2.1.

Cell Lines

Five human cancer cell lines were used in this study, all of them representative of a cancer type suitable for clinical HAL-PDT. The cell line A431 (ATCC CRL‐1555) is an epidermoid carcinoma and serves as a model system for skin cancer. The A549 cell line (ATCC CCL-185) is a lung carcinoma and HeLa S3 (ATCC CCL-2.2) is a cervical adenocarcinoma. WiDr (ATCC CCL-218) is a colorectal adenocarcinoma and T24 (ATCC HTB-4) is a transitional cell carcinoma from urinary bladder.

2.2.

Light Sources

To obtain a homogenous light field at appropriate wavelengths and within a range of irradiance values, light sources based on a light emitting diode (LED) array were used for this study.²³ Each lamp consists of an array containing 247 ($13\times 19$ diodes, 624 nm, illumination field $9\times 14\mathrm{cm}$) or 260 ($13\times 20$ diodes, 410 nm, illumination field $9\times 15\mathrm{cm}$) LEDs (Roithner LaserTechnik, Vienna, Austria) connected in parallel. The irradiance of these LEDs is adjusted by the current applied as described by Pieslinger et al.²³ The assembly of the LEDs and the distance from the LEDs to the illumination field are optimized to achieve a homogenous light field (maximal variation 10%). The LED light source for blue light illumination (LED type VL410-5-15, dominant wavelength 410 nm, spectral half-wave width 18 nm) allows for irradiance values up to $7.0\mathrm{mW}/\mathrm{c}{\mathrm{m}}^2$, and the red light illumination source (LED type R5CA5111P, dominant wavelength 624 nm, spectral half-wave width 20 nm) delivers up to $35.0\mathrm{mW}/{\mathrm{cm}}^2$. The emittance spectra can be found in Ref. 24 and the light viewing angle is 15 deg. Irradiance was measured in a grid pattern (covering the whole light field) by an LI-189 light meter equipped with a PY pyranometer detector (LI-COR, Lincoln, Nebraska, diameter 7.5 mm) and a LabMaster Ultima laser measurement system with a vis-sensor (Coherent Inc., Wilsonville, California, diameter 8 mm). Both light meters had a typical accuracy of $\pm 3\%$ for the total system. The light transmission of the microplates used is reported to be close to 100% at wavelengths $>350\mathrm{nm}$ according to information from the manufacturer. The transmission spectrum should, therefore, be close to that of the lamp itself.

2.3.

Cell Culture

All growth media were supplemented with 10% (v/v) fetal bovine serum, 2 mM l-glutamine, $100\mathrm{U}{\mathrm{ml}}^{-1}$ $\text{penicillin}/0.1\mathrm{mg}{\mathrm{ml}}^{-1}$ streptomycin, amphothericin B ($2.5\mu \mathrm{g}/\mathrm{ml}$), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), all obtained from Sigma (Oslo, Norway). For culture of T24 and A431, 1-mM Na pyruvate (BioWhittaker, Lonza, Belgium) was also added to the media. All cell lines used Dulbecco's modified eagle medium (DMEM) as a basic medium except for WiDr cells which were cultured in RPMI 1640 medium. Subcultivation was done by rinsing the cells twice with phosphate buffered saline (PBS) (37°C) before detachment by trypsinization. For all experiments, cells were seeded in appropriate cell culture dishes the day before treatment. Cell numbers (total, live, and dead) were determined by using the Countess™ system (Invitrogen™, Oslo, Norway) and the number of live cells was used for calculating the seeding.

2.4.

Hexyl 5-Aminolevulinate Photodynamic Therapy

A stock solution of 10 mM HAL dissolved in PBS (adjusted to pH 6.0) was freshly prepared on the day of the experiment and sterilized by filtration ($0.2\text{-}\mu \mathrm{m}$ filter). The growth medium was carefully removed from the dishes and replaced by medium without serum containing $20\mu \mathrm{M}$ HAL. After 3 h of incubation (dark conditions, 37°C, 5% $\mathrm{C}{\mathrm{O}}_2$, humidified atmosphere), the HAL medium was replaced by growth medium without HAL (with serum). The samples were illuminated from below using either of the LED arrays described above immediately after HAL removal and were subsequently placed in the incubator until further processing. All experimental steps after adding of HAL to the cells were done under subdued light conditions.

2.5.

Dose-Response

Cells were seeded (1 day before treatment) in 96-well cell culture microplates (black walls, clear bottom, Optilux, Falcon, VWR, Norway, see picture in Fig. 1) at the following densities, resulting in 50% to 60% confluency at the day of treatment: A431: 10,000 cells/well, A549: 10,000 cells/well, HeLa S3: 11,000 cells/well, T24: 9000 cells/well and WiDr: 11,000 cells/well. The standard filling volume was $100\mu \mathrm{l}/\mathrm{well}$. Six wells in each row contained cells while the outer ones only contained medium to minimize evaporation. Every experiment was accompanied by a “control” [no Photosensitizer (PS), no light], a “light-only” sample (no PS, maximal light dose), and a “HAL-only” sample (PS, kept in the dark). Please notice that all doses and controls necessary for the one dose–response curve were on the same microplate to ensure identical conditions except those of the varied photophysical parameters.

Fig. 1

A flow chart of the experimental design including the number of parallels and experiments is presented.

2.6.

Resazurin Assay

The resazurin assay is based on the conversion of the nonfluorescent resazurin to the fluorescing resorufin in viable cells. Approximately 22 h after illumination, 20-μl resazurin (2.5 mM, Sigma) was added into each well and incubated for 2 h in the dark incubator. Subsequently, resorufin fluorescence was determined using a microplate reader (BMG Labtech FluoStar Omega, Oslo, Norway, excitation wavelength 544 nm, emission wavelength 590 nm).

2.7.

PpIX Absorbance and Emission

Approximately 95% confluent $75{\mathrm{cm}}^2$ cell culture bottles of T24 and A431 cells were incubated with $20\mu \mathrm{M}$ HAL for 3 h. Cells were then rinsed once with PBS (37°C) and 5 ml of accutase (Sigma) was added. After a few minutes in the incubator, the detached cells were transferred into tubes. From each cell suspension, $10\mu \mathrm{l}$ were taken for cell counting by Bürker chamber. The suspensions were then centrifuged at 450 g (4°C, 5 min). The supernatants were removed and the pellets were resuspended in PBS to a final concentration of $4.0\times {10}^6\text{cells}/\mathrm{ml}$. All samples were kept on ice until spectra were read using a Fluorolog III fluorometer (Jobin Yvon-Spex, Horiba Group, Kjeller, Norway). For the fluorescence-excitation spectra, an LP700 filter was placed in front of the detector, the excitation slit was 3 nm, and the emission slit was 4 nm. Fluorescence emission was measured at 710 nm and excitation was performed in 1-nm steps from 350 to 700 nm in a signal/reference mode to make it comparable to absorption spectra. Emission spectra (550 to 750 nm, 1-nm step width) were excited at 405 nm with an excitation slit at 4 nm and an emission slit at 3 nm.

2.8.

Caspase 3/7 Activity

The lethal dose (LD) values as determined from the dose–response curves for A431 and T24 cells in Figs. 2 and 3 were used for this part of the study. For determination of the caspase activity, cells were seeded the day before treatment in Optilux-microplates (cell density: 9000/well for T24 10,000/well for A431). HAL-PDT was done as described above, but the cells were covered with only $30\mu \mathrm{l}$ medium (with serum) per well after illumination. At 0, 2, 5, and 8 h post illumination, a microplate was allowed to equilibrate to room temperature before $30\mu \mathrm{l}$ of Caspase-Glo® 3/7 Reagent (Promega, Oslo, Norway, room temperature, in accordance with the manufacturers’ protocol) was added to each well. The samples were mixed on a plate-shaker at 500 rpm for about 30 s and were subsequently incubated at room temperature for 1 h before luminescence was measured in the plate reader (BMG Labtech FluoStar Omega).

Fig. 2

Dose–response curves for five human cancer cell lines treated with blue light (410 nm) HAL-PDT ($20\mu \mathrm{M}$, 3 h, serum free). Three different irradiance values (0.7, 3.5 and $7.0\mathrm{mW}/\mathrm{c}{\mathrm{m}}^2$) were used for all cell lines. Cell viability is relative to an untreated control in the same microplate (no HAL, no light) and the $0\mathrm{J}/\mathrm{c}{\mathrm{m}}^2$ point is the “HAL no light” value. Each point is the average of two to five experiments (each in sextuplicate) with standard error of the mean (SEM) indicated by error bars.

Fig. 3

Dose–response curves for five human cancer cell lines treated with red light (624 nm) HAL-PDT ($20\mu \mathrm{M}$, 3 h). In (a) all five cell lines were illuminated at $35\mathrm{mW}/{\mathrm{cm}}^2$ and in (b) A431 cells were illuminated with $7\mathrm{mW}/\mathrm{c}{\mathrm{m}}^2$ in addition ($n=6$, small SEMs on error bars). Cell viability is relative to an untreated control in the same microplate (no HAL, no light) and the $0\mathrm{J}/\mathrm{c}{\mathrm{m}}^2$ point is the “HAL no light” value. Each point in (a) is the average of two to five experiments (each in sextuplicate) with standard error of the mean (SEM) indicated by error bars.

2.9.

Calculations and Statistics

All calculations for LD values are based on Figs. 2 and 3. The LD’s are simply read out, such as ${\mathrm{LD}}_{25}$ equals the dose needed to obtain 25% cell death, ${\mathrm{LD}}_{50}$ equals the dose needed to obtain 50% cell death and so on. Unfortunately, the exact same doses were not used for all experiments. This made it difficult to make a statistical comparison between the curves in Figs. 2 and 3. The reason that doses were sometimes slightly altered between the independent experiments was to improve the measurement points in order to show the expected sigmoidal shape of the curve. Normally one expects an experimental error of 10% to 15% in MTT and resazurin assays. For calculating the mean and standard error of the means (SEMs) in Figs. 2 and 3, the values from sextuplicate measurements in all three to five biological replicates were added together and used to calculate mean values and SEMs.

3.1.

Dose–Response Curves of Five Human Cancer Cell Lines after Blue Light HAL-PDT

To address the question of whether the cytotoxicity of blue light illumination of HAL-induced PpIX (abbreviated as $\mathrm{HAL}\text{-}{\mathrm{PD}\mathrm{T}}_{410}$ is dependent only on total light dose or on total light dose and irradiance, we measured dose–response curves of five human cancer cell lines using three different irradiance values (0.7, 3.5 or $7.0\mathrm{mW}/{\mathrm{cm}}^2$) under otherwise identical PDT procedures.

As shown in Fig. 2, the overall light dose required to kill close to 100% of the cells varies from 0.2 to $1.4\mathrm{J}/{\mathrm{c}\mathrm{m}}^2$, depending on the cell line. The most sensitive cell line is A431 both in terms of the irradiance and light dose required for complete cell killing, whereas the other cell lines are substantially more resistant. The shape of the dose–response curves differs among the different cell lines. The viability of T24 cells drops rapidly with increasing light dose, while the dose–response curve for HeLa S3 cells has a slight slope (depending on the irradiance) with a near complete cell kill at $>1.1\mathrm{J}/{\mathrm{cm}}^2$. WiDr displays a notable similarity to T24 cells regarding curve characteristics, excluding the HAL-only measurement. Unexpectedly, a less steep dose–response curve was observed when using a lower irradiance ($0.7\mathrm{mW}/{\mathrm{cm}}^2$) compared to a 10-fold higher irradiance ($7\mathrm{mW}/{\mathrm{cm}}^2$). This was observed for all five cell lines.

The dark toxicity of $20\text{-}\mu \mathrm{M}$ HAL varied from 35% (A549) to negligible (A431, T24). Blue light illumination without PS using the maximal light dose resulted in up to 40% cell death for A549 cells, and almost no cell killing for A431 and WiDr cells. Hyperthermia from illumination could potentially cause some of the differences between high and low irradiance. However, the temperature in growth medium did not change measurably for 410 nm ($7\mathrm{mW}/{\mathrm{c}\mathrm{m}}^2$, 30 min) illumination and increased only 2 deg for 624 nm ($35\mathrm{mW}/{\mathrm{cm}}^2$, 30 min), data not shown. Since illumination is carried out at room temperature, the cells will consequently not be exposed to hyperthermia during the PDT protocols employed.

3.2.

Dose–Response Curves of Five Human Cancer Cell Lines after Red Light HAL-PDT

Red light is commonly used for ALA-based PDT in the clinic. As a result of PpIX’s low absorption of red light compared to blue light and the irradiance range of the lamp, only the highest lamp output irradiance ($35\mathrm{mW}/{\mathrm{cm}}^2$) was used for red light HAL-PDT ($\mathrm{HAL}\text{-}{\mathrm{PDT}}_{624}$). An exception is the A431 cells which required the lowest light doses. Therefore it was possible to establish a dose–response curve for $7\mathrm{mW}/{\mathrm{c}\mathrm{m}}^2$ illumination as well. For an irradiance of $35\mathrm{mW}/{\mathrm{cm}}^2$, the illumination time required to achieve near complete cell destruction was below 30 min for all cell lines tested. Except for irradiance and wavelength, the PDT parameters were identical to those in the $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{410}$ experiments. Dose–response curves for all five cell lines after $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{624}$ are presented in Fig. 3(a).

Similar to the dose–response curves for $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{410}$, the dose–response curves of the individual cell lines to $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{624}$ differ in both shape and the light doses needed to achieve close to 100% cell killing. The required light doses for near complete cell kill varied from $12.6\mathrm{J}/{\mathrm{c}\mathrm{m}}^2$ for A431 to $>70\mathrm{J}/{\mathrm{c}\mathrm{m}}^2$ for WiDr and A549. Interestingly, the order of sensitivity of the cell lines to PDT estimated from ${\mathrm{LD}}_{\sim 100}$ values ($\mathrm{A}431>\mathrm{HeLa}$ $\mathrm{S}3>\mathrm{T}24>\mathrm{WiDr}>\mathrm{A}549$) is comparable for $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{410}$ and $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{624}$. Furthermore, A549 cells showed a biphasic response to $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{624}$ (and not a biphasic response to $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{410}$): a rapid drop in viability below $10\mathrm{J}/{\mathrm{cm}}^2$ is followed by a plateau up to $40\mathrm{J}/{\mathrm{cm}}^2$. At an even higher light dose ($70\mathrm{J}/{\mathrm{cm}}^2$), the viability of A549 cells drops to nearly zero. For the cell line with highest sensitivity to $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{624}$, A431, the influence of irradiance on the overall cytotoxicity was tested. The results from this experiment are presented again in Fig. 3(b), where lower irradiance ($7\mathrm{mW}/{\mathrm{cm}}^2$) results in less efficient cell destruction for intermediate doses when compared to the fivefold higher irradiance ($35\mathrm{mW}/{\mathrm{cm}}^2$).

As expected, the dark toxicity of $20\mu \mathrm{M}$ HAL in these experiments was about the same as in the $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{410}$ experiments. Furthermore, red light illumination without PS using the maximal light dose for each cell line induced no cell death within the experimental error, except for A549, which showed about a 30% drop in viability.

To make comparison between the different cell lines and photophysical parameters easier, we have assembled the values from Figs. 2 and 3 in Table 1. We have tabulated the values resulting in 25%, 50%, 75%, and 90% cell death, for ${\mathrm{LD}}_{25}$, ${\mathrm{LD}}_{50}$, ${\mathrm{LD}}_{75}$, and ${\mathrm{LD}}_{90}$, respectively. Since the slope of the dose–response curves is very flat when approaching 100% cell death, the definition of ${\mathrm{LD}}_{100}$ is somewhat subjective, and we chose to use the term “${\mathrm{LD}}_{\sim 100}$” instead. At ${\mathrm{LD}}_{\sim 100}$, close to 100% of the cells were dead in all experiments when measured by resazurin assay and there were no signs of viability on visual inspection of the cells.

Table 1

Calculated LD values based on Fig. 2 and Fig. 3, LD values are given in J/cm2. In the upper part of the table, LD values for HAL-PDT410 are presented and the corresponding LD-values for HAL-PDT624 are presented in the lower part of the table. For a few measurements, the dark toxicity was too high to make a LD25-calculation, the missing values are denoted “–”.

3.3.

Spectra of Intracellular HAL-Induced PpIX

In order to identify PpIX and possible its derivatives by spectroscopy, fluorescence excitation [Fig. 4(a)] and emission spectra [Fig. 4(b)] of HAL-induced intracellular PS were recorded in A431 and T24 cells.

Fig. 4

Spectra of intracellular HAL-induced ($20\mu \mathrm{M}$, 3 h) PpIX and possible derivatives thereof in A431 and T24 cells ($4\times {10}^6\text{cells}/\mathrm{ml}$). The spectra from untreated cells are subtracted from the presented PpIX spectra. (a) Fluorescence at 710 nm was measured for excitation wavelengths from 350 to 700 nm at 1 nm steps in signal/reference mode, making the spectra equivalent to absorption spectra. (b) Cells were excited at 410 nm and emission measured at 1 nm steps from 550 to 750 nm.

A431 and T24 were chosen as models for a PDT sensitive and a more resistant cell line, respectively. A431 is the most sensitive to HAL-PDT of all tested cell lines for both blue and red light illumination, while T24 is one of the most resistant cell lines when one compares doses required to achieve close to 100% cell killing. Both fluorescence excitation and emission spectra of untreated cells were measured (negligible values) and subtracted from the spectra measured from the incubated cells [Figs. 4(a) and 4(b)]. For both cell lines, the Soret peak is clearly visible at 410 nm and the maximum of the Q-band in the red wavelength range is close to 633 nm under the conditions used [Fig. 4(a)]. The ratio of the fluorescence excitation peaks (equivalent to absorption peaks) at 410 and 633 nm between the two cell lines A431 and T24 is 0.6 (T24:A431).

At 624 nm, the red light fluorescence excitation peak has dropped to 55% from its maximum at 633 nm. Therefore, the overall illumination period could be reduced by roughly one half if a light source with a maximum at 633 nm was used for illumination instead of the 624 nm as used in the present experiments.

The ratio between the peak heights of 410 and 624 nm is 56.3 for A431 and 56.4 for T24 cells, under the conditions used in this study [Fig. 4(a)].

T24 cells had lower fluorescence emission than A431 cells [Fig. 4(b)]. Red light fluorescence emission peaks at 636 nm for both cell lines, while a smaller maximum was determined at 706 nm. For both peaks, the overall fluorescence from T24 cells is about half of the fluorescence value from A431 cells.

3.4.

Caspase 3/7 Activity in A431 and T24 Cells Induced by HAL-PDT₄₁₀ and HAL-PDT₆₂₄

To test whether different illumination parameters influence apoptosis induction in A431 and T24 cells, caspase 3/7 activity was measured at 2, 5 and 8 h post HAL-PDT. These time points were chosen according to the results of a pilot study. Caspase 3/7 activity is considered a hallmark of apoptosis. Both cell lines were selected due to their property to show the full apoptotic phenotype, clear caspase induction, and comparable susceptibility toward HAL-PDT [see Figs. 2 and 3(a)]. A431 and T24 cells were again used to represent sensitive and more resistant cell lines to HAL-PDT, respectively. All LD values in this study are based on interpolated data from Figs. 2 and 3, therefore, biological and technical variations in these experiments are carried on into the LD values. In addition, the caspase measurements were first done based on the LD values from one dose-response experiment. The light doses were kept fixed in all three independent experiments, but it turned out after all dose-response experiments were done for Figs. 2 and 3 that the light doses used to obtain the bars in Figs. 5 and 6 did not result in identical LD values for all different photophysical parameters. As a result, the doses are assembled in subranges in Figs. 5 and 6. All values shown in Figs. 5 and 6 are relative to an untreated control (no light, no PS) set as 100%.

Fig. 5

Caspase 3/7 activity of A431 and T24 cells following HAL-PDT with 410-nm light and (a) $0.7\mathrm{mW}/\mathrm{c}{\mathrm{m}}^2$ and (b) $7.0\mathrm{mW}/\mathrm{c}{\mathrm{m}}^2$. Values are related to an untreated control, set as 100%. Caspase 3/7 activity was measured for different light doses at 2, 5, and 8 h after completed illumination. Bars represent averages from three independent experiments with triplicates in each experiment and SEMs are indicated.

Fig. 6

Caspase 3/7 activity in A431 and T24 cells following HAL-PDT with 624 nm light at $35.0\mathrm{mW}/{\mathrm{cm}}^2$. Values are related to an untreated control, set as 100%. Caspase 3/7 activity was measured for different light doses at 2, 5, and 8 h after completed illumination. Bars represent averages from three independent experiments with triplicates in each experiment and SEMs are indicated.

4.1.

Irradiance and Wavelength

Interestingly, our cell survival studies indicate that when using blue light and identical total light doses, high irradiance is more efficient in cell killing than low irradiance. This tendency is most strongly pronounced for A431 and HeLa S3 cells. In addition, at least for A431 cells, high irradiance red light induces a higher cytotoxicity than low irradiance at an identical total light dose. This observation may be (at least partially) ascribed to the counteracting effect of the cellular reactive oxygen species (ROS) defense systems, which have been shown to antagonize PDT when fractionated illumination is applied.³⁶ It seems likely that during longer illumination at lower irradiance, the ROS defense may have sufficient capacity to partially detoxify the PDT-induced oxidants. By contrast, high-irradiance PDT might overwhelm the antioxidative mechanisms.

The absorption ratios between 410 and 624 nm were $\sim 56$ for both cell lines, A431 and T24 [Fig. 4(a)]. This generally correlates quite well with killing efficiencies (Table 1), although there is some variation at different LD values. Wavelength-dependent light absorption by PpIX is probably a major parameter in PDT, but probably not the only one. Other factors likely to contribute are ROS detoxification, such as superoxide dismutase, as well as DNA repair and autophagy.

A difference in killing dependent on irradiance but with a constant dose has also been studied when using another sensitizer. Thus, a photofrin II PDT study on A549 cells demonstrated lower efficacy at lower irradiance values.³⁷ Although a lower irradiance may reduce killing in PDT treatment, that reduction might then to some extent be compensated by an improved oxygenation.³⁸

4.2.

Cell-Line Specific Responses

Essentially, the response to PDT treatment differs notably between cell lines, both regarding sensitivity and shape of survival curves (Figs. 2 and 3). Similar observations were made by others using different cell lines and PDT protocols.³⁹ Obviously, generation of increased amounts of PpIX is essential for efficient PDT. However, T24 has only half the amount of total PpIX when compared to A431 (Fig. 4), but is more than twice less sensitive to light (at least at wavelength 410 nm) than A431, again pointing to a complex basis for sensitivity to PDT treatment. Difference in the survival of different cell lines could possibly be explained by different intracellular distributions of PpIX. However, confocal microscopy of HAL-incubated cells ($20\mu \mathrm{M}$, 3 h) co-stained with MitoTracker Green performed in this study indicated that PpIX is mainly localized in mitochondria in all cell lines used here. After another hour of HAL-incubation, some PpIX is redistributed to the cell membrane, without notable differences between the cell lines (data not shown). To explore the basis for the different sensitivities would require substantial efforts beyond the scope of the present paper, but it is likely that cell-specific differences in response of cancer cell lines also apply to tumors.

4.3.

Apoptosis Induction

Apoptosis and necrosis are two major modes of cell death. We have specifically examined apoptosis and have assigned cell death that was not clearly apoptotic to most likely be necrotic. We consequently assume that the difference between the resazurin results and the caspase 3/7 activity measurements is caused by necrosis. However, there are additional mechanisms of cell death, including mitotic catastrophe and autophagy, both of which may, in fact, have a role in PDT.^21,40 The cell death mode apparently may depend both on the type of sensitizer and properties of the cells being treated. Apoptosis and mitotic catastrophe have some common and some unique properties.^41,42 PDT was shown to trigger immune suppression at high irradiances but immune stimulation at low irradiances in normal skin.²² This is interesting from the perspective of different death modes observed at high and low irradiance in our study. Stimulation of antitumor immunity after PDT is recognized, but the details/trigger mechanisms are still discussed.^43–46 Exploration of the effect of different illumination protocols on cell death mode in vitro may help in establishing a better basis for the choice of illumination parameters.

To examine the influence of different illumination parameters on the cell death mode, caspase 3/7 activity was measured at 2, 5 and 8 h post-illumination. These results demonstrate that the cellular response depends on the target cells as well as illumination protocol. The most pronounced difference in caspase 3/7 activity between blue and red light PDT was that $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{410}$ showed maximal caspase 3/7 activity at sub-full doses and $\mathrm{HAL}\text{-}{\mathrm{PDT}}_{624}$ maximum at full doses, which appears to be quite unique. For comparison, an ALA-PDT study on five different cell lines showed that the time course of apoptosis varied between the different cell lines and that the amount of apoptosis and necrosis varied with the cell line and dose, which is in line with the results presented in our study.⁴⁷

In conclusion, the shape of the dose survival curve, the LD values, and the amount of apoptotic cells vary significantly between the different cell lines examined. When comparing blue and red light illumination, the light absorbance of the respective wavelength corresponds well to the light sensitivity of the cells. Furthermore, there has been some uncertainty as to whether one could make a direct comparison or not between studies when different wavelengths or irradiances have been used. This study shows that if one compares results between studies with either different irradiances or wavelengths, one should be especially aware of the differences at sublethal doses.