2.1.

Tumor Model

All experiments were approved by the governmental review committee on animal care, and animals were kept under standard laboratory conditions.

Fresh fragments of tumor tissue of the experimental prostate adenocarcinoma sublines Dunning R3327-H, -HI, and -AT1¹⁰ were implanted subcutaneously in the distal right thigh of adult male Copenhagen rats (Charles River Laboratories Inc., Wilmington, Massachusetts). The well differentiated and hormone-sensitive H-tumor grows slowly with a volume doubling time (VDT) of $\sim 20\mathrm{days}$, while the differentiated but hormone-independent HI-tumor exhibits a VDT of $\sim 10\mathrm{days}$. The anaplastic AT1-tumor is hormone-independent showing a VDT of $\sim 5\mathrm{days}$.¹¹

During PAI, animals were anaesthetized with a mixture of 3% sevoflurane (Abbvie, Ludwigshafen, Germany) and air (ambient condition) at $1.0\mathrm{l}/\min$ if not stated differently. Animals were positioned on a heatable table, and ECG and respiration rate were monitored during imaging.

2.2.

Photoacoustic Imaging

Prior to PAI, the animals’ skin was thoroughly depilated by hair removal creme. Animals were placed on the heatable table and US gel was generously applied on the entire tumor volume surface. The present study was conducted with the commercial systems Vevo 2100 and Vevo 3100 (both Fujifilm VisualSonics Inc., Toronto, Canada) using a tunable Nd:YAG laser (Vevo LAZR, Fujifilm VisualSonics Inc.). The laser emits photons at a 20-Hz pulse rate with a peak energy of $\sim 30\mathrm{mJ}$ and 10 ns length.¹² Imaging was performed with the system-specific linear-array US transducers exhibiting a center frequency of 21 MHz (transducer LZ250 for Vevo 2100, transducer MX250 for Vevo 3100; both Fujifilm VisualSonics Inc.). For detection of deoxygenated and oxygenated hemoglobin, two excitation wavelengths (750 and 850 nm) were used. The PAI system was calibrated before each imaging session.

Settings for time gain compensation and signal gain were optimized for each measurement using the hemoglobin signal in the “HemoMeaZure” mode of the Vevo imaging software (Fujifilm, VisualSonics Inc.) to obtain a homogeneous signal distribution over the entire tumor. All PAI data were acquired in three-dimensional (3-D) “oxy-hemo” mode (Vevo imaging software) with a step size of 0.15 mm and low persistence if not stated differently. Tumor volumes were determined based on simultaneously acquired US B-mode images in VevoLab software (Fujifilm VisualSonics Inc.).

2.3.

Image Processing and Analysis

A protocol has been developed to assess pixel-based ${\mathrm{sO}}_2$ distributions over the entire 3-D tumor volumes using the beam-formed raw data of the signals at 750 and 850 nm, respectively. In the following, the measurement protocol is summarized.

I. Data filtering: A 3-D Gaussian filter (2 sigma) was applied to the beam-formed PA raw data for both wavelengths using the image processing software ImageJ.¹³

II. Oxygen saturation calculation: ${\mathrm{sO}}_2$ was calculated pixelwise from the PA signal at the corresponding wavelengths (${\mathrm{PA}}_{\lambda }$) using the molar extinction coefficients of deoxygenated and oxygenated hemoglobin (${ϵ}_{\mathrm{Hb}}^{\lambda }$ and ${ϵ}_{{\mathrm{HbO}}_2}^{\lambda }$, respectively¹⁴) according to

III. Data thresholding: The hemoglobin image was calculated in VevoLab¹² and was then converted into a likelihood-map representing the probability that the signal of one pixel originated from hemoglobin. If the probability of any given pixel in the likelihood-map was below a predefined threshold, the respective pixel in the ${\mathrm{sO}}_2$ image was set to zero and was excluded from further analysis.

IV. Region of interest analysis: Tumor ROIs were delineated manually according to simultaneously acquired US B-mode images.

V. ${\mathrm{sO}}_2$-histogram derivation: The ${\mathrm{sO}}_2$ distributions within the ROIs were condensed into normalized histograms using a bin size of 0.5%. For comparison of ${\mathrm{sO}}_2$ distributions, the median as well as the 25th and 75th percentiles (25/75) were extracted. Due to measurement uncertainty and noise, some of the measured ${\mathrm{sO}}_2$ distributions showed a negligible amount of histogram entries above 100% ${\mathrm{sO}}_2$.

VI. Earth mover’s distance calculation: To quantify the similarity of two histograms, the Earth mover’s distance (EMD),^15–17 a recognized measure of distance between two probability distributions,¹⁸ was calculated. It represents the minimal cost of transferring one distribution into another, when the cost is defined as the sum of the histogram entries times the distance they have to be moved.¹⁹ Recently, it has also been applied in various fields of biological and medical research.^20–22 For the present application in PAI, the EMD can be expressed as follows:

2.4.

Dependency of sO₂ Distributions on Signal Gain and Threshold

Measurements were performed at Vevo 2100 with Vevo-LAZR without persistence. Two animals were imaged with three different signal gains (36 dB, 38 dB, 40 dB). Data were analyzed for three different thresholds (0%, 15%, 30%). Both parameters were selected within a range applicable for in vivo measurements. Reproducibility of ${\mathrm{sO}}_2$ distributions was assessed by repeating this procedure after repositioning of animals. As ${\mathrm{sO}}_2$ distributions were essentially independent of the settings, all subsequent measurements were performed with an individually adjusted signal gain and a threshold of 20%. All following measurements were performed at the Vevo 3100 with the Vevo-LAZR at low persistence.

2.5.

Characterization of Tumor Sublines and Their Temporal Development

Two tumors of comparable volumes were imaged per subline (H, HI, AT1) and their ${\mathrm{sO}}_2$ distributions were pooled. Measurements were repeated at least three times over a period of up to 41 days.

2.6.

Sensitivity of sO₂ Distributions on External Changes in Oxygen Supply

The animals’ breathing gas supply was altered (Fig. 1): animals were sequentially imaged when breathing 100% oxygen (${\mathrm{O}}_2$), ambient air, and again 100% ${\mathrm{O}}_2$. After each alteration of oxygen conditions, a delay of 5 min was added allowing for adaption of the tumor to the new condition. Subsequently, an additional measurement was performed 10 min after clamping the tumor-supplying arteries with a transparent cable retainer, which induces acute hypoxic conditions in the tumors while the animals were still breathing 100% ${\mathrm{O}}_2$. Immediately thereafter, the cable retainer was removed and the animals were imaged again. This measurement procedure was performed for two tumors per subline. Measurements were repeated at the same time points as the measurement of the temporal development (Sec. 2.5). For comparison with well-oxygenated normal tissue, these measurements were performed also for the skin of two animals.

Fig. 1

Schematic illustration of the oxygenation conditions during PA measurements. Color-coded “oxyhemo” images show the same tumor ROI under different experimental conditions.

2.7.

Histology and Immunohistochemistry

After the final imaging, animals were injected intravenously with pimonidazole hydrochloride ($60\mathrm{mg}/\mathrm{kg}$, Hypoxyprobe™-1 Kit, NPI, Inc., Burlington, Massachusetts). After 1 h, Hoechst 33342 dye ($15\mathrm{mg}/\mathrm{kg}$, Merck, Darmstadt, Germany) was injected as a perfusion marker into the right ventricle of the heart 30 s before sacrificing the animals. Tumors were dissected and stored at $-80°\mathrm{C}$. Tumors were embedded in Tissue Tek (Sakura, Alphen aan den Rijn, the Netherlands), cut into $7\text{-}\mu \mathrm{m}$ thick slices, and fixated in methanol/acetone at $-20°\mathrm{C}$.

For evaluation of tumor tissue structures, cryo sections were stained with hematoxylin/eosin (H&E, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Briefly, sections were stained with hematoxylin for 5 min and washed in floating tap water for 10 min. Subsequently, they were immersed in eosin (0.5% aqueous solution) for 5 min and rinsed in distilled water before dehydration with ethanol and mounting in Eukitt (Kindler, Freiburg, Germany).

Immunofluorescence stainings were performed for pimonidazole, CD31, and carbonic anhydrase IX (CAIX). Cryo sections were incubated with signal enhancer (Life Technologies, Eugene, Oregon), blocked against unspecific binding (Dako North America, Inc., Carpinteria, California) and subsequently incubated with FITC-labeled mouse antipimonidazole antibody in 3% bovine serum albumin/phosphate buffered saline (BSA/PBS) (1:2000) over night at 4°C. The next day, sections were washed in PBS and incubated at room temperature (RT) with either goat anti-rat CD31 antibody (R&D Systems, Inc., Minneapolis, Minnesota) in 3% BSA/PBS (1:2000) for 1 h or rabbit polyclonal anti-CAIX antibody (Novus Biologicals, Littleton, Colorado) in 3% BSA/PBS (1:1000) for 2 h. After subsequent washing in PBS, sections were stained with donkey anti-goat AlexaFluor-555 antibody (1:3000, Invitrogen, Eugene, Oregon) or goat anti-rabbit AlexaFluor-555 antibody (1:1000, Invitrogen) in 3% BSA/PBS, respectively, at RT for 30 min. Sections were washed in PBS and mounted in Fluoromount^® (Dako). All sections were evaluated with an Axio Scan.Z1 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

3.1.

Dependency of sO₂ Distributions on Signal Gain and Threshold

Figure 2 and Table 1 display the ${\mathrm{sO}}_2$ distributions as well as the median and 25/75 percentiles of ${\mathrm{sO}}_2$ distributions for the different combinations of signal gains and thresholds for two HI-tumors.

Fig. 2

Normalized ${\mathrm{sO}}_2$ distributions of two HI-tumors for different signal gains and thresholds for two independent positionings of the animal. The ${\mathrm{sO}}_2$ distributions of tumor HI-1 were largely independent from signal gain, threshold, and positioning. The animal with the larger tumor HI-2 (rows 3 and 4) only exhibited identical ${\mathrm{sO}}_2$ distributions for a 0% threshold. With increasing threshold, the variability of the distribution increased due to the very low number of evaluated pixels.

Table 1

Medians and 25/75 percentiles of the sO2 distributions for the investigated combinations of signal gain and threshold. Values are given for two HI-tumors (small and large volume) and two independent positions of the animal (Repos = second positioning).

The tumor HI-1 exhibited a homogenous PA signal with a clearly detectable skin line. Visual inspection of the normalized ${\mathrm{sO}}_2$ distributions revealed only minor differences in the shape of the distributions for the different settings (Fig. 2, first and second row). Regarding the 25/75 percentile ranges, the distributions became slightly narrower with increasing threshold, however, without affecting the medians (Table 1). Repeating the imaging procedure after repositioning the animal did not reveal any differences in shape, medians, and 25/75 percentile ranges (Fig. 2 and Table 1). Depending on the signal gain during measurement and threshold during analysis, the number of pixels included into analysis ranged from 40% to 100%.

Tumor HI-2, exhibited a very weak PA signal with a hardly detectable skin line, even at a signal gain of 40 dB. Table 1 reveals that even though medians were not affected, the shape and 25/75 percentile ranges of the ${\mathrm{sO}}_2$ distributions changed somewhat depending on threshold and signal gain (Fig. 2, third and fourth row): While the histograms at 0% threshold were identical for all signal gains even after repositioning of the animal (Fig. 2 and Table 1), a large variability was observed for higher thresholds, especially in combination with a low signal gain. Under these conditions, only a negligible fraction of pixels with signals near the noise-level could be analyzed (e.g., only 10% of the pixels for all three signal gains at a threshold of 30%).

To obtain reliable and reproducible results, it was therefore important to adjust the measurement settings and especially the signal gain individually for each animal and each imaging session. The skin layer had been selected as a reliable reference for those adjustments.

3.2.

Characterization of Tumor Sublines

Figure 3 displays the pooled ${\mathrm{sO}}_2$ distributions of two tumors of comparable size per subline while the animals were breathing air. The well oxygenated H-tumor exhibited a narrow and well-defined peak with a median of 73% (25/75 percentile range: 69%/76%). Similarly, the HI-tumor exhibited a median of 71% with a somewhat wider distribution (25/75 percentile range: 62%/76%). By contrast, the distribution of the AT1-tumor was located around a median of 40% and exhibited a different shape, however, with a comparable 25/75 percentile range (34%/47%).

Fig. 3

Pooled ${\mathrm{sO}}_2$ distributions of two tumors per subline while animals were breathing air. The sublines show clearly distinguishable ${\mathrm{sO}}_2$ distributions.

3.3.

Temporal Development

PAI was repeated several times over a period of up to 41 days and as an example, ${\mathrm{sO}}_2$ distributions at different time points are displayed in Figs. 4(a)–4(c). Median values and 25/75 percentiles for all measurement time points are provided in Table 2.

Fig. 4

Temporal changes of the ${\mathrm{sO}}_2$ distributions for representative tumors of the three sublines: (a) H-2, (b) HI-3, and (c) AT1-1 tumor, imaged when the animals were breathing air. Results at other time points and for additional tumors are given in Table 2. (d) Development of tumor volume with time. As tumor HI-4 was only imaged once, it is not listed in (d). Black circles indicate imaging time points that are depicted in (a)–(c). (e) ${\mathrm{sO}}_2$ distributions and (f) representative ${\mathrm{sO}}_2$ color maps of tumor HI-3 when the animal was breathing 100% ${\mathrm{O}}_2$.

Table 2

Summary of oxygen challenge and clamping experiments for tumors and skin. Results are displayed as median and 25/75 percentiles for each tumor and skin for each oxygenation condition.

Repeating the measurement, the tumor H-2 [Fig. 4(a)] essentially maintained the shape of the distribution; however, the distributions shifted slightly toward lower ${\mathrm{sO}}_2$ values with time. During the observation period, the volume of this tumor did not change [Fig. 4(d)]. For tumor H-1, the width of the ${\mathrm{sO}}_2$ distribution increased slightly and the median values shifted slightly toward higher ${\mathrm{sO}}_2$ values (Table 2).

Tumor HI-3 exhibited a more heterogeneous ${\mathrm{sO}}_2$ distribution with a peak at high ${\mathrm{sO}}_2$ values at day 1, which then became broader with an increasing fraction of pixels at low ${\mathrm{sO}}_2$ values [$<40\%$ ${\mathrm{sO}}_2$, Fig. 4(b)]. While the peak of the tumor did not shift significantly, the fraction of low ${\mathrm{sO}}_2$ pixels changed considerably with time. Figure 4(e) illustrates the ${\mathrm{sO}}_2$ distributions of the same tumor when the animal was breathing 100% ${\mathrm{O}}_2$ rather than air. This tumor exhibited an increased heterogeneity as compared to the air-breathing condition. Distinct regions of different ${\mathrm{sO}}_2$ levels can be identified in the respective color maps [Fig. 4(f)]. The strong reduction of pixels with low ${\mathrm{sO}}_2$ values at day 41 correlates spatially with the occurrence of the central region without PA signal.

Although the volumes of the AT1-tumors increased up to sevenfold in 12 days [Fig. 4(d)], their ${\mathrm{sO}}_2$ distributions experienced only minor changes in ${\mathrm{sO}}_2$ medians and 25/75 percentile ranges [Fig. 4(c) and Table 2].

3.4.

Sensitivity of sO₂ Distributions on External Changes in Oxygen Supply

Figure 5 displays the changes in ${\mathrm{sO}}_2$ distributions for the three tumor sublines as well as for the skin when changing the external oxygen supply of the animals. For each of the cases, pooled data of two tumors (left column) and one exemplary full experiment performed in a single tumor (middle column) are shown. All medians and 25/75 percentiles are listed in detail in Table 2.

Fig. 5

Oxygen challenge and clamping experiment for each of the three sublines and the skin (reference tissue). Pooled ${\mathrm{sO}}_2$ distributions per subline and exemplary distributions of the full experiment for single animals: The left column displays the pooled ${\mathrm{sO}}_2$ distributions of two tumors of comparable volumes of the subline: (a) H (20 and $50{\mathrm{mm}}^3$), (c) HI (460 and $580{\mathrm{mm}}^3$), (e) AT1 (330 and $460{\mathrm{mm}}^3$, for the “clamped”-measurement, the distribution represents only AT1-1), and (g) the skin. Exemplary full oxygen challenge experiments performed in a single tumor per subline are shown in the middle column [(b), (d), (f), (h)]. The right column displays representative frames from 3-D PA ${\mathrm{sO}}_2$ measurements, which illustrate the characteristics of the response of the tumors after external changes in ${\mathrm{O}}_2$ supply. Note the highly nonuniform response of the HI-tumor exhibiting regions without changes during oxygen challenge (e.g., low ${\mathrm{sO}}_2$ region in the center of the tumor) and responding regions in the periphery of the tumor (with high initial ${\mathrm{sO}}_2$).

After switching the external gas supply from 100% ${\mathrm{O}}_2$ to air, the PA signal of the H-tumor immediately shifted to lower ${\mathrm{sO}}_2$ values while still maintaining its narrow and well-defined peak [Figs. 5(a) and 5(b), Table 2]. When clamping the tumor-supplying arteries, the ${\mathrm{sO}}_2$ distribution shifted to even lower values, while broadening significantly in spite of the fact that the animals were still breathing 100% ${\mathrm{O}}_2$. When executing the full oxygen challenge experiment [Fig. 5(b)], the initial ${\mathrm{sO}}_2$ distributions were restored after switching back from air to 100% ${\mathrm{O}}_2$ (“100% ${\mathrm{O}}_2$ post”) and after releasing the clamping (“100% ${\mathrm{O}}_2$ postclamping”), respectively.

When changing the external oxygen supply from 100% ${\mathrm{O}}_2$ to air, the broad ${\mathrm{sO}}_2$ distributions of the HI-tumors were shifted to lower values [Figs. 5(c) and 5(d)]. Clamping of tumors led to further shift and broadening of the peak. Again, when performing the full oxygen challenge experiment [Fig. 5(d)], the initial ${\mathrm{sO}}_2$ distributions were essentially restored after switching back from air to 100% ${\mathrm{O}}_2$ and after releasing the clamping, respectively.

In contrast to the H- and HI-tumors, essentially no differences were detected for the ${\mathrm{sO}}_2$ distributions of the AT1-tumors after switching from 100% ${\mathrm{O}}_2$ to air and after clamping [Figs. 5(e) and 5(f)].

The skin showed a broad distribution at high ${\mathrm{sO}}_2$ values when the animals were breathing 100% ${\mathrm{O}}_2$ [Figs. 5(g) and 5(h)]. When switching to air, the peak of the distribution was shifted to lower ${\mathrm{sO}}_2$ and clamping led to a further significant shift and broadening of the distributions. Again, when performing the full oxygen challenge experiment [Fig. 5(h)], the initial ${\mathrm{sO}}_2$ distributions were essentially restored after switching back from air to 100% ${\mathrm{O}}_2$ and after releasing the clamping, respectively.

Changes of the ${\mathrm{sO}}_2$ distributions during the oxygen challenge experiment were quantified by the similarity measure EMD (Fig. 6). Using the first measurement, where animals were breathing 100% ${\mathrm{O}}_2$, as a reference, it can be clearly seen that all other measurements with 100% ${\mathrm{O}}_2$ exhibit very low EMD values (i.e., high similarity), while air breathing and even more clamping exhibited much larger values (decreasingly low similarity). The AT1-tumor on the other hand showed no response to any external changes and their EMDs remained below 0.05 for all experiments.

Fig. 6

The similarity measure EMD for the oxygen challenge experiment: The EMD was calculated for all ${\mathrm{sO}}_2$ distributions measured for the three tumor sublines and the skin using the ${\mathrm{sO}}_2$ distributions of the first measurement with 100% ${\mathrm{O}}_2$ (“100% ${\mathrm{O}}_2$ pre”) as reference.

3.5.

Histology and Immunohistochemistry

Figure 7 displays representative stainings of the investigated tumors. The H-tumor showed to be highly differentiated with glandular structures comparable to normal prostate glands [Fig. 7(a)] and exhibited mature vessels [Figs. 7(d) and 7(g)]. The whole tumor was well perfused, indicated by a uniformly distributed Hoechst staining. Both H-tumors exhibited only a small, locally confined hypoxic region [Fig. 7(d)] with an increased CAIX expression [Fig. 7(j)]. Apart from those locally confined areas, both tumors were negative for pimonidazole and CAIX.

Fig. 7

Histology and immunofluorescence results of the H-, HI,- and AT1-tumor sublines (left, middle and right column, respectively): (a–c) H&E stainings, (d–f) immunofluorescence stainings for vessels (CD31 in red), hypoxia (pimonidazole in green), and perfusion (Hoechst in blue), (g–i) close-ups of the marked areas in (d–f), (j–l) immunofluorescence stainings for CAIX expression (red), hypoxia (green), and perfusion (blue). Scale bars: (a–c), (g–l): $500\mu \mathrm{m}$, (d–f): $1000\mu \mathrm{m}$.

The moderately differentiated HI-tumor contained mucin secreting glandular structures [Fig. 7(b)] with immature vessels, though not all of them were perfused [Figs. 7(e) and 7(h)]. Hypoxic areas were found to enclose vessels in a certain distance [Fig. 7(h)]. CAIX expression overlapped nearly completely with hypoxic areas [Fig. 7(k)].

The anaplastic AT1-tumor exhibited only capillaries, but no mature vessels and showed to be completely undifferentiated without any prostate-specific cells [Fig. 7(c)]. The entire tumor was pervaded by very short and thin capillaries of which only a minority was perfused [mainly at the periphery of the tumor; Figs. 7(f) and 7(i)]. Additionally, prominent hypoxic areas were found [Figs. 7(f) and 7(i)]. The entire AT1-tumor expressed CAIX, with slightly stronger expression at the border of hypoxic areas [Fig. 7(l)].

4.1.

Dependency of sO₂ Distibutions on Signal Gain and Threshold

As expected, the resulting ${\mathrm{sO}}_2$ distributions were mostly independent of signal gain, applied threshold, and positioning of the animals. The higher variability of the ${\mathrm{sO}}_2$ distributions observed for HI-2 can be explained by the overall weak PA signal as the low number of pixels that remained after thresholding cannot be considered as being representative for the tumor. Hence, we decided to adjust the signal gain individually per animal and imaging session taking the strong and distinct PA signal of the well-oxygenated healthy skin as reference. This approach is justified by the fact that tumors were always transplanted at the same location according to a fixed protocol while environmental factors, such as RT, temperature of animal heating table, and gel temperature, were kept constant. Based on these measures, the skin can be considered as reliable and reproducible reference tissue for the selection of individual measurement parameters.

4.2.

Characterization of Tumor Sublines, Temporal Changes, and Response to External Changes in Oxygen Supply

The three tumor sublines H, HI, and AT1 are know to differ with respect to several parameters, most important the oxygenation status.^10,34,35

The highly differentiated and slowly growing H-tumor is most similar to normal prostate tissue. The narrow peaks of the ${\mathrm{sO}}_2$ distributions at high ${\mathrm{sO}}_2$ values indicate homogenously oxygenated tumors, which were confirmed by immunohistochemistry (IHC), revealing mature vessels ensuring a sufficient blood supply. The shape of the ${\mathrm{sO}}_2$ distributions was maintained during the observation period without developing a shoulder at lower ${\mathrm{sO}}_2$ values as found for the HI-tumor.

In addition, PAI was sensitive enough to detect the acute changes in oxygenation induced by changing the breathing gas from 100% ${\mathrm{O}}_2$ to air as well as by clamping and the response of different animals was highly uniform. A similar finding was described by Zhao et al.³⁶ The small hypoxic fraction found in IHC stainings of the H-tumors is also in accordance with previously published work³⁷ and is negligible in comparison to the other sublines.

The ${\mathrm{sO}}_2$ distributions of small HI-tumors exhibited narrow ${\mathrm{sO}}_2$ peaks comparable to those of the H-tumors but showed an additional shoulder in the low ${\mathrm{sO}}_2$ region. HI-tumors are known to develop hypoxia with time,³⁷ which was also confirmed by the IHC staining and by the increasing fraction of low-${\mathrm{sO}}_2$ pixels during the long-term temporal observation. The immediate response to external changes in oxygen supply and the presence of responding and nonresponding tumor regions has also been described by Zhao et al.³⁸ Tumor HI-3 especially illustrates the potential of the established protocol: During the 41 days of observation, this tumor underwent major morphological changes, which were reflected by the changing shapes of the corresponding ${\mathrm{sO}}_2$ distributions. This makes the developed protocol especially interesting for longitudinal studies (e.g., after treatment), where changes in oxygenation are expected.

The anaplastic AT1-tumor exhibited narrow peaks in the low ${\mathrm{sO}}_2$ region, which remained essentially unchanged with respect to time point and oxygenation conditions ($\mathrm{EMD}<0.05$ for all comparisons). IHC staining revealed that the immature and hardly perfused capillaries could not sufficiently supply the AT1-tumors, which explains the chronically low ${\mathrm{sO}}_2$ values.³⁷ While the results of the oxygen challenge experiment are in accordance with those of Mason et al.³⁹ and Zhao et al.,³⁶ other investigators found a very small but statistically significant change in perfusion after changing the external oxygen supply from 100% ${\mathrm{O}}_2$ to air.⁴⁰ As our results were reproducible over a period of 12 days and since the comparison of different tumor sublines is based on measurements performed on the same day, any PAI system-related artifacts can be ruled-out.

Considering the different responses of the tumor sublines, PAI measurements performed during oxygen challenge experiments may be a suitable method to differentiate chronic from acute hypoxic tumors (e.g., H/HI versus AT1).

4.3.

Comparison of Tumor Sublines and Normal Tissue (Skin)

Changing the breathing gas from 100% ${\mathrm{O}}_2$ to air showed comparable responses in ${\mathrm{sO}}_2$ distributions of the H- and HI-tumors as well as the normal tissue (skin). Similar results were found by Smith et al.,⁴¹ who investigated the response of several vessel types to different breathing conditions by PAI. Using clamping, tumors and normal tissue showed different responses in our study: while the skin was strongly affected by the acute hypoxic situation, showing a dramatic decrease in ${\mathrm{sO}}_2$, the distributions of the H- and HI-tumors were shifted to less extreme ${\mathrm{sO}}_2$ values after clamping, however, the distributions were significantly broadened. The different responses of normal tissues and tumors suggest that the clamping method reduces perfusion uniformly to very low ${\mathrm{sO}}_2$ values in normal tissue but to very different degrees in different areas of the tumor. The reason for this may be the irregular and chaotic vascular structure of tumors as well as the less clearly defined access of tumor vessels to the large normal arteries. Another possible hypothesis could be a different adaption of tumor and normal cells to oxygen changes leading to various ${\mathrm{sO}}_2$ changes depending on the tissue composition and the level of dedifferentiation of tumor cells.

For detailed biological investigation of tumor hypoxia, the small number of animals per subline may be considered as a limitation. The main purpose of this study, however, was to establish the quantitative pixel-based PAI method to measure the oxygenation status of experimental tumors. Using three different tumor models with different characteristics, we demonstrated suitability, sensitivity, and feasibility of the established protocol. In the future, multimodal imaging studies may allow further validation of this PAI method and may also generate complementary data allowing for a more complete characterization of the tumors.