Photosensitizer Activation Drives Apoptosis by Interorganellar Ca2+ Transfer and Superoxide Production in Bystander Cancer Cells

In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca2+) and other second messengers, resulting in cell demise. Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called “bystander effects”. To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca2+ transfer and the production of mitochondrial superoxide anion (O2−∙) and hydrogen peroxide (H2O2) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca2+ uptake. Efficient Ca2+ efflux from the ER was required to promote mitochondrial O2−∙ production in these bystander cells. Our results support a key role for ER–mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing.


Introduction
PS photoactivation is a well-established therapeutic approach used to promote cell killing based on the interaction between visible light and matter [1,2]. The use of PS drugs in the clinic is termed "photodynamic therapy" (PDT), which is successfully targeted to cutaneous cancers, infections, and other pathologies [3][4][5][6][7]. In PDT, wavelength-selective light exposure of cells loaded with a PS promotes the inert PS molecule to a relatively long-lived excited state in which it interacts with molecular oxygen (O 2 ). Primary products of PS activation are singlet oxygen ( 1 O 2 ) and O 2 − ·, which in turn initiate a cascade of secondary reactions, leading to the formation of H 2 O 2 , hydroxyl radical (OH·), and oxidation of substrates [8,9]. These events are followed by activation of numerous cellular pathways, including signaling by Ca 2+ and nitric oxide (NO), terminating with cellular damage or demise [10]. The damage caused by PS excitation propagates from directly irradiated cells to surrounding nonexposed cells, a phenomenon that occurs also in radiotherapy and in both cases is referred to as the Bergamo II architecture (Thorlabs, Inc., Newton, NJ, USA). In both cases, the 671 nm laser light emitted from a multimode fiber optics was recollimated using an achromatic doublet, and the beam was injected into the microscope optical path just above the objective via reflection at 45 • off a dichroic mirror (650 nm shortpass dichroic, Cat. No. 69-217, Edmund Optics, Barrington, NJ, USA, for the spinning disk microscope, 600-750 nm notch dichroic custom-made by Semrock, Rochester, NY, USA, for the two-photon microscope). The recollimated laser beam was focused by the objective into a 10 µm diameter spot (see Supplementary Materials, Figure S1), which allowed spatially confined irradiation of a single cell in the culture (i.e., "focal" irradiation). Photostimulation consisted in delivering a series of photoactivation flashes in the time interval between consecutive frames. To this end, laser emission was controlled electronically by transistor-transistor logic (TTL) commands generated by a programmable electronic platform (Arduino Uno Rev3, Cat. No. A000066, Arduino, Somerville, MA, USA, code available in Supplementary Materials). The overall duration of the irradiation protocol was 100 s. Standard laser exposure was set at 250 ms per second, corresponding to a total exposure of 25 s and a total optical energy of 100 mJ (corresponding to~3·10 17 photons per irradiated cell) at an irradiance f~5·10 6 mW/cm 2 . Hereafter, we refer to these irradiation conditions as standard stimulation (SS) conditions. All the results described in this article were obtained in SS conditions, except for data shown in Figure 3, Figure 4, and Video 1 (see Supplementary Materials), where we modulated the delivered optical energy by tuning both laser power and photoactivation flash duration. We defined three other stimulation conditions that we refer to as: high irradiance (HI) conditions,~7·10 6 mW/cm 2 (~6·10 17 photons per irradiated cell), medium irradiance (MI) conditions,~4·10 6 mW/cm 2 (~2.5·10 17 photons per irradiated cell), and low irradiance (LI) conditions,~7·10 3 mW/cm 2 (~6·10 14 photons per irradiated cell).
Hereafter, to describe bystander effects triggered by focal PS photoactivation, we refer to the nearest neighbors of the irradiated cell as "cells of the 1st order", to the second neighbors as "cells of the 2nd order", and so on. The single-cell analysis was limited to cells of the 5th order by the size of the field of view (~200 µm in diameter).

Cell Viability Assays
PS-loaded B16-F10 cell cultures were focally irradiated using the spinning disk microscope setup. After PS activation, cells were incubated at room temperature with cell viability assay solutions, as detailed hereafter.

Live/Dead Assay
At different time points from the end of photostimulation, cultures were incubated for 15 minutes in NES supplemented with the components of the LIVE/DEAD Viability/Toxicity Kit (Cat. No. L3224, Thermo Fisher Scientific), therefore, green fluorescent Calcein acetoxymethyl ester (AM, 5 µM) and red fluorescent Ethidium homodimer-1 (8 µM). Thereafter, the staining medium was replaced by NES, and fluorescence images were acquired.

Apoptosis Assay
For this assay, we used the Polarity Sensitive Indicator of Viability Apoptosis (pSIVA) Microscopy Kit (Cat. No. NBP2-29382, Novus Biologicals, Centennial, CO, USA). The assay solution consisted of pSIVA conjugated to the green fluorescent IANBD dye (pSIVA-IANBD) and red fluorescent propidium iodide (PI, 5 µM) dissolved in NES. Time-lapse fluorescence microscopy was performed for 2 h after the end of laser irradiation.  Figure S5; average time constant τ = 206 ± 58 s, n = 6 cells).

Imaging of Ca
PS-loaded cells (expressing G-CEPIA1er or CEPIA2mt or loaded with MitoSOX Red) were superfused with Tg-containing NES for 6 minutes and then focally irradiated and imaged as described above.

Simultaneous Two-Photon Imaging of ER and Mitochondrial Ca 2+ in Cells Co-Expressing R-CEPIA1er and CEPIA2mt Biosensors
Two-photon excitation of both biosensors was provided by the beam of an optical parametric oscillator (MPX Chameleon Compact OPO, Coherent, Inc., Santa Clara, CA, USA) tuned to 1025 nm, coupled to the Bergamo II microscope (Thorlabs, Inc.) and fed by a femtosecond pulsed Titanium-sapphire pump laser (Chameleon Ultra II Laser, Coherent, Inc. No. H7422-40, Hamamatsu Photonics K.K., Shizuoka, Japan). Images were acquired simultaneously in these two emission channels at 3 frames/s (Hz).

Data Analysis and Statistics
Image processing and data analysis were carried out using Matlab (R2019a, The MathWorks, Inc., Natick, MA, USA) and the open-source software ImageJ/Fiji (ImageJ-win64). Fluorescence signals were extracted from sequences of recorded frames as the average pixel values within selected regions of interest (ROIs) after uniform background subtraction. Image background was computed as the average pixel value within a square ROI, placed in a region of the image where there were no detectable fluorophores. Fluorescence traces were computed as relative changes of the instantaneous fluorescence emission intensity (F(t)) with respect to the average prestimulus value (F 0 ), that is as: For the irreversible indicator MitoSOX Red, the change in mitochondrial O 2 − · concentration was evaluated as the time derivative of the acquired dF/F 0 fluorescence trace using the Matlab function diff. Photobleaching correction was performed by fitting a single exponential function to the prestimulus time course (baseline) of each trace, and extrapolating the fitting function to the overall acquisition time interval. In the graphs, mean fluorescence traces are shown as point-by-point mean ± standard error of the mean (s.e.m.) for the indicated number of independent experiments. In histograms, mean values are quoted ± s.e.m. In order to construct the optimal experimental design and estimate the sample size of the groups for each type of experiment, we set a probability α = 5% for the type I error in the ANOVA test. Then, fixing β = 4α = 20% so as to obtain a test power of 1 − β = 80%, we computed the number n of each of the two samples to be compared using the formula: with z α/2 = 1.96 and z β = 1.28. We quantified the variability of the data (variance, σ 2 ) and established the minimum difference ∆ = µ 1 − µ 2 between averages that had a biological significance. Statistical comparisons of means were made by ANOVA (independent samples) or by paired sample t-test (dependent samples), where p-value (P) < 0.05 indicates statistical significance. Asterisks were used to indicate significant differences as follows: *, P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Focal PS Activation Induces Apoptosis in Bystander Cells
To investigate the effects of PS activation, we exposed B16-F10 cell cultures to focal irradiation under SS conditions (see Materials and Methods, Section 2.2). At the end of photostimulation, we used a live/dead colorimetric assay to test the effectiveness of the photoactivation protocol. We detected impairment of plasma membrane integrity within 15 min both in the directly exposed cell and in the surrounding nonirradiated (i.e., bystander) cells (see Supplementary Materials, Figure S2). Next, we performed time-lapse confocal fluorescence microscopy to investigate the occurrence of apoptotic processes using the pSIVA-IANBD polarity sensitive probe, which binds to phosphatidylserine exposed on the surface of apoptotic cells, and propidium iodide (PI), which selectively stains the nuclei of damaged cells. As shown in Figure 1, the irradiated cell and surrounding bystander cells showed detectable pSIVA green fluorescence signals~30 min after PS excitation was terminated. After one hour, PI nuclear staining was detectable in five orders of bystander cells (i.e., within an area of radius~80 µm from the irradiated cell). Dead or late apoptotic cells were revealed in the whole field of view (radius 100 µm) within two hours of photostimulation. In a separate set of experiments, we used B16-F10 cell cultures expressing GANLS-DEVD-BNES (see Materials and Methods, Section 2.1), a genetically encoded fluorescent biosensor that decreases its fluorescence emission upon activation of caspase-3 [26]. As shown in Supplementary Materials, Figure S3, the fluorescence emission of GANLS-DEVD-BNES decreased by 44 ± 1% in bystander cells of the 1st order, by 32 ± 1% in the 2nd order, by 30 ± 1% in the 3rd order, and 28 ± 1% in the 4th order.
Together, these results indicate that PS activation in a single B16-F10 cell triggered pro-apoptotic stimuli that were transmitted to bystander nonexposed cells, in accord with prior work in different tumor cell lines [16].

Focal PS Activation Triggers an Intercellular Cytosolic Ca 2+ Wave that Spreads Radially across Bystander Cells Both in the Presence and in the Absence of Extracellular Ca 2+
In prior work, we showed that changes of cytosolic Ca 2+ concentration ([Ca 2+ ]c) in C26GM and MCA-203 tumor cells propagate as a wave from the irradiated cell to bystander cells, while NO diffuses rapidly out of the irradiated cell [16]. Here, we monitored cytosolic Ca 2+ signaling with Fluo-4 in B16-F10 cultures. As shown in Figure 2, cytosolic Ca 2+ responses were detected in all cells of the field of view (i.e., at least up to the 5th order of bystander cells) within one minute of PS photoactivation under SS conditions. To determine whether these [Ca 2+ ]c elevations were due to Ca 2+ influx and/or release from intercellular Ca 2+ stores, we repeated focal PS activation experiments in a Ca 2+ -free extracellular medium (see Materials and Methods, Section 2.2). Data analysis showed no statistically different [Ca 2+ ]c peaks up to the 4th order of bystander cells (Figure 2b), indicating that the increase of [Ca 2+ ]c was mainly due to the mobilization of Ca 2+ from the internal sources.
However, the kinetics of wave propagation was significantly different in the two experimental conditions. Namely, in 2 mM extracellular Ca 2+ concentration ([Ca 2+ ]e), the propagation speed was larger (5.4 ± 0.8 μm/s, n = 6 independent experiments) within cells of the 3 rd order (i.e., ∼50 μm from the irradiated cell) and lower in bystander cells of higher orders (2.8 ± 0.4 μm/s, n = 6 independent experiments, P = 0.006). In 0 mM [Ca 2+ ]e, the difference between the wave speeds in 3rd and 5th order cells approached statistical significance (3.8 ± 0.7 μm/s and 2.1 ± 0.2 μm/s, n = 6 independent experiments, P = 0.06). To a first approximation, the kinetics of wave propagation were fitted to a

Focal PS Activation Triggers an Intercellular Cytosolic Ca 2+ Wave that Spreads Radially across Bystander Cells Both in the Presence and in the Absence of Extracellular Ca 2+
In prior work, we showed that changes of cytosolic Ca 2+ concentration ([Ca 2+ ] c ) in C26GM and MCA-203 tumor cells propagate as a wave from the irradiated cell to bystander cells, while NO diffuses rapidly out of the irradiated cell [16]. Here, we monitored cytosolic Ca 2+ signaling with Fluo-4 in B16-F10 cultures. As shown in Figure 2, cytosolic Ca 2+ responses were detected in all cells of the field of view (i.e., at least up to the 5th order of bystander cells) within one minute of PS photoactivation under SS conditions. To determine whether these [Ca 2+ ] c elevations were due to Ca 2+ influx and/or release from intercellular Ca 2+ stores, we repeated focal PS activation experiments in a Ca 2+ -free extracellular medium (see Materials and Methods, Section 2.2). Data analysis showed no statistically different [Ca 2+ ] c peaks up to the 4th order of bystander cells (Figure 2b), indicating that the increase of [Ca 2+ ] c was mainly due to the mobilization of Ca 2+ from the internal sources.
However, the kinetics of wave propagation was significantly different in the two experimental conditions. Namely, in 2 mM extracellular Ca 2+ concentration ([Ca 2+ ] e ), the propagation speed was larger (5.4 ± 0.8 µm/s, n = 6 independent experiments) within cells of the 3 rd order (i.e.,~50 µm from the irradiated cell) and lower in bystander cells of higher orders (2.8 ± 0.4 µm/s, n = 6 independent experiments, P = 0.006). In 0 mM [Ca 2+ ] e , the difference between the wave speeds in 3rd and 5th order cells approached statistical significance (3.8 ± 0.7 µm/s and 2.1 ± 0.2 µm/s, n = 6 independent experiments, P = 0.06). To a first approximation, the kinetics of wave propagation were fitted to a logarithmic function for the 2 mM condition and to a linear function for the 0 mM condition (Figure 2c). Moreover, in cells of the 5th order, the peak amplitude of the Fluo-4 signal, the area subtended by the signal, and the slope of the rising phase of the signal were all significantly larger in 0 mM [Ca 2+ ] e than in 2 mM [Ca 2+ ] e (Figure 2b; see also Supplementary Materials Figure S4).
In summary, the amplitude of cytosolic Ca 2+ signals in 2 mM [Ca 2+ ] e decreased with distance from the source, whereas in 0 mM [Ca 2+ ] e we observed an opposite trend. This paradoxical effect supports the notion that intracellular Ca 2+ stores play a critical role in the bystander signals triggered by PS activation.  Figure S4). In summary, the amplitude of cytosolic Ca 2+ signals in 2 mM [Ca 2+ ]e decreased with distance from the source, whereas in 0 mM [Ca 2+ ]e we observed an opposite trend. This paradoxical effect supports the notion that intracellular Ca 2+ stores play a critical role in the bystander signals triggered by PS activation.

PS Activation Triggers Ca 2+ Release from the ER
To determine whether the ER is the main source of the observed cytosolic Ca 2+ rise upon PS activation, we used B16-F10 cells expressing R-CEPIA1er, a red-fluorescent Ca 2+ biosensor targeted to the ER [27]. Imaging of PS-loaded cells revealed a rapid drop of the [Ca 2+ ] ER following photostimulation under HI conditions (Figure 3a). This characteristic drop was never observed in cells not loaded with the PS, indicating that the effect was not due to photodamage of either the cell or the R-CEPIA1er biosensor by the laser used to activate the PS (Figure 3b, black traces). R-CEPIA1er signals from different subcellular ROIs were superimposable (Figure 3a), indicating homogeneity of Ca 2+ release from the ER in these conditions. We tested also different irradiation levels and determined that HI conditions compromised irreversibly the refilling capability of the ER (Figure 3b). Partial replenishment of ER Ca 2+ content occurred under MI conditions. LI conditions still induced Ca 2+ release from the ER, albeit at significantly diminished rates. ER Ca 2+ release rates under HI and MI conditions were not significantly different, whereas the rate measured under LI conditions was about 10 times lower ( Figure 3c). of Ca 2+ signals as a function of cell order in 2 mM [Ca 2+ ]e (blue bars) or 0 mM [Ca 2+ ]e (green bars) condition (irradiated cell = 0 order, ** P = 0.006). (c) Average distance of the given bystander cell order from the irradiated cell vs. average time-to-half-peak. The latter was set to zero (on the x-axis) in the irradiated cell. Interpolating curves were obtained by logarithmic (2 mM

PS Activation Triggers Ca 2+ Release from the ER
To determine whether the ER is the main source of the observed cytosolic Ca 2+ rise upon PS activation, we used B16-F10 cells expressing R-CEPIA1er, a red-fluorescent Ca 2+ biosensor targeted to the ER [27]. Imaging of PS-loaded cells revealed a rapid drop of the [Ca 2+ ]ER following photostimulation under HI conditions (Figure 3a). This characteristic drop was never observed in cells not loaded with the PS, indicating that the effect was not due to photodamage of either the cell or the R-CEPIA1er biosensor by the laser used to activate the PS (Figure 3b, black traces). R-CEPIA1er signals from different subcellular ROIs were superimposable (Figure 3a), indicating homogeneity of Ca 2+ release from the ER in these conditions. We tested also different irradiation levels and determined that HI conditions compromised irreversibly the refilling capability of the ER (Figure 3b). Partial replenishment of ER Ca 2+ content occurred under MI conditions. LI conditions still induced Ca 2+ release from the ER, albeit at significantly diminished rates. ER Ca 2+ release rates under HI and MI conditions were not significantly different, whereas the rate measured under LI conditions was about 10 times lower (Figure 3c).

Mitochondria Attempt to Buffer the Ca 2+ Released from the ER Following PS Activation
Release of Ca 2+ ions from the ER is expected to activate mechanisms that buffer Ca 2+ to avoid overloading the cytoplasm [28]. Figure 4 shows that LI photostimulation was sufficient to induce a coordinated mitochondrial Ca 2+ uptake which paralleled Ca 2+ release from the ER within the first 20 s of laser exposure. For these experiments, we used CEPIA2mt, a green fluorescent mitochondrial Ca 2+ biosensor [27], in combination with multicolor multiphoton imaging of PS-loaded B16-F10 cells co-expressing R-CEPIA1er and CEPIA2mt (see Materials and Methods, Section 2.5). Note that, after an initial increase of Ca 2+ concentration in the mitochondrial matrix, mitochondria started to leak out Ca 2+ ions, as shown by the dimming of the CEPIA2mt signal (which was not due to photobleaching; see also Supplementary Materials, Video V1).
Next, we monitored mitochondrial Ca 2+ to determine whether or not uptake occurred in bystander cells under SS conditions. As shown in Figure 5, the mean mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) increased in each order of bystander cells within the field of view and was progressively delayed with increasing distance from the site of photostimulation (see also Supplementary Materials, Video V2). In the irradiated cell, [Ca 2+ ] m rose to a peak within~6 s from the onset of laser irradiation, decreased toward the baseline level in~11 s, and thereafter kept following below this level. In cells of the 1st order, [Ca 2+ ] m fell towards the prestimulus level by the end of observation time window (80 s). At this time point, [Ca 2+ ] m was still elevated in cells of higher orders ( Figure 5).

Mitochondrial Ca 2+ Uptake and O2¯¯•Production Depend on Ca 2+ Release from the ER
The results reported above suggested a dependence of mitochondrial Ca 2+ uptake on Ca 2+ release from the ER. In order to test this hypothesis, we performed PS activation under SS conditions in cells that had been previously treated with Thapsigargin (Tg), a specific and irreversible inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pumps (see Materials and Methods, Section 2.4.1, and Supplementary Materials, Figure S5). Ca 2+ release from the ER was significantly reduced after the exposure to Tg compared with control conditions (Figure 6a). Importantly, mitochondrial Ca 2+ uptake was suppressed and fluorescence emission from the mitochondrial Ca 2+ biosensor decreased below the prestimulus level ∼5 s (n = 3 cells) after the onset of photostimulation.
Cytotoxic effects due to PS excitation are mainly attributed to downstream ROS cascades [9]. To characterize the dynamics of ROS production under SS conditions, we loaded B16-F10 cells with MitoSOX Red, a fluorescent indicator selective for mitochondrial O2¯¯•. The response of MitoSOX Red was extremely rapid in the irradiated cell, as expected, since O2¯¯• is a product of primary (type I) photochemical reactions triggered by PS activation [29]. Surprisingly, we detected O2¯¯• signals also in bystander cells up to the 4th order within seconds (see Supplementary Materials, Video V3), indicating that O2¯¯• production in these cells proceeded by a different pathway, independent of direct PS activation. Of note, mitochondrial O2¯¯• production was considerably inhibited, both in the irradiated cell and in bystander cells, by pretreatment with Tg, indicating that it depended on Ca 2+ release from the ER (Figure 6b,c).
Together, these results support the notion that mitochondrial Ca 2+ uptake spreads cytotoxic effects triggered by PS activation in the irradiated cell by promoting additional mitochondrial ROS production in bystander cells. Ca 2+ release from the ER is key to this process, and ultimately leads to the spreading of apoptosis to bystander cells, as shown in Figure 1.

Mitochondrial Ca 2+ Uptake and O 2 − · Production Depend on Ca 2+ Release from the ER
The results reported above suggested a dependence of mitochondrial Ca 2+ uptake on Ca 2+ release from the ER. In order to test this hypothesis, we performed PS activation under SS conditions in cells that had been previously treated with Thapsigargin (Tg), a specific and irreversible inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pumps (see Materials and Methods, Section 2.4.1, and Supplementary Materials, Figure S5). Ca 2+ release from the ER was significantly reduced after the exposure to Tg compared with control conditions (Figure 6a). Importantly, mitochondrial Ca 2+ uptake was suppressed and fluorescence emission from the mitochondrial Ca 2+ biosensor decreased below the prestimulus level~5 s (n = 3 cells) after the onset of photostimulation.
Cytotoxic effects due to PS excitation are mainly attributed to downstream ROS cascades [9]. To characterize the dynamics of ROS production under SS conditions, we loaded B16-F10 cells with MitoSOX Red, a fluorescent indicator selective for mitochondrial O 2 − ·. The response of MitoSOX Red was extremely rapid in the irradiated cell, as expected, since O 2 − · is a product of primary (type I) photochemical reactions triggered by PS activation [29]. Surprisingly, we detected O 2 − · signals also in bystander cells up to the 4th order within seconds (see Supplementary Materials, Video V3), indicating that O 2 − · production in these cells proceeded by a different pathway, independent of direct PS activation.
Of note, mitochondrial O 2 − · production was considerably inhibited, both in the irradiated cell and in bystander cells, by pretreatment with Tg, indicating that it depended on Ca 2+ release from the ER (Figure 6b,c). Together, these results support the notion that mitochondrial Ca 2+ uptake spreads cytotoxic effects triggered by PS activation in the irradiated cell by promoting additional mitochondrial ROS production in bystander cells. Ca 2+ release from the ER is key to this process, and ultimately leads to the spreading of apoptosis to bystander cells, as shown in Figure 1.

Focal PS Activation Promotes H2O2 Production in Bystander Cells
In cells, spontaneous dismutation or enzymatic reactions convert O2¯¯• to H2O2 [30]. To determine whether this pathway was activated following PS excitation, we used B16-F10 cells expressing HyPerRed, a red fluorescent selective biosensor which can track H2O2 concentration changes [31]. Figure 7a shows instantaneous production of H2O2 in the irradiated cell following focal PS activation. Similar to mitochondrial Ca 2+ biosensors, also HyPerRed fluoresce emission fell below prestimulus level after rising to a peak soon after PS activation. Within few seconds, H2O2 production proceeded with peculiar bimodal kinetics also in bystander cells from the 1st to the 4th order (Figure 7b).

Focal PS Activation Promotes H 2 O 2 Production in Bystander Cells
In cells, spontaneous dismutation or enzymatic reactions convert O 2 − · to H 2 O 2 [30]. To determine whether this pathway was activated following PS excitation, we used B16-F10 cells expressing HyPerRed, a red fluorescent selective biosensor which can track H 2 O 2 concentration changes [31]. Figure 7a shows instantaneous production of H 2 O 2 in the irradiated cell following focal PS activation. Similar to mitochondrial Ca 2+ biosensors, also HyPerRed fluoresce emission fell below prestimulus level after rising to a peak soon after PS activation. Within few seconds, H 2 O 2 production proceeded with peculiar bimodal kinetics also in bystander cells from the 1st to the 4th order (Figure 7b).

Discussion
In the present study, we explored selected signaling pathways triggered by focal PS activation and the propagation of signals related to these pathways to surrounding nonexposed (bystander) cells. Our results can be summarized as follows: (i) focal excitation of the PS (Aluminum Phthalocyanine Chloride) promoted apoptosis in both irradiated and bystander cells; (ii) the cytosolic Ca 2+ rise triggered by photoactivation of the PS was mainly due to Ca 2+ release from intercellular Ca 2+ stores both in the irradiated cell and in the bystander cells up to a distance of ∼80 μm; (iii) Ca 2+ ions released by the ER following PS activation were partially transferred to the mitochondrial matrix; (iv) pre-emptying of ER Ca 2+ store by the irreversible SERCA pump inhibitor Thapsigargin prevented both photoactivation-induced Ca 2+ uptake and O2¯¯• production in mitochondria; (v) H2O2 levels rose with a bimodal kinetics in bystander cells within seconds of PS excitation.

PS Activation-Induced Ca 2+ Signaling in the ER
The excitation of photosensitized cells causes the rise of [Ca 2+ ]c in a plethora of cellular systems [16,[32][33][34][35]. Surprisingly, in our system, peak amplitudes of cytosolic Ca 2+ signals were systematically, albeit not significantly, higher in 0 mM [Ca 2+ ]e compared with 2 mM [Ca 2+ ]e in all cells. In addition, the peak amplitude, the area subtended by the signal, and the slope of the linear rising signal in furthest bystander cells were all significantly larger in 0 mM [Ca 2+ ]e than in 2 mM [Ca 2+ ]e., indicating that the propagation of the cytosolic Ca 2+ wave was strengthened in the absence of extracellular Ca 2+ .
It is well known that different mechanisms contribute to the spatiotemporal shaping of cytosolic Ca 2+ signals [28,36]. Among them, store-operated Ca 2+ entry (SOCE) is the major route of Ca 2+ influx in nonexcitable cells [36,37]. When the ER Ca 2+ store is depleted, extracellular Ca 2+ ions enter the cell through plasma membrane (PM) channels at ER-PM contacts and are rapidly redistributed to the whole ER lumen [38]. It has been reported that in the presence of a prolonged Ca 2+ -mobilizing stimulus, external Ca 2+ influx by SOCE serves as "driving force" for continuous Ca 2+ release in the cytoplasm [39]. This process might play a role in cellular response to photodynamic insult. In fact, ER could function as a bypass route for Ca 2+ from extracellular space to cytosol. If this was the case, the extracellular space would provide an additional Ca 2+ source that would allow the cell to partially save the ER Ca 2+ store. On the contrary, in 0 mM [Ca 2+ ]e ER stress would be stronger, resulting in delayed reactions towards recovery of Ca 2+ homeostasis, larger cytosolic Ca 2+ signals, and possibly enhanced cellular damage. Several studies support the idea that Ca 2+ concentration in the ER controls cellular sensitivity to apoptosis depending on the amount of Ca 2+ released following a Ca 2+ -mobilizing

Discussion
In the present study, we explored selected signaling pathways triggered by focal PS activation and the propagation of signals related to these pathways to surrounding nonexposed (bystander) cells.
Our results can be summarized as follows: (i) focal excitation of the PS (Aluminum Phthalocyanine Chloride) promoted apoptosis in both irradiated and bystander cells; (ii) the cytosolic Ca 2+ rise triggered by photoactivation of the PS was mainly due to Ca 2+ release from intercellular Ca 2+ stores both in the irradiated cell and in the bystander cells up to a distance of~80 µm; (iii) Ca 2+ ions released by the ER following PS activation were partially transferred to the mitochondrial matrix; (iv) pre-emptying of ER Ca 2+ store by the irreversible SERCA pump inhibitor Thapsigargin prevented both photoactivation-induced Ca 2+ uptake and O 2 − · production in mitochondria; (v) H 2 O 2 levels rose with a bimodal kinetics in bystander cells within seconds of PS excitation.

PS Activation-Induced Ca 2+ Signaling in the ER
The excitation of photosensitized cells causes the rise of [Ca 2+ ] c in a plethora of cellular systems [16,[32][33][34][35]. Surprisingly, in our system, peak amplitudes of cytosolic Ca 2+ signals were systematically, albeit not significantly, higher in 0 mM [Ca 2+ ] e compared with 2 mM [Ca 2+ ] e in all cells. In addition, the peak amplitude, the area subtended by the signal, and the slope of the linear rising signal in furthest bystander cells were all significantly larger in 0 mM [Ca 2+ ] e than in 2 mM [Ca 2+ ] e ., indicating that the propagation of the cytosolic Ca 2+ wave was strengthened in the absence of extracellular Ca 2+ .
It is well known that different mechanisms contribute to the spatiotemporal shaping of cytosolic Ca 2+ signals [28,36]. Among them, store-operated Ca 2+ entry (SOCE) is the major route of Ca 2+ influx in nonexcitable cells [36,37]. When the ER Ca 2+ store is depleted, extracellular Ca 2+ ions enter the cell through plasma membrane (PM) channels at ER-PM contacts and are rapidly redistributed to the whole ER lumen [38]. It has been reported that in the presence of a prolonged Ca 2+ -mobilizing stimulus, external Ca 2+ influx by SOCE serves as "driving force" for continuous Ca 2+ release in the cytoplasm [39]. This process might play a role in cellular response to photodynamic insult. In fact, ER could function as a bypass route for Ca 2+ from extracellular space to cytosol. If this was the case, the extracellular space would provide an additional Ca 2+ source that would allow the cell to partially save the ER Ca 2+ store. On the contrary, in 0 mM [Ca 2+ ] e ER stress would be stronger, resulting in delayed reactions towards recovery of Ca 2+ homeostasis, larger cytosolic Ca 2+ signals, and possibly enhanced cellular damage. Several studies support the idea that Ca 2+ concentration in the ER controls cellular sensitivity to apoptosis depending on the amount of Ca 2+ released following a Ca 2+ -mobilizing stimulus [40,41], including PS activation [35,42]. Hence, it would be interesting to investigate the amount of Ca 2+ released, as well as the extension of Ca 2+ wave propagation, in 0 mM [Ca 2+ ] e and whether inhibition of external Ca 2+ influx could enhance cytotoxic stimuli transmission.
Our findings also revealed that Ca 2+ release from the ER following PS activation was nonlinearly dependent on the irradiance. In fact, the lowest irradiance we tested (~7·10 3 mW/cm 2 ) was comparatively more efficient at depleting ER Ca 2+ store than the medium irradiance, which was 3 orders of magnitude higher (~4·10 6 mW/cm 2 ), albeit in the first case ER emptying proceeded far more slowly. The simplest explanation is that efficiency of photochemical reactions triggered by PS excitation depends on O 2 availability, which limits the rate of 1 O 2 and O 2 − · production, and, ultimately, sets an upper boundary for photodynamic cell-killing efficiency [9,43]. Together, our data suggest a central role for intracellular Ca 2+ stores in the bystander signals triggered by PS activation. Recent work in intact cells by Joseph and colleagues demonstrated that "sensitization of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca 2+ release is associated with oxidative stress" [44]. In particular, both O 2 − · and its breakdown product H 2 O 2 directly activate IP 3 Rs [45,46]. Therefore, we speculate that ROS produced in primary and secondary photochemical reactions [29] triggered interorganellar Ca 2+ signaling by direct activation of IP 3 Rs in the ER membrane. Of note, ER-refilling mechanisms were inhibited at the highest irradiance we used (~7·10 6 mW/cm 2 ), which is consistent with the notion that ROS inhibits SERCA pumping activity [47,48].

PS Activation-Induced Ca 2+ Signaling in Mitochondria
Besides ER and extracellular space, mitochondria are known to shape the spatiotemporal profile of cytoplasmic Ca 2+ signals, acting as Ca 2+ sinks [28,49]. In our experiments, a fluorescent biosensor targeted to the inner mitochondrial matrix (IMM) signaled Ca 2+ uptake, implying a role for ER-mitochondria tethering in photoactivation-dependent Ca 2+ signaling. At ER-mitochondria contacts, physical membranous structures named mitochondrial-associated membranes (MAMs) form bridges which allow dynamic interactions between the two organelles [36]. During IP 3 R-mediated Ca 2+ release from the ER, Ca 2+ concentration at MAMs can locally reach even one order of magnitude higher values (~10 µM) than average [Ca 2+ ] c , overcoming the opening threshold of the mitochondrial Ca 2+ uniporter (MCU), the main Ca 2+ -selective channel responsible for fast Ca 2+ accumulation in the IMM [36,50]. We have demonstrated here that mitochondrial Ca 2+ uptake is involved in bystander Ca 2+ signaling, generating an intercellular mitochondrial Ca 2+ wave. That is, the increase in [Ca 2+ ] m progressively propagated to nonexposed bystander cells up to a distance of~80 µm within 30 s of PS activation. The [Ca 2+ ] m increase lasted more than 60 s in bystander cells. In contrast, it rose to a peak and subsequently displayed a descending phase decreasing below prestimulus levels in the irradiated cell. Both Ca 2+ overload and ROS elevation in mitochondria promote the opening of mitochondria permeability transition pores (mPTPs) [51,52]. The mPTP is a high-conductance nonselective channel in the IMM, the persistent opening of which induces IMM depolarization, cessation of oxidative phosphorylation, ROS production, Ca 2+ efflux, and finally matrix swelling and outer mitochondrial membrane rupture, with consequent release of cytochrome c and other pro-apoptotic factors [52][53][54]. In prior work, we found that activation of the same PS used in the present studies causes cytochrome c release in bystander cells [16]. The mitochondrial permeability transition (PT) promotes apoptosis or necrosis, depending on the severity of the insult it mediates [53]. Our findings showed that apoptotic processes were activated in the irradiated and bystander cells. Hence, we hypothesize that PS excitation involved the mitochondrial PT and triggered the intrinsic or "mitochondrial" apoptotic pathway, which involves cytochrome c release from mitochondria and activation of caspase-9/-3 [55]. Further experiments tracking the kinetics of caspases activation in bystander cells are required to clarify the connection between interorganellar Ca 2+ signaling and bystander cell killing.

Ca 2+ Signaling and ROS Interplay in PS Activation-Induced Bystander Responses
Ca 2+ and ROS signaling pathways are tightly interconnected in a mutual interplay: Ca 2+ increase in the mitochondrial matrix drives ROS formation as a product of upregulated oxidative metabolism [30,48] and, in turn, ROS modulate Ca 2+ signaling through interactions with PM Ca 2+ channels, activation of intercellular Ca 2+ release channels and inhibition of Ca 2+ ATPases [48,56,57]. Moreover, they are both involved in cellular responses to PS excitation in cells and are key regulators of cell death processes. Nonetheless, the signaling scheme leading to the activation of cell death pathways following photostimulation remains unclear, especially the propagation of cytotoxic stimuli in bystander effects [21,58]. Several studies proposed ROS as mediators of bystander responses [19,23]. Here, we showed that an intercellular wave of mitochondrial O 2 − · production spread from the irradiated cell towards nearby bystander cells within seconds of PS excitation. Importantly, we determined that Ca 2+ efflux from the ER was required for mitochondrial Ca 2+ overload and O 2 − · production in both the irradiated and bystander cells. Therefore, our data indicate that ER-mitochondria Ca 2+ transfer is crucial for the spreading of cytotoxic effects triggered by PS activation by promoting additional mitochondrial ROS production in bystander cells. It is even more likely to expect exacerbation of oxidative stress following PS activation because of the hypoxic condition due to photodynamic O 2 consumption. In fact, recently published data reported that MCU is targeted by ROS in hypoxia conditions, stimulating mitochondrial Ca 2+ accumulation [59]. Accordingly, we tracked a bimodal kinetics in the increase of H 2 O 2 level in bystander cells after PS activation.

Conclusions
Collectively, our data support the hypothesis of a central role for ER-mitochondria communication in the induction of a mitochondrial Ca 2+ -driven second generation of ROS, which can be crucial for the induction of apoptotic pathways in bystander cells. In this process, ER stress is key to the modulation of Ca 2+ signaling triggered by PS activation and, therefore, to the propagation of cytotoxic stimuli to nonexposed bystander cells.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/8/10/1175/s1, Figure S1: Photoactivation area; Figure S2: Cell death process in PS-loaded B16-F10 cell cultures exposed single-cell irradiation; Figure S3: Focal PS excitation triggers Caspase-3 activation in the irradiated and bystander cells. Figure S4: Analysis of cytosolic Ca 2+ signals triggered in bystander cells following single-cell PS activation in the presence or in the absence of extracellular Ca 2+ ; Figure S5: Kinetics of ER Ca 2+ store empting by Thapsigargin (Tg); Video V1: Simultaneous two-color confocal multiphoton imaging of interorganellar Ca 2+ transfer following PS activation; Video V2: Propagation of intercellular mitochondrial Ca 2+ wave following focal PS activation; Video V3: Mitochondrial O 2 − · production in bystander cells following focal PS activation.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.