The panel of images in illustrates the level of hsp70 activation in the hsp70-GFP/EMT6 cells as reported by GFP fluorescence for a range of treatment fluences from 0.1 to 1 J cm−2
. PDT treatment with a fluence of 0.5 J cm−2
caused a significantly higher GFP expression compared to the untreated control cells (P
< 0.05). summarizes the extent of GFP expression for the different treatment fluences. The GFP intensities are normalized to the control. The data shows that the level of hsp70 promoter-driven GFP expression, relative to the low constitutive basal levels in control cells, was maximally induced when cells were subjected to 0.5 J cm−2
fluence, with a ~1.7-fold higher GFP amplitude. Pair-wise t
-tests showed that fluorescence levels in these cells were significantly higher than in the untreated controls, mTHPC-only group and the two PDT treatment groups of 0.1 and 1 J cm−2
< 0.05). The GFP intensities diminished when the cells were irradiated with 1 J cm−2
, and these levels were not different from control, mTHPC only and 0.1 J cm−2
irradiated cells at the P
= 0.05 significance. These results are qualitatively comparable with those reported in our earlier molecular imaging study, where we established this cell line and used it to characterize the mTHPC-PDT induced hsp70 promoter activation in vitro
]. Although the goal of the current study was to investigate the spatial and temporal kinetics of hsp70 activation in PDT-treated tumors in vivo
, an important additional focus was to further characterize the cell line by examining the relationship among PDT-induced hsp70 activation as reported via
GFP expression, HSP70 protein levels, and cell cytotoxicity as reported by a clonogenic cell survival assay for the range of treatment fluences.
Fig. 1 Representative images of hsp70 promoter-driven GFP fluorescence in hsp70-GFP/EMT6 cell monolayers subjected to the following conditions: control ((−) mTHPC (−) light) and 0.3 mg ml−1 mTHPC-sensitized cells irradiated with fluences (more ...)
Fig. 2 Normalized mean GFP fluorescence intensity for various treatment conditions in vitro. Error bars represent SEM. The intensities were calculated from analysis of GFP fluorescence in hsp70-GFP/EMT6 cells and normalized to those measured in untreated controls (more ...)
Western blot assays of protein quantification in this study were performed to test the validity of using a fluorescent reporter model system to examine the expression profile of intracellular hsp70 promoter activation in vitro and in vivo in response to PDT. In order to investigate the relationship between GFP expression and HSP70 protein levels, at 7 hours after irradiation, cells were lysed and protein extracts were analyzed by Western blotting. As shown in , mTHPC photosensitization induces an increase in HSP70 expression in response to 0.5 J cm−2. summarizes the intensity levels of the HSP70 protein bands, which were quantified by densitometry and normalized against levels of β-actin, serving as an internal loading control. Each data point is calculated from measurements performed in at least three independent samples. We observed a ~1.8-fold increase in HSP70 after 0.5 J cm−2 PDT treatment compared to both untreated cells and cells treated with mTHPC alone. The protein levels at 1 J cm−2 dropped and at the P = 0.05 significance were not different from those measured in untreated and mTHPC-only sensitized cells. This trend in HSP70 protein levels closely reproduces the pattern of PDT-mediated hsp70 promoter-induced GFP fluorescence shown in , thereby establishing the validity of promoter-driven GFP as reporter of PDT-induced HSP expression in tumors in vivo.
Fig. 3 a: hsp70-GFP/EMT6 cells were lysed and assayed for HSP70 protein levels using Western blotting. Actin levels were used to monitor protein loading. b: Analysis of the Western immunoblots showed increased expression of HSP70 in 0.5 J cm−2 irradiated (more ...)
shows the measured clonogenic survival of mTHPC-PDT-treated hsp70-GFP/EMT6 cells. Each data point is a mean of at least three independent experiments. All of the data are normalized to the plating efficiency of control ((−) drug, (−) light) cells. Clonogenic survival measured in cells subjected to overnight incubation with 0.3 μg ml−1
mTHPC, but not irradiated was 98%. Survival for light-only controls ((−) mTHPC, (+) 1 J cm−2
), and ((+) mTHPC, (−) light) cells were similar (data not shown). The objective of presenting the clonogenic data was to offer a qualitative interpretation for the correlation between reduced cell survival and irradiation-induced decrease in GFP at higher fluences. We find that optimal hsp70 activation reported via
GFP and HSP70 protein expression are associated with in vitro
treatment fluences corresponding to 30% cell survival. At the higher fluence of 1 J cm−2
, the significantly lower level of GFP fluorescence and HSP70 expression correlates well with <1% cell survival. This is qualitatively consistent with our earlier findings, where we showed that reduced cell viability, measured by trypan blue exclusion assay, is responsible for the reduction in GFP intensity with increasing PDT doses [11
]. Our results are in qualitative agreement with a report by Luna et al. [9
], in which the authors observed that CAT expression, under the control of an hsp70 promoter in RIF cells, initially increased with increasing NPe6-PDT doses and then decreased. The authors attributed the decrease in CAT expression to the PDT cytotoxic response.
Fig. 4 Clonogenic survival of hsp70-GFP/EMT6 cells sensitized with 0.3 μg ml−1 mTHPC for 24 hours and subjected to no light, and fluences of 0.1, 0.25, 0.5, 0.75, and 1 J cm−2. All of the data are normalized to the plating efficiency (more ...)
We next used tumor growth control to compare the therapeutic efficacy of a range of treatment protocols in BALB/c mice bearing ear tumors derived from the stably transfected hsp70-GFP/EMT6 cells. Tumors were treated when they reached ~3 mm in diameter. Tumor volume doubling was the end point used as a measure of treatment failure, and cures were defined as no evidence of tumor 90 days after PDT. The Kaplan–Meier plots of demonstrate that the tumor response to mTHPC-PDT was dramatically enhanced with fluences of 2 and 4 J cm−2 relative to that observed with 0.1 J cm−2. Among the eight mice treated with 4 J cm−2, 100% were cured as defined by no evidence of tumor 90 days after irradiation. Hundred percent cures was also observed in tumors treated with 10 J cm−2 (data not shown). Among the 16 mice treated with 2 J cm−2, 15 (93%) were cured. However, no cures were observed in the 0.1 J cm−2 group (n = 8), where the median tumor volume doubling time was 7 days and not significantly different from untreated controls ((−) light, (−) mTHPC), which had a median tumor doubling time of 6 days (n = 8).
Fig. 5 Kaplan–Meier curves of tumor responses to mTHPC-PDT. Laser irradiation at 658 nm was performed at an irradiance of 20 mW cm−2 for a range of fluences from 0.1 to 10 J cm−2 at 24 hours after intravenous administration of 0.3 mg (more ...)
With the mTHPC-PDT treatment conditions that result in non-curative and curative tumor responses established, the stable transfection of EMT6 cells with a reporter GFP gene construct under the control of an hsp70 promoter allowed us to evaluate in vivo the response of the hsp70 promoter to these treatment protocols, using wide-field stereofluorescence and confocal fluorescence imaging. The stereomicroscope system was used for the long-term analysis and for quantification of the GFP fluorescence intensities in vivo. Shown in is a representative panel of images illustrating the time course of GFP expression in the same field of view (FOV) of an EMT6 tumor before mTHPC administration (control), prior to PDT irradiation (mTHPC only, no light), and at 7, 24, 30, and 48 hours after treatment with 0.1 J cm−2. The images illustrate that the expression at 7 and 24 hours post-PDT is higher than control and mTHPC-only levels. There is a slight decrease in GFP at the 30 hours time-point followed by a rebound in fluorescence at 48 hours. This phenomenon of an oscillation in GFP levels following an initial increase was observed in some but not all cases. It may be hypothesized that the initial increase in GFP expression is due to direct PDT-induced oxidative stress, and the second wave is associated with the onset of a sub-sequent insult initiated by therapy-induced perfusion deficits. This observation of oscillatory behavior in hsp70-promoter induction demonstrates the complex temporal heterogeneity of molecular responses that can be visualized, in the same animal, using a fluorescent reporter protein, such as GFP. The expression pattern of GFP as a function of time after treatment with a curative fluence of 10 J cm−2 is shown in . Qualitatively, we observe that, unlike the 0.1 J cm−2 case where GFP signal increases after treatment and remains sustained at high levels until at least 48 hours post-PDT, GFP expression peaks at 24–30 hours time and then declines. The decrease at longer time-points is likely associated with cellular damage and lysis.
Fig. 6 In vivo time course analysis of hsp70 promoter-driven GFP expression in an individual hsp70-GFP/EMT6 ear tumor subjected to mTHPC-PDT with 0.1 J cm−2. All images were acquired using wide-field stereofluorescence microscopy under the identical (more ...)
Series of stereofluorescence images illustrating hsp70-promoter driven GFP fluorescence up to 48 hours in an individual ear tumor treated with a curative fluence of 10 J cm−2. The FOV in the images is 9.5 mm × 7 mm.
A quantitative analysis of the average fluorescence intensities in these mice at the indicated time-points for the different treatment fluences is summarized in . The GFP levels in unsensitized tumors illuminated with irradiation fluences of 0.1–10 J cm−2 were similar to those observed in control tumors that received neither mTHPC administration nor light (data not shown). The GFP intensities in each of the plots are normalized to the value obtained from analysis of control tumors (no mTHPC, no light). The intensity of the hsp70 promoter-induced GFP levels is highest with 0.1 J cm−2, with a maximum at 48 hours. Average GFP signals at 48 hours are ~2.5-fold higher than those in untreated tumor controls. Treatment fluences of 2 and 4 J cm−2 also resulted in induction of hsp70, but the relative increase in maximum GFP fluorescence with respect to controls is more modest at ~1.8- and 1.4-fold, respectively. Consistent with the panel of images shown in , tumors subjected to 10 J cm−2 showed a trend of initial increase in GFP expression up to 24 hours followed by a decrease. Relative to mTHPC-only tumor controls, at 24 hours the tumors irradiated with 10 J cm−2 exhibited a statistically significant (P < 0.05) increase of ~23% higher GFP fluorescence, while the levels at 48 hours were not statistically different from those in the untreated control and mTHPC-only groups. The GFP levels at 24 hours post-irradiation with 4 and 10 J cm−2 were not statistically different. The analyses of the wide-field imaging measurements in conjunction with the tumor growth control studies therefore demonstrate a lack of positive correlation between PDT-mediated hsp70 promoter activation and tumor response. Tumor treatment conditions that induce a strong and prolonged expression of a transgene, such as GFP, under the control of the hsp70 promoter do not result in tumor cures.
Fig. 8 Quantitative analyses of GFP fluorescence intensity in hsp70-GFP/EMT6 ear tumors subjected to fluences of 0.1, 2, 4, and 10 J cm−2. For each treatment group, the mean GFP values were normalized to that obtained from control tumors, prior to mTHPC (more ...)
It is interesting to note that 0.3 mg kg−1
mTHPC administration induced a small but significant 10–20% increase in GFP fluorescence compared to that in nonsensitized tumors. The mTHPC-induced stress response in vivo
was not totally unexpected because previous studies have shown enhanced stress protein levels in two cell lines in response to Photofrin incubation alone [28
]. The same authors reported increased HSF binding in response to SnET2 incubation, although minimal HSF binding and no HSP70 induction was observed after Photofrin incubation [4
]. Our own studies have shown mTHPC incubation induced concentration-dependent GFP expression [11
]. These varied results reported in the literature in addition to our own findings thus suggest that stress responses due to drug exposure are specific and strongly sensitizer dependent.
An interesting study by Korbelik et al. [29
] showed that PDT induces cell surface expression and release of HSPs. The extent of the cell surface HSP70 expression was related to PDT dose, with higher doses causing increased extracellular exposure. They also found that, to a lesser degree, surface HSP70 was evident on tumor cells, in vivo
and in vitro
, that were subjected to lower doses and that remained viable. As extracellular and released HSPs are considered to be an important component in eliciting immune responses [14
], we became interested in correlating PDT-induced hsp70 promoter activation as reported via
GFP fluorescence with that of extracellular HSP70. In order to perform these experiments, we adopted an in vivo
antibody labeling technique recently demonstrated by our laboratory, in which we directly injected PE-conjugated antibodies against HSP70, and were thereby able to visualize in vivo
the distribution of extracellular HSP70. As noted in Materials and Methods Section, both hsp70-promoter driven GFP fluorescence and anti-HSP70-PE were excited efficiently by 488 nm light and detected simultaneously by two appropriately filtered detectors. are in vivo
images obtained at three depths in the same tumor. The images are comprised of GFP (green) or PE (red) fluorescence, and were acquired 24 hours post-irradiation with the curative fluence of 4 J cm−2
. The images at all three depths exhibit patterns of extracellular HSP70 accumulation that are not associated with regions of the tumor that express high levels of GFP. The panel of images in demonstrates the spatial distribution of extracellular HSP70 in relation to GFP expression for the non-curative PDT dose of 0.1 J cm−2
and the curative dose of 10 J cm−2
. Each image is comprised of nine 800 μm × 800 μm individual fields, which were stitched to create a montage with a FOV of 2.4 mm × 2.4 mm, as described in the Materials and Methods Section. This ability to create large FOV mosaics of in vivo
confocal images allowed us to visualize heterogeneities in extracellular HSP70 distribution. For both treatment doses, the images were acquired 24 hours post-irradiation. In , which is a merged image of GFP and PE-conjugated anti-HSP70, with the exception of sparse regions at the tumor boundary, there is almost complete absence of extracellular HSP70. This suggests that at this non-lethal dose of 0.1 J cm−2
, even at 24 hours where hsp70 activation in these tumors is ~1.7-fold higher than in untreated controls (), the great majority of the tumor cells do not undergo translocation to the cell surface and/or release of HSP70. In , which is a merged image from a tumor irradiated with 10 J cm−2
, a significantly higher degree of labeled anti-HSP70 is observed, thus indicating that at these curative doses a large fraction of the stressed cells release or expose their HSPs. lists the approximate fraction of the total pixels that are anti-HSP70 positive for the four treatment fluences that were examined. We found that while ~7% of the pixels in the 0.1 J cm−2
treated tumors exhibited anti-HSP70 positive pixels, the corresponding numbers for 2, 4, and 10 J cm−2
were ~2-, 3-, and 6-fold higher, respectively.
Fig. 9 In vivo two-color confocal images of hsp70 promoter-induced GFP fluorescence (green) and extracellular HSP70 (red) in an hsp70-GFP/EMT6 tumors subjected to mTHPC-PDT. Extracellular HSP70 was labeled using a PE-conjugated antibody against HSP70. a–c (more ...)
Fraction of Pixels That Were Anti-HSP70 Positive in Imaged Tumor Fields Corresponding to PDT Doses From 0.1 to 10 J cm−2
In summary, the in vivo
imaging studies have allowed us to explore a range of mTHPC-PDT conditions and establish treatment regimes that result in the intracellular activation of HSP70 and its extracellular release. To the best of our knowledge, we have presented the first in vivo
images of extracellular HSP70 in response to PDT-mediated oxidative stress. Results from in vivo
wide-field and microscopic scale confocal imaging indicate that maximal and sustained activation of hsp70 promoter correspond to PDT doses that are non- or sub-curative, while conditions that lead to complete tumor cures exhibit lower levels of hsp70 promoter-induced GFP expression, and produce significant accumulation of surface-exposed or extracellular HSP70. Hsp70-driven GFP expression and accumulation of extracellular HSP70 exhibit minimal spatial correlation in tumors treated with a non-or sub-curative dose. A dichotomy exists between the effects of HSP based on its relative location, intra- versus extracellular. Findings from several studies have shown that the activation of intracellular HSP70 induces the cell’s anti-apoptotic mechanisms [30
] and is anti-inflammatory [31
], while extracellular HSP70 stimulates cytokine and chemokine synthesis, up-regulates co-stimulatory molecules and enhances anti-tumor surveillance [32
]. Our finding that treatment conditions that induce maximal promoter activation of HSP70 do not result in tumor cure is therefore consistent with the cytoprotective role of activated intracellular HSP70. At higher PDT doses, the positive correlation between tumor cure and the extent of extracellular HSP70 content is also in agreement with the hypothesis that cellular necrosis causes the discharge of intracellular contents into the extracellular milieu, thereby liberating immune-stimulatory “danger” molecules such as HSPs that contribute to long-term tumor growth control.