2.1.

Cell Lines

The F98 glioma cell line (American Type Culture Collection) used in all cell monolayer experiments was originally derived from transformed fetal CD Fisher rat brain cells following exposure to ethyl-nitrosourea on the 20th day of gestation.^17,18 The human grade IV glioma cell line (ACBT- G. Granger, University of California, Irvine) was used in the experiments on MTS. The cells were cultured in Dulbecco’s Modified Eagle Media (DMEM, Gibco, Carlsbad, CA) with high glucose and supplemented with 2 mM L-glutamine, gentamycin ($100\mathrm{mg}/\mathrm{m}$l), and 2% heat-inactivated fetal bovine serum (Gibco). Cells were maintained at 37°C in a 7.5% ${\mathrm{CO}}_2$ incubator.

2.2.

Spheroid Generation

MTS were formed by a modification of the centrifugation method first described by Ivascu and Kubbies.¹⁹ $5\times {10}^3$ cells in 200 μl of culture medium per well were alloquated into the wells of ultra-low attachment surface 96-well round-bottomed plates (Corning Inc., NY). The plates were centrifuged at 1000 g for 10 min. Immediately following centrifugation, the tumor cells formed into a disk shape. The plates were maintained at 37°C in a 7.5% ${\mathrm{CO}}_2$ incubator for 48 h to allow them to take on the usual three-dimensional spheroid form.

2.3.

PDT Monolayer Toxicity

F98 cells were incubated in $1\mu \mathrm{g}/\mathrm{ml}$ ${\mathrm{AlPcS}}_{2a}$ (Frontier Scientific Inc., Logan, UT) and DMEM for 18 h and washed out 4 h before illumination. Following incubation, cells were washed three times with phosphate buffered saline (PBS), and 5 ml of fresh medium was added. Irradiation was done using 670 nm light from a diode laser (Intense, North Brunswick, NJ). The cells were exposed to a range of radiant exposures (0.75 to $6\mathrm{J}/{\mathrm{cm}}^2$) delivered at a light irradiance of $5\mathrm{mW}/{\mathrm{cm}}^2$. Following irradiation, cells were harvested with trypsin, counted using a Coulter Counter (Beckman Coulter, Model Z, Fullerton, CA) and then incubated at various cell densities in complete media for seven days to allow for colony growth. Colonies that grew from the surviving cells were stained with crystal violet and counted ($>50\mathrm{cells}/\mathrm{colony}$) A total of four experiments were performed.

2.4.

PDT Spheroid Toxicity

Forty-eight hours after generation, spheroids were transferred from the micro plate wells into 35 mm Petri dishes, 16 to 24 per dish, and incubated in $1\mu \mathrm{g}/\mathrm{ml}$ ${\mathrm{AlPcS}}_{2a}$ and DMEM for 18 h. Following incubation, spheroids were irradiated ($\mathrm{irradiance}=5\mathrm{mW}/{\mathrm{cm}}^2$; radiant $\mathrm{exposure}=1.5\mathrm{J}/{\mathrm{cm}}^2$) with 670 nm light from a diode laser. Light was coupled into a 200 μm diameter optical fiber containing a microlens at the output end. Following irradiation, individual spheroids were placed into separate wells of a 48-well culture plate and monitored for growth. Determination of spheroid size was carried out by averaging two measured perpendicular diameters of each spheroid using a microscope with a calibrated eyepiece micrometer and their volume calculated mathematically assuming a perfect sphere. Typically, 16 to 24 spheroids were followed in each trial. Since each trial was performed three times, a total of 48 to 72 spheroids were followed for a given set of parameters. Spheroids were followed for up to 29 days.

2.5.

Bleomycin Toxicity

F98 cells were incubated in varying concentrations (0.1 to $10\mu \mathrm{g}/\mathrm{ml}$) of BLM (Sigma, St. Louis, MO) and DMEM for 4 h and then washed out. Following incubation, cells were harvested and prepared for colony assay or spheroid growth as described previously. A total of six experiments were performed.

2.6.

PCI Toxicity

F98 monolayers and ACBT spheroids were incubated in $1\mu \mathrm{g}/\mathrm{ml}$ ${\mathrm{AlPcS}}_{2a}$ for 18 h followed by 4 h in various concentrations of BLM. ${\mathrm{AlPcS}}_{2a}$ was washed out 4 h before illumination. Following incubation, cells or spheroids were irradiated with 670 nm light ($\mathrm{irradiance}=5\mathrm{mW}/{\mathrm{cm}}^2$; radiant $\mathrm{exposure}=1.5\mathrm{J}/{\mathrm{cm}}^2$). Following irradiation, cells were washed, harvested, and prepared for colony growth, while individual spheroids were placed into separate wells of a 48-well culture plate and monitored for growth as described previously.

2.7.

Two Photon Microscopy

Forty-eight hours after PDT or PCI treatment, the spheroids were stained using a combination of Hoechst 33342 and Ethidium Homodimer 1 (Invitrogen H1399, Carlsbad, CA) and were visualized using an inverted Zeiss laser-scanning microscope (LSM 410, Carl Zeiss, Jena, Germany) with an excitation wavelength of 488 nm. The Zeiss LSM 510 NLO Meta microscopy system is based on an Axiovert 200 M inverted microscope equipped with standard illumination systems for transmitted light and epifluorescence detection and a standard set of visible light lasers (an Argon laser $458/477/488/514\mathrm{nm}/30\mathrm{mW}$, a Helium: Neon laser $543\mathrm{nm}/1\mathrm{mW}$ and a Helium: Neon laser $633\mathrm{nm}/5\mathrm{mW}$) for confocal microscopy. It is equipped with a femtosecond Titanium: Sapphire laser excitation source (Chameleon-Ultra, Coherent) for multi-photon excitation with the exceptional tunability range from 690 to 1040 nm. The microscope platform is equipped with a motorized X-Y scanning stage and long-working distance and high numerical aperture objectives (10, 20, 40, and $100\times$). This system allows the differential visualization of cell nuclei using two-photon microscopy. Simultaneously detected blue and red emissions were isolated by using BP 390-465 infrared (IR) and BP 565-615 IR band pass filters, respectively. Fluorescent images were pseudo-colored blue (live) and red (dead).

2.8.

Flow Cytometry

Flow cytometry was used to determine the fraction of viable, apoptotic, and necrotic cells in treated and control spheroids. The two different fluorescent labels used were Annexin V-FITC (Beckton, Dickson and Company, Franklin Lakes, NJ) to distinguish apoptotic cells and propidium iodide (PI: Sigma, St. Louis, MO) to label necrosis. Unlabeled cells were assumed to be viable. The spheroids were incubated in 15 mL tubes for 20 min in 500 μL of a one-to-one mixture of 1% collagenase (Invitrogen Corp., Carlsbad, CA) in Hanks’ Balanced Salt Solution $1\times$ (HBSS with ${\mathrm{Ca}}^{2+}$ and ${\mathrm{Mg}}^{2+}$, Invitrogen Corp., Carlsbad, CA) to dispase (Beckton, Dickson and Company, Franklin Lakes, NJ). After incubation, the spheroids were vortexed for 5 sec, and 5 mL of PBS was added. The tubes were then centrifuged in a Beckman GS-6 centrifuge (Beckman Coulter Inc., Fullerton, CA) for 5 min at 1000 rpm. The PBS was then decanted, 5 mL of PBS was added, and the tubes were vortexed for 5 sec. The tubes were centrifuged again, the PBS was decanted, and 500 μL of PBS was added. For a final time, the tubes were centrifuged, the PBS was decanted, and $1\times$ binding buffer (Beckton, Dickson and Company, Franklin Lakes, NJ) was added to achieve a final concentration of ${10}^6\mathrm{cells}/\mathrm{mL}$. Next, 100 μL of the solution was transferred along with 400 μL of $1\times$ binding buffer to a 5 mL BD Falcon tube. The two labels were then added with 5 μL of Annexin V-FITC and 10 μL of $100\mu \mathrm{g}/\mathrm{mL}$ concentration PI. The solutions were gently agitated and then incubated for 15 min at room temperature in the dark. For each experiment, a set of control solutions was also prepared. One control remained unlabeled, the next labeled only with Annexin V-FITC, and the third labeled only with PI. Finally, after 15 min of incubating in the dark, each tube was analyzed in a Beckton Dickson FACSCalibur (Beckton, Dickson and Company, Franklin Lakes, NJ) flow cytometer along with CellQuest software.

2.9.

Statistical Analysis

All data were analyzed and graphed using Microsoft Excel. The arithmetic mean and standard deviation were used throughout to calculate averages and errors. Statistical significances were calculated using the student’s t-test, as well as the Welch’s t-test. Two values were considered distinct when their p-values were below 0.05.

Synergism was calculated when analyzing PCI treatments. The equation shown below was used to determine if the PCI effect was synergistic, antagonistic, or additive, where $\alpha$ is the ratio of the cumulative effect of two therapies administered independently to the net effect of combining the two therapies at a given dose.

In this equation, ${\mathrm{SF}}^i$ represents the survival fraction for a specific treatment. If two treatments are to be compared, the survival fractions of each separate treatment are multiplied together and then divided by the survival fraction when both treatments were applied together. The interaction is calculated based on the dose of each treatment. The resulting number $\alpha$ describes the summative effect. If $\alpha >1$, the result is synergistic (supra-additive). If $\alpha <1$, the result is antagonistic, and if $\alpha =1$, the result is simply additive.²⁰

3.1.

PDT and PCI on F98 Monolayers

In order to determine the optimal drug and light fluence levels for evaluating the effects of PCI on the F98 monolayers, titration of both drug and light dose were performed. The results are shown in Fig. 2(a) and 2(b) for increasing light dose and drug concentration, respectively. As expected, the data show an increase in PDT efficacy with increasing radiant exposure. The cells appeared to be sensitive to ${\mathrm{AlPcS}}_{2a}$-PDT as indicated by the low 50% survival dose (${\mathrm{LD}}_{50}$). As seen in Fig. 2(b), F98 cells are also sensitive to BLM exposure. The ${\mathrm{LD}}_{50}$ for BLM was approximately $0.5\mu \mathrm{g}/\mathrm{ml}$; nearly all cells were killed at a concentration of $10\mu \mathrm{g}/\mathrm{ml}$.

Fig. 2

(a) Effects of PDT on F98 colony formation. F98 cells incubated in $1\mu \mathrm{g}/\mathrm{ml}$ ${\mathrm{AlPcS}}_{2a}$ for 18 h. Wavelength of 670 nm; light fluence 0.75 to $6\mathrm{J}/{\mathrm{cm}}^2$; irradiance of $5\mathrm{mW}/{\mathrm{cm}}^2$. (b) Effects of 4-h incubation with increasing bleomycin (BLM) concentration ($\mu \mathrm{g}/\mathrm{ml}$). Each data point represents the mean of four experiments. Error bars denote standard errors.

3.2.

${\mathrm{AlPcS}}_{2a}$-PCI Efficacy on F98 Monolayers

There was a statistically significant difference in survival between cells exposed to single modality treatment (BLM or PDT) and cells subjected to PCI (Fig. 3). Greater PCI effects were observed at higher BLM concentrations. The p-values of treatment modalities compared to each other are shown in Table 1. Since PCI is a technique which relies on the combination of drug and photosensitizer, the resultant toxicities should show more than an additive effect between BLM and AlPcS2a-PDT. The degree of synergism was calculated by comparing the survival fractions of BLM and PDT alone with that of the PCI treatment for a given dose. As evidenced from the calculated $\alpha$ values, PCI demonstrated a synergistic effect, especially at the higher BLM concentration: $\alpha =1.5$ and 2.7 for 0.1 and $0.25\mu \mathrm{g}/\mathrm{ml}$ BLM, respectively (the higher the $\alpha$ value, the greater the degree of synergism).

Fig. 3

Effects of BLM, PDT, and BLM PCI on F98 colony formation. F98 cells incubated in $1\mu \mathrm{g}/\mathrm{ml}$ ${\mathrm{AlPcS}}_{2a}$ for 18 h. Wavelength of 670 nm; radiant exposure of $1.5\mathrm{J}/{\mathrm{cm}}^2$; irradiance of $5\mathrm{mW}/{\mathrm{cm}}^2$ and BLM concentrations of 0.1 and $0.25\mu \mathrm{g}/\mathrm{ml}$. *designates significant differences ($p<0.05$); each data point represents the mean of four experiments. Error bars denote standard errors.

Table 1

p-values for various F98 monolayer PCI toxicity groups. (Bleomycin concentrations 0.1 or 2.5  μg/ml; light dose 1.5 J).

3.3.

Spheroid Growth Assays

The monolayer results clearly demonstrated a highly significant PCI effect in the F98 rat glioma cell line. The next step was to determine whether such an effect could be elicited in a more complex in vitro model. This was done by performing growth assays on MTS of the human glioma cell line, ACBT. Experiments were performed on four groups: (1) control, (2) AlPcS2a- PDT, (3) BLM, and (4) AlPcS2a-PCI (PDT+BLM) with and without light irradiation. In all experiments, $1.5\mathrm{J}/{\mathrm{cm}}^2$ radiant exposure and $1\mu \mathrm{g}/\mathrm{mL}$ ${\mathrm{AlPcS}}_{2a}$ were used. Based on the monolayer results (Figs. 2 and 3), sub-optimal BLM and light doses, representing 70% to 80% cell survival, were chosen in order to optimize the PCI effect. Figure 4(a) shows the average growth kinetics measured over a four-week period. BLM concentrations of 0.1 and $0.25\mu \mathrm{g}/\mathrm{ml}$ were used in these experiments. Three identical experiments were performed with 16 spheroids in each group per experiment. Since the growth kinetics of the ${\mathrm{AlPcS}}_{2a}$ dark control was not significantly different from control cultures and the effects of light treatment on the BLM only (i.e., no photosensitizer) cultures were insignificant, they are omitted from the figure. As seen in the figure, both PDT and BLM induced a clear spheroid growth delay, but by week 4, the average size in these two treatment groups had reached control levels. In contrast, the average size of the PCI-treated spheroids increased only slightly during the four-week measurement interval. The $\alpha$ values for the PCI effects on spheroid survival at four weeks was 1.4 and 4.6 for BLM concentrations of 0.1 and $0.25\mu \mathrm{g}/\mathrm{ml}$, respectively. The toxic effects on spheroid volume growth, evaluated after four weeks in culture, of PCI of BLM were compared to the effects of BLM alone over a concentration range of 0.1 to $10\mu \mathrm{g}/\mathrm{ml}$. As can be seen from Fig. 4(b), the spheroids were much more resistant to the effects of BLM compared to cell monolayers [Fig. 2(b)]. PCI greatly enhanced the effects of the drug, and the effects of PCI with $0.1\mu \mathrm{g}/\mathrm{ml}$ BLM were equivalent to those observed at $10\mu \mathrm{g}/\mathrm{ml}$ of drug alone ($p<0.05$). Figure 5 shows the average percentage (from triplicate experiments) of the spheroids that were viable four weeks following the different treatment types. Neither BLM nor AlPcS2a, with or without irradiance, had a significant effect on overall spheroid survival after four weeks. On the other hand, there was a statistically significant ($p<0.05$) diminution in survival of spheroids subjected to PCI ($0.25\mu \mathrm{g}/\mathrm{ml}$ BLM, $1.5\mathrm{J}/{\mathrm{cm}}^2$ irradiation) compared to those exposed to either drug or PDT, with less than 20% of the spheroids remaining viable.

Fig. 4

Effects of BLM, PDT and BLM PCI on ACBT MTS growth. (a) Average volume growth measured in μl as a function of incubation time with 0.1 and $0.25\mu \mathrm{g}/\mathrm{ml}$ BLM. (b) Average spheroid volume measured following four weeks of incubation as a function of BLM concentration, 0.1 to $10\mu \mathrm{g}/\mathrm{ml}$. $1\mu \mathrm{g}/\mathrm{ml}$ ${\mathrm{AlPcS}}_{2a}$ incubation for 18 h. Wavelength of 670 nm; radiant exposure of $1.5\mathrm{J}/{\mathrm{cm}}^2$; irradiance of $5\mathrm{mW}/{\mathrm{cm}}^2$. Each data point represents the mean of three experiments. Error bars denote standard errors.

Fig. 5

Viability of control and treated ACBT MTS after four weeks in culture. $1\mu \mathrm{g}/\mathrm{ml}$ ${\mathrm{AlPcS}}_{2a}$ incubation for 18 h. Wavelength of 670 nm; radiant exposure of $1.5\mathrm{J}/{\mathrm{cm}}^2$; irradiance of $5\mathrm{mW}/{\mathrm{cm}}^2$; and 1-h BLM incubation at a concentration of $0.25\mu \mathrm{g}/\mathrm{ml}$. Each data point represents the mean of three experiments. Error bars denote standard errors.

3.4.

Two-Photon Fluorescence Microscopy

The results of live/dead assay employing two-photon fluorescence images demonstrated enhanced toxicity of PCI-treated spheroids compared to PDT-only treatment. This was inferred from the high proportion of red fluorescing cells observed from the PCI-treated spheroids [Fig. 6(c)]. By comparison, a smaller number of red fluorescing cells were observed from PDT-only spheroids [Fig. 6(d)], and these were mainly at the spheroid periphery. As illustrated in Fig. 6(a), virtually no dead cells were observed in control spheroids, since spheroids with a diameter of less than 400 μm do not generally have a necrotic core.

Fig. 6

Live/dead assay of control and treated spheroids. Two-photon fluorescence microscopy images of ACBT spheroids stained with Hoechst 33342 (blue: live) and Ethidium Homodimer (red: dead). Images were taken at a depth of 80 μm. Spheroids were fixed 48 h after treatment: (a) control, (b) PDT 1.5 J, (c) PCI with $0.25\mu \mathrm{g}/\mathrm{mL}\mathrm{bleomycin}+1.5\mathrm{J}/{\mathrm{cm}}^2$. Field of view for all images was $400\times 400\mathrm{\mu m}$.

3.5.

Flow Cytometry

Determination of ACBT spheroid cell viability by flow cytometry was utilized to examine the effects of treatment on cellular viability. This was performed by examining the percentages of viable, necrotic, and apoptotic cells as a function of treatment for the control cultures and each of the three treatment groups. Forty-eight hours following BLM, PDT, or PCI treatment, the spheroids were disaggregated by collagenase treatment into a single cell suspension labeled with Annexin V-FITC to distinguish apoptotic cells and propidium iodide to label necrotic cells. The labeled cells were then passed through the cell sorter. In the PDT- and BLM-treated MTS, the majority of the cells were still viable. In sharp contrast, only 25% of the cells from the dissociated spheroids were viable following PCI treatment (Fig. 7). At the drug and light levels employed, the main mode of cell death was apoptotic.

Fig. 7

Flow cytometry results. Percent composition (viable, necrotic, apoptotic) following various spheroid treatments are shown. As is evident from the figure, following PCI, cell death occurs by apoptosis as well as necrosis. However, the predominant mechanism of cell death is apoptotic.