contrast-enhanced T1-weighted images showing a partial or complete lack of contrast medium uptake, which implies devascularization, with the consequence of developing tissue necrosis [18]. In that study, contrast changes were present immediately after treatment and lasted the entire follow-up period [18]. In our study, we found the same MRI signal intensity pattern reported.

Applying the continuous HIFU mode after 4 hours, no contrast medium uptake could be seen as a sign of devascularization in the continuous HIFU mode; however, a small transitional zone of higher contrast medium uptake between devascularized and normal-appearing tissue was seen. This zone was also seen in histology and immunohistology, in which the cells appeared to be compact and dense on H and E staining. The TUNEL staining in this zone showed the most apoptotic cells, and on immunohistochemistry, HSP70 could be detected. Because of the findings on MRI, histology, and immunohistology, we can assume that in this zone a lot of the genes got up- or down-regulated. The up-regulated genes in the continuous HIFU mode are related to ischemia, immune response, and repair mechanisms.

In our experience, the most highly up-regulated genes were HSP70 and HSP40; MHC; IGFBP; the {delta}-aminolevulinic acid synthase (ALA synthase); and the cytochrome c oxidase, subunit VIIc. The heat shock proteins are a family of molecular chaperones induced by environmental stresses such as oxidative injury. They contribute to protection from and adaptation to cellular stress [19, 20]. The up-regulation of the HSP70 gene led to a production of the HSP70 protein itself, which could be confirmed by immunohistochemistry and Western-blot analysis. MHC plays a central role initiating both humoral and cell-mediated immunity [21, 22]. Immediately after tissue injury, genes are up-regulated, promoting tissue restoration. For example, a major anabolic-related gene is the insulinlike growth factor 1 (IGF1). IGF1 has similarities to insulin and possesses potent anabolic and cell growth effects [23].

The lack of contrast uptake in the continuous HIFU mode-treated tumors is a sign of devascularization leading to ischemia of the tissue. The tissue responds with the up-regulation of genes to overcome the ischemia. In our study, two genes were up-regulated related to heme synthesis and related to ischemia, the {delta}-aminolevulinic acid synthase and the ß-1-globin gene. All these different processes require energy, with the result of the up-regulation of the cytochrome c oxidase, subunit VIIc gene. Other genes such as the histamine receptor H2, the interferon-inducible GTPase, the guanine nucleotide binding protein, and T-cell specific GTPase are related to immune response and intracellular signal transduction [24–26].

The down-regulated genes such as the mast cell protease 2, lectin, galactose binding, soluble 1, and mouse 3 mRNA for ß-galactoside–specific lectin (14kDa) regulate immune and inflammation processes and show that under external stress certain metabolic processes are down-regulated.

In short-pulse HIFU mode MRI, histology, and microarray analysis showed consistent results. In MRI, the contrast medium uptake in the tumors before and after treatment with short-pulse HIFU did not differ. MRI did not show any signs of tumor devascularization; however, a slight increase in the diffusion coefficient could be found. In the H and E staining, the cells were swollen compared with the nontreated tumor. In the microarray analysis, none of the genes related to ischemia or immune response were highly up-regulated as in the continuous HIFU mode-treated tumors. This is also in accordance with the results of immunohistochemistry and the Western-blot analysis, which also did not show HSP70 protein production.


Figure 11
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  		Fig. 3A —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. Untreated SCCVII tumors show few apoptotic cells in TUNEL staining (B) and no stain for heat shock protein 70 (HSP70) (C).

 


Figure 12
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  		Fig. 3B —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. Untreated SCCVII tumors show few apoptotic cells in TUNEL staining (B) and no stain for heat shock protein 70 (HSP70) (C).

 


Figure 13
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  		Fig. 3C —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. Untreated SCCVII tumors show few apoptotic cells in TUNEL staining (B) and no stain for heat shock protein 70 (HSP70) (C).

 


Figure 15
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  		Fig. 3E —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. After application of continuous-wave high-intensity focused ultrasound (HIFU), necrosis in tumor (arrows) was seen. In TUNEL staining (E), apoptotic cells and in immunohistochemistry, positive stain for HSP70 (F) was seen.

 


Figure 18
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  		Fig. 3H —Histology of SCCVII (squamous cell carcinoma) tumors. From left to right, images are stained with H and E, terminal deoxynucleotidyl transferase-mediated dUTP (deoxyuridine triphosphate) nick-end labeling (TUNEL), and HSP70. In pulsed-wave mode, note many apoptotic cells (arrow, H) in TUNEL staining (H) but no stain for HSP70 (I) in immunohistochemistry.

 

The results show that the short-pulse HIFU mode does induce a variety of genes activating different cellular and metabolic processes. The activation of the T-cell system in tumor tissue by up-regulation of prothymosin alpha, cytotoxic T lymphocyte-associated protein 2 alpha, and the T-cell specific GTPase [27] could be observed. The up-regulation of annexin A1–lipocortin 1 in the tumors treated with short-pulse focused ultrasound beams and the up-regulation of the CD14 antigen and other genes support the hypothesis that application of short-pulse focused ultrasound beams does induce apoptosis [28, 29]. The other up-regulated genes induce a variety of cellular processes, such as genes regulating the replication of DNA, transcription, and protein biosynthesis or regulation of the cell cycle and cell proliferation.

The most down-regulated genes are also related to metabolic processes, showing that the metabolism of the cells is decreased. The pyruvate dehydrogenase E1 alpha 1, the glycerol-3-phosphate acyltransferase, mitochondrial, and the carnitine acetyltransferase are enzymes catalyzing important reactions in the glucose, amino acid, and fat acid metabolism [30–32]. The activation of these genes led to changes in the cell, either to recovery or death of the cell. Cell death has two morphologic expressions: necrosis and apoptosis. On the basis of previous studies [33, 34], it is possible that apoptosis may be induced by low-power-threshold HIFU exposure. Our results in histology and the results of microarray analysis support this hypothesis. In TUNEL staining, apoptotic cells were found in the short-pulse HIFU-treated areas but not in the nontreated tumor. Mild hyperthermia has been shown both in vitro and in vivo to inhibit tumor growth and induce apoptosis in a variety of cells, including glioma and glioblastoma cells [35].

The results presented here show that differences in gene expression can be seen depending on the applied mode of HIFU. Microarray analysis in HIFU treatment protocols allows the investigation of which genes get up- or down-regulated at which HIFU intensity. This opens the opportunity to directly influence any gene up- or down-regulation at a certain mode or certain intensity of HIFU. The induction or the down-regulation of genes can influence tissue growth to induce either tissue growth or apoptotic processes.

However, with our system, we were not able to perform temperature measurement in the tumors. Therefore, we do not know if the greater change in expression of certain genes reflects thermal differences from treatment or some other factors. In further planned studies, we want to proceed with a tumor regression study to see if the change in gene expression in the tumors treated with continuous-wave HIFU and those treated with short-pulse wave HIFU has a significant biologic effect and corresponds to greater tumor regression. We found that functional genomics can detect cellular changes induced by HIFU that cannot be visualized by standard T1- and T2-weighted MRI, diffusion-weighted MRI, or histologic analysis. These studies, therefore, show that genomic analysis can provide valuable information to characterize tissue after interventional radiologic procedures and might be included when designing studies that investigate changes in tissue morphology after interventional therapy.


Acknowledgments
 
We gratefully acknowledge Corrine Davis (Department of Comparative Medicine, Stanford University) for interpretation and Pauline Chu (Department of Comparative Medicine, Stanford University) for technical preparation of the histology slides.


References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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