Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cells in vitro

Objective. Glioblastoma (GBM) is the most common and lethal type of high-grade adult brain cancer. The World Health Organization have classed GBM as an incurable disease because standard treatments have yielded little improvement with life-expectancy being 6–15 months after diagnosis. Different approaches are now crucial to discover new knowledge about GBM communication/function in order to establish alternative therapies for such an aggressive adult brain cancer. Calcium (Ca2+) is a fundamental cell molecular messenger employed in GBM being involved in a wide dynamic range of cellular processes. Understanding how the movement of Ca2+ behaves and modulates activity in GBM at the single-cell level is relatively unexplored but holds the potential to yield opportunities for new therapeutic strategies and approaches for cancer treatment. Approach. In this article we establish a spatially and temporally precise method for stimulating Ca2+ transients in three patient-derived GBM cell-lines (FPW1, RN1, and RKI1) such that Ca2+ communication can be studied from single-cell to larger network scales. We demonstrate that this is possible by administering a single optimized ultra-violet (UV) nanosecond laser pulse to trigger GBM Ca2+ transients. Main results. We determine that 1.58 µJ µm−2 is the optimal UV nanosecond laser pulse energy density necessary to elicit a single Ca2+ transient in the GBM cell-lines whilst maintaining viability, functionality, the ability to be stimulated many times in an experiment, and to trigger further Ca2+ communication in a larger network of GBM cells. Significance. Using adult patient-derived mesenchymal GBM brain cancer cell-lines, the most aggressive form of GBM cancer, this work is the first of its kind as it provides a new effective modality of which to stimulate GBM cells at the single-cell level in an accurate, repeatable, and reliable manner; and is a first step toward Ca2+ communication in GBM brain cancer cells and their networks being more effectively studied.


Introduction
World Health Organization (WHO) grade IV Glioblastoma (GBM) [1] is the most common of the aggressive types of human brain cancer.GBM is also the most lethal form of brain cancer and is characterized by its extensive invasiveness being most prevalent in adults aged 45-75 with an incidence of 3.5 per 100 000 people [2].Treatment typically involves combined surgical resection, chemotherapy and radiotherapy [3].Without treatment a patient has a typical prognosis of 6 months which can be marginally extended to 15 months with treatment [4,5] with ⩽5% of patients surviving 5 years or more [6,7].
GBM is a grade IV astrocytoma thought to originate from the mutation of astrocyte and glial precursors [8] which are non-electrical cells.Their predominant form of communication is through calcium (Ca 2+ ) ion signalling, which plays a vital role in many cellular functions found to be significantly altered in GBM [9].Ca 2+ signals are generated by ion flux through ion channels which exist in the cellular membrane and the internal Ca 2+ stores, and between neighbouring cells gap junction [10].These channels are gated by different mechanisms such as membrane voltage, ligand expression, signalling molecule expression, or even ion concentration [9].In healthy astrocytes, ion channels play essential roles in facilitating and limiting four key cellular processes which are cell proliferation, cell size, cell migration, and intercellular coordination [9].However, in GBM there is upregulation of ion channel expression which promotes abnormal control of the aforementioned four key cellular processes, the superposition of which contribute to the survival of GBM and its resistance to treatment [11][12][13][14][15].Additional recent evidence has suggested that synchronised Ca 2+ signalling in GBM tumour networks plays a vital role in the maintenance and resistance of the GBM networks [16].Furthermore, neuronal input can modulate this network Ca 2+ signalling [17] as well as the Ca 2+ signalling observed in single GBM [18] cells that are thought to lead GBM invasion [19].
The stimulation of Ca 2+ transients allows for controlled studies of intracellular and intercellular communication.Common methods used to generate Ca 2+ transients have included mechanical stimulation [20][21][22], chemical stimulation [23][24][25][26], uncaging of signalling molecules [27], and optogenetics [28,29].Mechanical stimulation typically involves using pressure applied by a micropipette to generate a signalling response [20][21][22].However, using mechanical stimulation requires physical contact and is thus limited to stimulation of the cell surface.Chemical stimulation is performed via the application of receptor agonists such as glutamate, ATP and adenosine.The agonists then generate Ca 2+ transients through the related signalling pathways [23][24][25][26].Chemical stimulation is limited spatially, as a chemical typically diffuses throughout a cell culture media, and temporally, as the chemical will persist in the cell culture media until removed or sequestered.The uncaging of signalling molecules is another method which directly activates molecular signalling pathways that are involved in Ca 2+ signalling.To achieve this, inactive caged molecules are preloaded into live cells.These are then activated through pulses of laser light which interact with the light sensitive cage [27].The uncaged signalling molecules are then free to interact with signalling pathways.This method becomes limited due to the requirement of the preloading of the caged molecules via microinjection or, if the molecule is membrane permeable, by passive loading [30].Finally, optogenetics allows for the activation of light sensitive specific ion channels which then generate ion fluxes [28,29].However, the limitation of optogenetics is that it requires the genetic modification of the host's cells.
Laser stimulation is a method which overcomes some limitations posed by the above-mentioned methods.Laser stimulation is achieved by targeting a cell with a coherent pulsed beam of light.The pulse is steered by beam optics within a microscope's field of view, allowing for parallel stimulation and imaging of cells with high spatial resolution.Femtosecond laser pulses were initially used to stimulate Ca 2+ transients in HeLa cells [31] and were later applied to rodent astrocytes [32][33][34] and neurons [35] to evoke Ca 2+ responses.The laser pulses used in the studies of [31][32][33][34][35] had pulse durations of ∼100 fs and wavelengths of ∼800 nm.Longer pulses have also been used to achieve stimulation [36][37][38].250 µs duration laser pulses with wavelengths of ∼1900 nm were used to stimulate rodent astrocytes and neurons [36], 4 ms pulses were successful in stimulating rat ganglion neurons [37], and 8 ms pulses were also successful in stimulating astrocytes [38].
In general, lasers have been mainly used clinically to ablate and debulk brain tumours with benefits such as reduced pain and recovery time over traditional debulking methods [39][40][41] and laser irradiation has been used as a therapeutic [42,43] to reduce proliferation and mitosis.To date there have been limited studies using laser stimulation for the basic science of understanding communication in GBM.Infra-red laser pulses at 1470 nm with durations greater than 100 ms have been used to elicit Ca 2+ transients in single U87 human GBM cells [44].Hence [44], demonstrated that infra-red laser pulses did not disrupt the cell membrane and produced reliable Ca 2+ transients.However, no work to date has been performed using nanosecond ultra-violet (UV) laser pulses to elicit reliable and repeatable Ca 2+ transients in single GBM cells and networks of GBM cells.
Our group has previously demonstrated that nanosecond UV laser stimulation is a reliable and repeatable method to induce Ca 2+ transients in human NTERA2/D1 (hNT) astrocytes [45] in vitro and in small networks of hNT astrocytes [46,47].Our aim in this article is to expand on this work to generate Ca 2+ elevations in three patient-derived GBM cell-lines.This technique has the potential to serve as a valuable first tool to enable the study of Ca 2+ transients in single and networks of GBM cells in vitro.To achieve our aim, we first determined the relationship between laser pulse energy density (PED) to the number of pulses that elicits a single Ca 2+ response.Secondly, we assessed GBM cell viability after single laser stimulation across a logarithmic range of PEDs and demonstrated that GBM cells remained functional after laser stimulation with ATP.Thirdly, we measured the average temporal profile of the Ca 2+ transient produced by the GBM cell-lines in response to laser stimulation at the optimal PED.Fourthly, we demonstrate that laser stimulation of Ca 2+ transients allowed for repeatable stimulation of the GBM celllines and report the ideal recovery time necessary for a GBM to recover between pulses.Finally, we highlight how a GBM cell stimulated by an optimal energy laser pulse could also elicit Ca 2+ transients to its neighbouring cells and hence communicate to a network of GBM cells in vitro.

Methods
In this section, we describe: the methods employed in the cell culture of the three GBM patient-derived cell-lines, the UV nanosecond laser stimulation of the cultured GBM cells across a range of laser energies, and the subsequent recording of the Ca 2+ responses obtained.

Patient-derived GBM cell culture
The three curated patient-derived GBM cell-lines used in this work were produced using the methods of Stringer et al [48].Ethics approval and permission to use these cells for this work was granted on 21 March 2021 through the Auckland Health Research Ethics Committee, The University of Auckland, New Zealand (Ref.AH22121).The cell-lines used in this article were the FPW1, RN1, and RKI1 cell-lines, the morphology of these cells grown on Matrigel coated Petri dishes are shown in figure 1.It was observed that the cell-lines FPW1 and RN1 displayed a more singlecell morphology whereas the RKI1 cell-line displays a more closely packed morphology.The FPW1, RN1, and RKI1 cell-lines were patient-derived, low-passage and serum-free cell-lines.Stringer et al [48] used a detailed phenotypic characterisation and molecular subtyping of these cell-lines and concluded that they were all classified to be of mesenchymal subtype of GBM which is the most aggressive subtype [49].
Following the thawing of the GBM cells, they were carefully reconstituted in StemPro NSC SFM media (Gibco, Cat# A1050901) after removal by centrifugation of the cryopreservation media.The cell suspension was then added to a pre-prepared Matrigel (Corning, Cat# 354234) coated T75 flask.Incubation of cells was performed in 5% CO 2 at 37 • C. Cells were grown until 80% confluent with StemPro media being changed every 2-3 d as required.Once confluency was reached the cells were dissociated for seeding.Dissociation was performed by gently rinsing the cells with 5 ml of Ca 2+ and Mg 2+ free PBS (Gibco, Cat# 10010023).The PBS was removed and 1 ml of Accutase (Sigma-Aldrich, Cat# A6964) was used to dissociate the cells from the T75 flask.StemPro media was then used to stop the action of the Accutase.The cells were then counted and seeded into Matrigel coated 35 mm Petri dishes (Thermo Scientific, Cat# 150318) at a density of 100 000 cells per 35 mm Petri dish.

Live cell imaging of GBM cells
Changes in internal Ca 2+ concentration were measured by Fluo-4 (Invitrogen, Cat# F14201) imaging.
Fluo-4 is a molecule which exhibits an increase in fluorescence upon binding to Ca 2+ .Firstly, cells were loaded with Fluo-4 by incubating the Petri dishes with 1 ml of StemPro media containing Fluo-4 at a concentration of 1.5 µM for 30 min (i.e. 1 ml of StemPro media with 1.5 µl of 1 mM Fluo-4).The Fluo-4/StemPro media mix was then removed and the cells were gently rinsed twice with 1 ml imaging media.Imaging media was made by supplementing HBSS (Gibco, Cat# 14025092) with 1% Glutamax (Gibco, Cat# 35050061)) (10 µl Glutamax per 1 ml HBSS).Finally, 3 ml of imaging media was added, and the cells were left to incubate for 15 min.

UV nanosecond laser stimulation of GBM cells & comparison to spontaneous Ca 2+ transients
Petri dishes were then transferred to an Olympus BX53 upright microscope equipped with an incubator and imaged using a 20× (NA = 0.5) or 40× (NA = 0.8) water immersion lens.The 40× lens was used when interrogating single cells with laser stimulation (sections 3.1-3.5 and 3.8) and provided a field of view of 224 µm × 167 µm.The 20× objective was used to measure network response to laser stimulation (section 3.7) and provided a field of view of 449 µm × 335 µm.Laser stimulation was performed using a MicroPoint Laser Illumination and Ablation System (Andor Technology ©) coupled to the microscope.This system was comprised of a pulsed nitrogen tuneable dye laser capable of producing 3 ns 200 µJ pulses at 340 nm.Using a dye cell, the wavelength was tuned to 365 nm.The optical path of the laser was then controlled by galvanometers which allow for steering of the laser pulse to the precise location of interest.PED was controlled by the UV laser's transmission attenuator with logarithmically spaced transmission steps.The spot size of the laser was estimated by ablating a first-surface mirror and then measuring the area of the ablated spot.The spot size had an area that was 9 µm 2 .Laser pulses were directed at the cell nuclei as visualised by the NucBlue dye.We only wished to direct the laser to the cell nucleus as this could consistently be identified through the live nuclei stain.
Laser stimulation and imaging were performed simultaneously.Time-series fluorescent images were captured with an Andor Clara-C cooled CCD camera ©.Images were captured with a period of 300 ms, and an exposure time of 250 ms (dark intervals of 50 ms) with 2 × 2 pixel binning (sections 3.1, 3.3-3.5,3.7, and 3.8) used to increase the sample rate.Initially, Fluo-4 imaging was performed for 15 s to establish a baseline.After which, a laser pulse was fired and the Ca 2+ fluorescent response was recorded for 180 s.
The excitation light was produced by a standard Olympus 100 W halogen bulb (12V100WHAL-L) operated at 70% power.This light then passed through a Chroma Technology GFP filter (D480/20X) and was reflected through a beam combiner (MM-7239-30-70) (used to combine the laser pulse and illuminating light).The light then passed through the BX53 microscope, followed by a Semrock filter cube (FL-007139), and finally the objective lens.The Semrock filter cube used allowed the laser pulse to pass without interference while also having equivalent spectral properties to an Olympus U-FBW filter cube.
During the laser stimulation imaging sequence the wavelength of excitation light was 480 nm with an illumination time of 0.25 s and dark intervals of 0.05 s. 2 × 2 pixel binning (sections 3.1, 3.3-3.5,3.7, and 3.8) was used to increase the sample rate.Initially, Fluo-4 imaging was performed for 15 s to establish a baseline.After which, a laser pulse was fired and the Ca 2+ fluorescent response was recorded for 180 s.The number of images per imaging sequence was 640 (total imaging time 195 s).The size of the images was 449 µm × 335 µm when using the 20× lens and 224 µm × 168 µm when using the 40× lens.The power of excitation light at the sample level was measured using a Thor Labs PM400 Optical Power Meter and S121C Photodiode Power Senor.The power of excitation light, as defined by Laissue et al [50], at the sample level was found to be 10.30µW for the 20× lens and 6.14 µW for the 40× lens.The intensity of excitation light at the sample level was calculated to be 120 µW cm −2 for the 20× lens and 305 µW cm −2 for the 40× lens.The radiant exposure was calculated to be 30 µJ cm −2 for the 20× lens and 76 µJ cm −2 .This is a much lower radiant exposure than is experienced by the cells when subject to UV laser stimulation which is between 0.50 × 10 −8 µJ cm −2 -3.43 × 10 −8 µJ cm −2 (0.50 µJ µm −2 -3.43 µJ µm −2 ).
A brightfield image of the cells was captured before and after the imaging protocol to assess cell membrane integrity before and after the laser stimulation.For these images, higher resolution (1392 × 1040) images could be captured without pixel binning (section 3.2).When determining the optimal PED, the brightfield images were used to assess viability.At the optimal PED the viability of the stimulated cells was assessed by repeat stimulation of a cell with an additional ATP stimulation being administered to demonstrate cell functionality after laser stimulation.The criteria by which we assessed the phototoxicity was to assess the images taken before and after the laser stimulation of the cell for typical phototoxic signs, such as: an increase in autofluorescence, photobleaching, or the morphological signs (cell swelling, blebbing or appearance if vacuoles).In all cases, these signs were not observed [50].
In order to measure spontaneous Ca 2+ transients the GBM cell-lines were grown following the methods described in section 2.1.Imaging was performed using the same method as described in section 2.2 with the exception of the laser system being turned off.Individual cells displaying Spontaneous Ca 2+ transients were identified and the Ca 2+ transients were isolated and temporally aligned so that an average transient could be calculated.Crosscorrelation and auto-correlation was performed between the spontaneous Ca 2+ transients and the laser induced Ca 2+ transients.

Assessing long term viability after UV laser stimulation
To examine the long term viability after UV laser stimulation GBM cells were grown in Petri dishes with 500 µm × 500 µm square gridlines (Ibidi, Cat# 81166).The cells were stained with NucBlue, SYTOX Orange (Invitrogen, Cat# S11368), and Fluo-4.The number of live cells within each square was then counted, with dead cells (labelled with SYTOX Orange) not counted.All cells within each square were targeted with a laser pulse of no PED, optimal PED, or high PED (ablation of all targeted cells).The cells were then placed back into the incubator for 24 h.After 24 h, the cells were restained with NucBlue and SYTOX Orange and again the number of live cells per square was counted.The percentage change (%change) in the number of live cells per square 24 h after UV laser stimulation was then calculated.%change was used as the GBM cells continued proliferating and migrating after laser stimulation so each individual targeted cell could not be identified after 24 h.The high and no PED experiments were performed to control for any GBM cell proliferation and migration.

Pharmacological & ion removal treatments
The GBM cell-lines were exposed to four different treatments.These were Ca 2+ free media, 100 µM suramin which is a purinergic receptor antagonist, 1 µM thapsigargin (Tg) which is a potent inhibitor of the ER Ca 2+ ATPase pump, and Ca 2+ free media + 1 µM Tg.Tg was prepared as a stock solution at 1 mM in DMSO.
Under all treatments, including the control, the cells grown in 35 mm Petri dishes were incubated in 1 ml StemPro media supplemented with 1.5 µM Fluo-4 for 30 min.After 30 min the StemPro media was removed and the cells were rinsed with imaging media three times.Finally, 3 ml of the imaging media was added to the Petri dish and left to incubate for a further 15 min.
For the Ca 2+ free and Ca 2+ free + Tg treatments Ca 2+ free HBSS (Gibco, Cat# 14175095) supplemented with 1% Glutamax was substituted for the imaging media at all stages.For the Tg and Ca 2+ free + Tg treatments 1 µM Tg was added to all media that the cells were exposed to.For the control and Ca 2+ free treatment 1 µl of DMSO per 1 ml of media was added to all media that the cells were exposed to act as a vehicle control.

ATP stimulation of GBM cells
GBM cell functionality after laser stimulation was tested by first staining the cells with Fluo-4.Next the cells were transferred to the microscope and connected to a Gilson™ MINIPULS Evolution™ peristaltic pump via microfluidic tubes.The peristaltic pump was set at a flow rate of 1 ml min −1 .Initially, Fluo-4 imaging was performed for 15 s while imaging media was perfused over the cells.After 15 s a cell was targeted with a laser pulse and then allowed to recover for a further 200 s while imaging media was perfused over the cells.After ∼200 s, ATP solution (imaging media containing 20 µM ATP) was perfused over the cells for 15 s before it was changed back to imaging media for a further 180 s.

Image processing & data analysis
The obtained Fluro-4 fluorescence images were then processed using MATLAB © (R2020b) developed software.Using ImageJ, the individual GBM cells were identified manually and each was labelled as a region of interest (ROI).The mean fluorescence within each ROI was then calculated.The mean fluorescence time-series data was then filtered and normalised using the method described by Jia et al [51].This method worked by first calculating a timedependent baseline within a sliding window which was subtracted from the data.Finally, an exponentially weighted sliding average was used to filter the data.This method was robust to drift in the signal as well as faster oscillatory noise [51].The filtered and normalised value was represented as ∆F/F 0 and served to improve the signal to noise (S/N) ratio of the Ca 2+ transients.
The effects of different treatments were assessed using GraphPad Prism 9. First a Brown-Forsythe and Welch ANOVA was performed and if a significant difference between the groups was indicated the multiple comparisons of treatments was assessed using Dunnett's T3 multiple comparison test.The critical p-value used was 0.05.

Results
In this section, the effects of the UV nanosecond laser stimulation on the GBM cells from three different cell-lines are described.Firstly, we determine the relationship between laser PED to number of pulses that elicits a single Ca 2+ response.Secondly, we assessed GBM cell viability after single laser stimulation across a logarithmic range of PEDs and demonstrated that GBM cells remained functional after laser stimulation with ATP.Thirdly, we measured the average temporal profile of the Ca 2+ transient produced by a typical GBM cell in response to laser stimulation at the optimal PED.Fourthly, we demonstrate that laser stimulation of Ca 2+ transients allowed for repeatable stimulation of GBM cells and report the ideal recovery time necessary for a GBM to recover between pulses.Fourthly, we highlight how a GBM cell stimulated by an optimal energy laser pulse could then also elicited Ca 2+ transients to its neighbouring cells and hence communicate to a network of GBM cells in vitro.Finally, we measured the effect of different treatments on the ability of GBM cells to produce Ca 2+ transients in response to UV laser stimulation.Controls are also provided that demonstrate that GBM cells only elicited Ca 2+ responses when stimulated on the GBM cell as opposed to elsewhere in the media.

Relationship between laser PED & the probability a pulse elicits a single Ca 2+ response
In this section we determine the average number of pulses that were required to generate a single GBM Ca 2+ transient over a broad range of laser PEDs in the three GBM cell-lines that were examined, figure 2. The range of laser PEDs was logarithmically spaced between 0.50 µJ µm −2 and 3.43 µJ µm −2 with n = 15 for all three cell-lines at all PEDs.From figure 2, it was observed that for all three cell-lines the average number of pulses to generate a Ca 2+ transient decreased with increasing laser PED.At the lowest PED (0.50 µJ µm −2 ) the average number of pulses to elicit a Ca 2+ transient was 8.3 ± 5.7 for the FPW1 cellline, 6.2 ± 3.2 for the RN1 cell-line, and 7.3 ± 5.7 for the RKI1 cell-line.At a PED of 0.73 µJ µm −2 the average number of pulses to elicit a Ca 2+ transient was 6.7 ± 5.4 for the FPW1 cell-line, 2.5 ± 1.3 for the RN1 cell-line, and 4.1 ± 4.1 for the RKI1 cell-line.At a PED of 1.07 µJ µm −2 the average number of pulses to elicit a Ca 2+ transient was 4.2 ± 3.9 for the FPW1 cell-line, 1.5 ± 0.8 for the RN1 cell-line, and 2.9 ± 1.8 for the RKI1 cell-line.At a PED of 1.58 µJ µm −2 the average number of pulses to elicit a Ca 2+ transient was found to be 1.5 ± 1.3 for the FPW1 cell-line, 1.2 ± 0.4 for the RN1 cell-line, and 1.4 ± 0.9 for the RKI1 cellline.At the highest two PED of 2.33 µJ µm −2 and 3.43 µJ µm −2 the average number of pulses to elicit a Ca 2+ transient was found to be 1.0 ± 0.0 for all three cell-lines.

Determining GBM cell viability after UV laser stimulation
The range of UV nanosecond laser PEDs that permitted the GBM cells to remain viable after stimulation was then determined.
Cell viability was assessed by capturing brightfield images of the targeted cell immediately after the end of the imaging protocol, 180 s after laser stimulation.The brightfield images allowed for a visual assessment of the cell viability by the operator.Figure 3 Figure 4, shows the probability of maintaining cell viability after a single laser pulse for the three celllines.In general, it was found that as PED increased there was a decrease in the probability of the cell maintaining viability across all three cell-lines.It was determined that at a PED of 1.07 µJ µm −2 or less the FPW1 and RKI1 cell-lines always maintained an intact cell membrane.The RN1 cell-line was determined to always maintain an intact membrane at a PED of 0.50 µJ µm −2 .However, the probability of The optimal PED should allow reliable stimulation of a Ca 2+ transient with a single pulse while also requiring a high probability of maintaining cell viability.From figure 4, it was determined that the optimal PED for stimulating Ca 2+ transients in all three patient-derived GBM cell-lines was 1.58 µJ µm −2 .
Since, the aim for this work was to achieve a reliable stimulation of Ca 2+ transients with a single pulse laser whilst maintaining the highest cell viability, we felt that a 5% attrition rate was justified as it maintained the highest viability whilst being able to optically stimulate all three patient-derived cell-lines consistently with the same power.
It should be noted that all results that follow were obtained using this optimal PED of 1.58 µJ µm −2 .

Determining the Ca 2+ profile of GBM response to an optimum UV laser pulse & comparison to GBM spontaneous Ca 2+ profiles
In this section, we determine the mean Ca 2+ transient profile produced by the three GBM cell-lines when The average Ca 2+ transient temporal profile for the three GBM cell-lines Ca 2+ was calculated (n = 15 for all three cell-lines) and is shown in figure 5(d).Figure 5(d) demonstrates the characteristic Ca 2+ transient seen in the three GBM cell-lines where a rapid increase in ∆F/F 0 is followed by a slower recovery to the baseline.The time after laser stimulation to reach maximum ∆F/F 0 was found to take on average ∼15 s for all three cell-lines.This was then followed by a recovery to baseline that took ∼50 s on average for the FPW1 cell-line and ∼60 s for the RN1 and RKI1 cell-lines.
Next we compared UV laser induced Ca 2+ transients to spontaneous Ca 2+ transients.In general, the spontaneous Ca 2+ transients were found to display a lower peak and shorter duration than the laser stimulated cell, figure 6.This is most apparent in the RKI1 cell-line where the spontaneous Ca 2+ wave has a duration of ∼10 s compared to ∼70 s for the laser elicited wave.The correlation analysis shows that the peak correlation between the UV laser induced and spontaneous Ca 2+ transients is ∼0.5 for the FPW1 and RN1 cell lines.Thus it is unlikely that these are mistaken.

Repeatability of UV laser stimulation & recovery period of a GBM cell
We proceeded to assess the repeatability of laser stimulation at the optimal PED of the three GBM cell-lines by retargeting the same cell multiple times recording the Ca 2+ transient for 180 s and then allowing a dark recovery period of 120 s (the microscope shutter was closed during this period).This would demonstrate that the target cell remains viable and functional after laser stimulation.Figure 7, shows consecutive Ca 2+ transients displayed by a single GBM cell from each of the three cell-lines after successive UV laser stimulation delivered after the recovery period of ∼300 s.It was found in general that individual cells could be stimulated up to 11 times.We observed the response of multiple cells after several trials across all three cell-lines, shown in figure 7 to be consistent within experimental variation within the individual cell-lines as well as across all three cell-lines examined.
The GBM cells were stimulated once every 5 min and allowed to recover before the subsequent laser pulse was delivered.We found over all three cell-lines that the maximum transmission peak could be reattained later.Most Ca 2+ transients displayed by the cells show the characteristic form however the fourth transients displayed by the FPW1 cell-line is smaller.We believe this is due to experimental variability and could be dependent on what other cell processes may be going on at the time of stimulation.The average    While Ca 2+ transients typically took 50 s to recover it was observed that 300 s was required between laser pulses in order to achieve repeatable laser stimulation of individual cells.

Validating GBM cell functionality with ATP after a UV laser stimulation
To validate GBM cell functionality after laser stimulation the GBM cells were exposed to the neurotransmitter ATP.ATP (20 µM, for 15 s) was perfused over GBM cells ∼200 s after a laser pulse induced Ca 2+ transient to show that further Ca 2+ transients could be evoked through neurotransmitter application.Figure 8(a) shows how a typical GBM cell (RN1 cell-line) remained functional after a laser pulse by producing a Ca 2+ transient in response to ATP application.Hence, demonstrating GBM cell functionality after a laser pulse.There is a time-lag between when ATP perfusion began at 250 s and when the Ca 2+ transient was observed at ∼275 s.This was because the ATP was perfused into the Petri dish at 1 ml min −1 .Thus, the concentration of ATP in the Petri dish increased over time until it reached a concentration high enough to stimulate the GBM cells.Figures 8(b) and (c) show the fluorescent images corresponding to laser stimulation and ATP stimulation, respectively.During laser stimulation only the targeted cell responded with a Ca 2+ transient whereas during ATP stimulation neighbouring cells were also observed to be stimulated.This highlights the spatial precision of UV nanosecond laser stimulation.
Figures 8(d)-(f), show the average ATP induced Ca 2+ transient for cells which have previously been laser pulse stimulated (red) and those that have not (blue) for all three cell-lines (FPW1, RN1, and RKI1 respectively) (n = 5 for each cell-line and each group).For the FPW1 cell-line (figure 8(d)) the average max(∆F/F 0 ) reached was 0.028 ± 0.011 for cells that had been previously stimulated with a laser pulse compared to 0.015 ± 0.009 for those which had not previously been laser pulse stimulated.For the RN1 cell-line (figure 8(e)) the average max(∆F/F 0 ) reached was 0.026 ± 0.018 for cells that had been previously stimulated with a laser pulse compared to 0.020 ± 0.013 for those which had not previously been laser stimulated.For the RKI1 cell-line (figure 8(f)) the average max(∆F/F 0 ) reached was 0.010 ± 0.007 for cells that had been previously stimulated with a laser pulse compared to 0.010 ± 0.007 for those which had not previously been laser stimulated.

Validating long term viability after UV laser stimulation
It was observed that UV laser pulse stimulation at the optimal PED did not have any visible effect on the cell morphology of the three cell-lines 24 h after stimulation or on the number of dead cells.Cells from the FPW1, RN1, and RKI1 cell-lines targeted with no PED (0 µJ µm −2 ) before stimulation and 24 h after stimulation are shown in figures 9(a), (d), and (g) respectively, before stimulation on the left-hand side (LHS) and 24 h after stimulation on the right-hand side (RHS).Cells from the FPW1, RN1, and RKI1 cell-lines targeted with optimal PED (1.58 µJ µm −2 ) before stimulation and 24 h after stimulation are shown in figures 9(b), (e), and (h) respectively, before stimulation on the LHS and 24 h after stimulation on the RHS.Cells from the FPW1, RN1, and RKI1 celllines targeted with high PED (11 µJ µm −2 ) before stimulation and 24 h after stimulation are shown in figures 9(c), (f), and (i) respectively, before stimulation on the LHS and 24 h after stimulation on the RHS.In general, there was no visible difference between no PED and optimal PED images across all three cell-lines.However, at high PED, there is a reduction in the number of cells and an increased number of dead (red) cells.However, even at the high PED after 24 h there were cells within the square suggesting that cells had migrated into the square.
The %change in each cell-line at no PED, optimal PED, and high PED are shown in figure 9(d).The %change for the FPW1 cell-line at no PED, optimal PED, and high PED were 40 ± 15%, 36 ± 18%, and −56 ± 8% respectively.The %change for the RN1 cell line at no PED, optimal PED, and high PED were 32 ± 12%, 41 ± 14%, and −43 ± 7% respectively.The %change for the RKI1 cell line at no PED, optimal PED, and high PED were 18 ± 9%, 23 ± 7%, and −87 ± 4% respectively.The differences between no PED and optimal PED were not statistically significant for the FPW1, RN1, and RKI1 cell-lines (p = 0.9437, p = 0.1547, and p = 0.7552 respectively).The difference between the optimal PED and high PED for all cell-lines was statistically significant (p < 0.05).

Demonstration of how an initial UV stimulated GBM cell triggers subsequent GBM Ca 2+ transients in a GBM network
This article aims to determine the optimum UV nanosecond laser PED of a single pulse that can elicit a Ca 2+ transient in a GBM cell whilst maintaining its viability and functionality such that it can be stimulated many times in an experiment for Ca 2+ communication to be triggered in a network of GBM cells.This highlights the potential for network studies to be performed using such a method.In this section, we demonstrate how the optimum UV nanosecond single pulse laser energy can successfully trigger subsequent Ca 2+ transients between neighbouring GBM cells in a network for each of the three different GBM cell-lines.Figures 10(a)-(c) highlights how the UV laser stimulated GBM cell (red filled circle) initially displayed a Ca 2+ transient at 5 s.It was observed that the Ca 2+ transient of cell 1 triggered a subsequent Ca 2+ transient from its neighbouring cells at 14 s, similarly triggering cells in the wider neighbourhood.In addition, it should be noted that in these images we observed that there is propagation of Ca 2+ through the cell processes and cell bodies.
The heat-map of the Ca 2+ transients, figures 10(d)-(f), are shown for the three celllines in the GBM networks corresponding to figures 10(a)-(c) respectively.In general, cells which are more distant from the target cell were found to display Ca 2+ transients later than those that are closer in proximity, this is most apparent in figure 10(d).However, as displayed by cell 7 and cell 9 in figure 10(f) more distant cells can be stimulated earlier.

Controls: off target & on target responses of a GBM cell
To determine whether the network response shown in figure 10 was caused by Ca 2+ communication and

Determining the main initiation pathway for laser pulse stimulated Ca 2+ transients
Next, we determined the main initiation pathway (i.e. the source) of the UV laser stimulated Ca 2+ transients by performing pharmacological blocking and Ca 2+ ion removal treatments and assessing its effect on the maximum laser pulse stimulated fluorescent Ca 2+ transient (max(∆F/F 0 )), figure 12(a).Experiments were performed using an optimal PED of 1.58 µJ µm −2 under control treatment, Ca 2+ free treatment, suramin exposure treatment, Tg exposure treatment, and Ca 2+ free and Tg treatment, with n = 10 for all three GBM cell-lines and conditions.For all three cell-lines the Brown-Forsythe and Welch ANOVA indicated there were differences between the groups (p < 0.0001 for all three cell-lines).
For the FPW1 cell-line, the average max(∆F/F 0 ) under control conditions was 0.035 ± 0.025.Under Ca 2+ free treatment the average max(∆F/F 0 ) for the FPW1 cell-line was 0.017 ± 0.007 which was a statistically significant (p = 0.011) difference from the control.Under suramin treatment the average max(∆F/F 0 ) for the FPW1 cell-line was 0.031 ± 0.020 which was not a statistically significant (p = 0.999) difference from the control.Under Tg treatment the average max(∆F/F 0 ) for the FPW1 cell-line was 0.007 ± 0.006 which was a statistically significant (p < 0.001) difference from the control.Under Ca 2+ free + Tg treatment the average max(∆F/F 0 ) for the FPW1 cell-line was 0.002 ± 0.001 which was a statistically significant (p < 0.001) difference from the control.
For the RN1 cell-line the average max(∆F/F 0 ) under control conditions was 0.081 ± 0.044.Under Ca 2+ free treatment the average max(∆F/F 0 ) for the RN1 cell-line was 0.049 ± 0.021 which was not a statistically significant (p = 0.266) difference from the control.Under suramin treatment the average max(∆F/F 0 ) for the RN1 cell-line was 0.083 ± 0.028 which was not a statistically significant (p = 0.999) difference from the control.Under Tg treatment, the average max(∆F/F 0 ) for the RN1 cell-line was 0.004 ± 0.003 which was a statistically significant (p = 0.001) difference from the control.Under Ca 2+ free + Tg treatment, the average max(∆F/F 0 ) for the RN1 cell-line was 0.002 ± 0.001 which was a statistically significant (p = 0.001) difference from the control.
For the RKI1 cell-line, the average max(∆F/F 0 ) under control conditions was 0.064 ± 0.044.Under Ca 2+ free treatment the average max(∆F/F 0 ) for the RKI1 cell-line was 0.037 ± 0.018 which was not a statistically significant (p = 0.284) difference from the control.Under suramin treatment the average max(∆F/F 0 ) for the RKI1 cell-line was 0.053 ± 0.035 which was not a statistically significant (p = 0.997) difference from the control.Under Tg treatment, the average max(∆F/F 0 ) for the RKI1 cell-line was 0.004 ± 0.003 which was a statistically significant (p < 0.001) difference from the control.Under Ca 2+ free + Tg treatment, the average max(∆F/F 0 ) for the RKI1 cell-line was 0.003 ± 0.002 which was a statistically significant (p < 0.001) difference from the control.
The corresponding average Ca 2+ transients under the different treatments from figure 12  duration and reached a much lower max(∆F/F 0 ).Under Tg + Ca 2+ free treatment the average Ca 2+ transient was almost non-existent.

Discussion
This work demonstrates for the first time how to reliably and repeatably stimulate Ca 2+ transients in three different adult patient-derived mesenchymal GBM brain cancer cell-lines using UV nanosecond laser stimulation.
The relationship of increasing PED leading to a decrease in the average number of pulses needed to elicit a Ca 2+ transient was displayed by all three cell-lines.The RN1 cell-line was found to be more responsive to laser stimulation as it required on average fewer laser pulses to elicit a Ca 2+ transient up to a PED of 1.58 µJ µm −2 .The FPW1 cell-line was the least responsive at PEDs <1.58 µJ µm −2 .At PEDs of >1.58 µJ µm −2 all cell-lines were equally responsive.
The relationship of increasing laser PED leading to a decrease in the probability of a cell remaining viable after laser stimulation was displayed by all three cell-lines.The FPW1 and RKI1 cell-lines displayed the same relationship for PEDs up to and including 2.33 µJ µm −2 .The RN1 cell-line was found to be more susceptible to laser stimulation at PEDs of 0.73 µJ µm −2 , and 1.07 µJ µm −2 .At the highest PED the RN1 cell-line was found to be the most resistant to ablation while the FPW1 cell-line was the most susceptible to ablation.
The average Ca 2+ transients displayed by the FPW1, RN1, and RKI1 cell-lines in response to UV laser stimulation have similar dynamics.Where a slower recovery followed a rapid increase in ∆F/F 0 .There is a difference in the max(∆F/F 0 ) reached with the RN1 cell-line displaying the largest max(∆F/F 0 ) and the FPW1 cell-line displaying the smallest max(∆F/F 0 ).It should be noted, that we do not observe microdomain events in the end of the processes of the cells as was observed by Venkataramani et al [19] in the BG5 human patient-derived GBM cell-line.Instead, we observed laser stimulation eliciting Ca 2+ transients that occurred throughout the cell body and cell processes.
We demonstrated that UV laser stimulation at the optimal PED did not ablate the targeted cells and that the cells remained functional and viable immediately after and 24 h after laser stimulation.We showed that cells targeted at the optimal PED displayed the same long term viability as cells targeted with no laser stimulation.
Laser stimulation has primarily been applied to GBM as a potential therapeutic with tumour debulking [39][40][41] and reducing tumour proliferation [42,43] as the two main study avenues.To our knowledge Moreau et al have performed the only study on the basic science of eliciting Ca 2+ transients in GBM cells using infra-red laser stimulation [44].Moreau et al interrogated single cells from the human U87 GBM cell-line using an infra-red laser with a wavelength of 1470 nm, using a spot diameter of 114 µm, and multiple pulses over an exposure duration of 500 ms.It was reported that U87 GBM cells stimulated using these parameters maintained intact cell membranes after displaying Ca 2+ transients.After laser stimulation the U87 GBM cells of Moreau et al displayed rapid increases in ∆F/F 0 with a maxima being reached in <10 s followed by a much slower recovery to the baseline over ∼100 s.A difference between our study and that of Moreau et al is that our work was performed in adult patient-derived GBM cell-lines which closely represents the tumour from which they were derived and unlike the U87 GBM cell-line will not contain cell culture artefacts.An additional difference is that the infra-red laser of Moreau et al will have a heating effect which is the proposed mechanism of activation of the Ca 2+ transients they observed.This is different to our UV nanosecond laser which is ionising radiation and operates with 3 ns single pulses and with a spot size with a diameter of 7.5 µm.
Due to the lack of laser stimulation studies involving GBM cells and because GBM is an astrocytoma, it would be sensible to compare this work to the healthy astrocyte Ca 2+ transients which have been elicited by laser stimulation.Zhao et al reported that astrocytes purified from Wistar rats exposed to a laser with a wavelength of 800 nm, a pulse width of 90 fs, modulated at 80 MHz for ∼1 ms, and a total energy delivered of 48 µJ displayed Ca 2+ transients [34].In [34], it was reported that the elicited Ca 2+ transients had durations of ∼140 s.Additionally, it was reported that the Ca 2+ transients were able to be evoked up to five times successfully.Furthermore, the Ca 2+ transients observed by Zhao et al [34] were found to be communicated to neighbouring cells within a ∼170 µm radius [34] which was a similar distance as we observed.
Cayce et al investigated infrared laser stimulation in vivo using intact brains of anesthetised Sprague-Dawley rats to induce Ca 2+ transients [36].The infra-red laser had a wavelength of 1.875 µm and the stimulation was performed with a pulse width of 250 µs for a duration of 500 ms.Additionally, the spot size of the laser had a diameter of 400 µm which is significantly larger than the spot size formed by the UV nanosecond laser we employed and is larger than the GBM cells we examined.As this work was performed in live intact brains in vivo, the Ca 2+ transients in response to laser stimulation were documented as having contributions from both neurons and astrocytes.The authors determined that the Ca 2+ transients had a slow and fast component and that the astrocytes were the primary cell type responsible for the slow component of the observed Ca 2+ transients.These slow components reached a peak in 2.4 s on average and had a total duration of 7.5 s on average which is a shorter time course than the Ca 2+ transients that we observed in GBM cells in response to UV nanosecond laser stimulation.Furthermore, it was observed in [36] that the elicited Ca 2+ transients propagated away from the initial stimulated site in a manner consistent with intercellular Ca 2+ transients in astrocytes.Cayce et al observed that the Ca 2+ transients propagated up to 300 µm from the centre point of the laser stimulation which itself had a diameter of 400 µm.As our field of view was 449 µm × 335 µm and in general we targeted cells near the centre of the frame the maximum distance a cell could be from the target cell was ∼300 µm.We observed Ca 2+ transient propagation up to 289 µm from the target cell, consistent with Cayce et al [36].
Zhao et al [34], Cayce et al [36], and Borrachero-Conejo et al [38] performed their work with rat primary astrocytes which are known to be smaller, propagate Ca 2+ transients more slowly, and be less structurally complex and diverse than human astrocytes [52].Thus, studies of adult human astrocytes are valuable for comparison as GBM is an adult brain cancer.A study by our group, Raos et al, in adult human hNT astrocytes reported that nanosecond UV laser stimulation was a reliable method to elicit Ca 2+ transients [45].hNT astrocytes are an adult astrocyte differentiated from the human carcinoma NTERA2/D1 cell-line and which have been validated as a suitable model for primary human astrocytes [53,54].Our observed Ca 2+ transients in GBM cells are of similar durations and profiles to those seen in the hNT astrocytes.hNT astrocytes stimulated with UV nanosecond laser pulses displayed rapid ∼5 s increases in Ca 2+ followed by ∼100 s recovery to the baseline.The optimal PED presented by Raos et al for stimulation of hNT astrocytes was 0.9 µJ µm −2 -1.4 µJ µm −2 which maintained cell viability of above 90%.This is consistent with what we determined to be the optimal laser PED for GBM cells which was 1.58 µJ µm −2 with a viability of 93%.Network communication over ∼200 µm via intercellular Ca 2+ transients was also observed in UV laser pulse stimulated hNT astrocyte networks similar to that seen in GBM cell networks [45].
To determine the source of the Ca 2+ in UV laser pulse stimulated Ca 2+ transients we examined the effect of removing the extracellular Ca 2+ (Ca 2+ free media), depleting the Ca 2+ content of the ER (Tg) and of removing the extracellular Ca 2+ and depleting the ER (Ca 2+ free media + Tg).We found that removal of the extracellular Ca 2+ reduced the observed max(∆F/F 0 ) by 40%-60%, when compared to the control, across the three cellline, with the difference being statistically significant for only the FPW1 cell-line.We found the primary source of Ca 2+ in UV laser pulse stimulated Ca 2+ transients to be the ER.This is because treatment with the ER Ca 2+ depleting molecule Tg significantly affected the ability for GBM cells, from all three cell-lines, to produce Ca 2+ transients and reduced the max(∆F/F 0 ) by ∼95% compared to the control.Additionally, Ca 2+ free media + Tg treatment had a similar size effect to Tg treatment alone on max(∆F/F 0 ) when compared to the control.
Our results suggest that extracellular Ca 2+ is influencing our observed UV laser pulse stimulated Ca 2+ transients.However, we hypothesis that the observed differences in Ca 2+ transients between the control and Ca 2+ free media are due to the loss of Ca 2+ by the cells to the media and not due to an influx of extracellular Ca 2+ through the cell membrane for two reasons: (1) Because the effect of an influx of extracellular Ca 2+ after UV laser stimulation should be observed when comparing the control to the Ca 2+ free media treatment, and when comparing the Tg treatment to the Ca 2+ free media + Tg treatment.However, both the Tg and Ca 2+ free media + Tg treatments almost eliminate any Ca 2+ transients in response to UV laser stimulation, suggesting that the ER is the only major contributor to the observed Ca 2+ transients.(2) When the cells are exposed to Ca 2+ free media Ca 2+ ions in the cytosol will experience a chemical gradient directed out of the cell and be forced from the cytosol and into the extracellular media through membrane channels such as the Na + /Ca 2+ exchangers [55].The lack of Ca 2+ in the extracellular media will then mean that Ca 2+ cannot be bought into the cell to replace the lost Ca 2+ .Thus, the total amount of Ca 2+ within the cell will decrease.Additionally, as Ca 2+ leaks from the ER into the cytosol [56] this Ca 2+ will also be lost to the extracellular media thereby reducing the total amount of Ca 2+ in the ER and reducing the magnitude of any Ca 2+ transients.
We examined if UV laser stimulation activated the purinergic receptor pathway to elicit Ca 2+ transients by employing suramin, a widely used purinergic receptor antagonist [16,57], to block the action of the purinergic receptors.We found that suramin did not significantly change UV laser stimulated Ca 2+ transients in any of the three cell-lines.So, these results suggest that the purinergic receptors do not mediate Ca 2+ release from the ER during UV laser stimulation.
Previous work using U87 GBM cells in vitro found that Ca 2+ free media + Tg treatment eliminated any IR laser stimulated Ca 2+ transients while Ca 2+ free media alone had no effect [44].Additionally, Tg treatment significantly reduced the magnitude of UV laser elicited Ca 2+ transients displayed by hNT astrocytes in vitro [58].Furthermore, Ca 2+ free media had a small but not significant effect on the magnitude of UV laser elicited Ca 2+ transients displayed by hNT astrocytes in vitro [58].Thus, our result that the ER is responsible for the majority of the Ca 2+ released during UV laser induced Ca 2+ transients agrees and IR laser stimulation of U87 cells and UV laser stimulation of hNT astrocytes.However, it should be noted that the IR laser stimulation was performed with a wavelength of 1470 nm and an exposure time of 500 ms, compared to our UV laser stimulation with a wavelength of 365 nm with single 3 ns pulses.For hNT astrocytes however, we must note that while the experimental setup was the same the cell type was different with hNT astrocytes being a healthy adult astrocyte compared to the three patient-derived mesenchymal GBM cell-lines used in this work.
With these results in mind, previous work by Moreau et al [44] hypothesised that the mechanism of action of IR stimulated Ca 2+ transients was mediated by a heating effect of the laser pulse due to relaxation from a vibrational transition within an electronic energy level [59].Whereas, UV laser pulses involve relaxations between electronic energy level transitions [59].We hypothesise that the single nanosecond UV laser energy pulse used here does not disrupt the cell membrane or the ER (as the pulse was optimised to preserve the viability of cells).This was validated as the cells were not observed to bleb or die after stimulation and the ER remained functional and able to be repeatably stimulated.Thus, together with our observations for Tg we believe that the UV laser pulse activates the ER solely to release Ca 2+ without causing damage to it or the cell membrane.
Applying laser stimulation to in vivo and ex vivo contexts is an interesting possibility.However, whilst outside the scope of this study, this approach in general could be applied to both ex vivo and in vivo.Optogenetics [28,29], an alternate method of optically stimulating cells, is a rapidly expanding current field of research that permits large ensembles of genetically modified cells to be optically noncoherent triggered.Our approach avoids genetic modification of cells and also triggers single individual cells which is an advantage over optogenetics at the surface of the tissue.However, laser stimulation may have difficulty reaching deeper tissue with large coherent focussed powers, in comparison to optogenetics, without significantly affecting cells closer to the surface.

Conclusions
This article aimed to determine the optimum UV nanosecond laser PED of a single pulse that can elicit a Ca 2+ transient in adult patient-derived mesenchymal GBM cells whilst maintaining their viability and functionality such that they can be stimulated many times in an experiment in order for Ca 2+ communication to be triggered in a network of GBM cells and more effectively studied.
In this article, we determined the relationship between laser PED to number of pulses that elicits a single Ca 2+ response.At a PED of 0.50 µJ µm −2 the average number of laser pulses required to stimulate a Ca 2+ transient was 8.3, 6.2, and 7.3 for the FPW1, RN1, and RKI1 cell-lines respectively.At a PED of 0.73 µJ µm −2 the average number of laser pulses required to stimulate a Ca 2+ transient was 6.7, 2.5, and 4.1 for the FPW1, RN1, and RKI1 cell-lines respectively.At a PED of 1.07 µJ µm −2 the average number of laser pulses required to stimulate a Ca 2+ transient was 4.2, 1.5, and 2.9 for the FPW1, RN1, and RKI1 cell-lines respectively.At a PED of 1.58 µJ µm −2 the average number of laser pulses required to stimulate a Ca 2+ transient was 1.5, 1.2, and 1.4 for the FPW1, RN1, and RKI1 cell-lines respectively.At a PED of 2.33 µJ µm −2 and 3.43 µJ µm −2 the average number of laser pulses required to stimulate a Ca 2+ transient was 1 for all cell-lines.
Secondly, we assessed GBM cell viability after single laser stimulation across a logarithmic range of PEDs and demonstrated that GBM cells remained functional after laser stimulation with ATP.It was found that: 100% of FPW1 and RKI1 cells remained viable after laser stimulation when the laser PED was 1.07 µJ µm −2 or lower.100% of RN1 cells remained viable after stimulation with a laser PED of 0.5 µJ µm −2 , this dropped to 93% and 87% at an energy density of 0.73 µJ µm −2 and 1.07 µJ µm −2 respectively.93% and 87% of cells from all three cell-lines remained viable at energy densities of 1.58 µJ µm −2 and 2.3 µJ µm −2 respectively.
We thus determined that the optimal UV nanosecond laser PED for stimulation of adult patientderived mesenchymal GBM cells was 1.58 µJ µm −2 .This optimal PED resulted in a cell viability of 93%.
Thirdly, we measured the average temporal profile of the Ca 2+ transient produced by the three GBM cell-lines in response to laser stimulation at the optimal PED.It was found that the average time to reach a max(∆F/F 0 ) was ∼15 s for all three celllines.The max(∆F/F 0 ) reached after laser stimulation differed between the cell-lines with a max(∆F/F 0 ) of 0.043 ± 0.037, 0.103 ± 0.029, and 0.078 ± 0.073 for the FPW1, RN1, and RKI1 cell-lines respectively.
Fourthly, we demonstrate that laser stimulation of Ca 2+ transients at the optimal PED allowed for repeatable stimulation of GBM cells and report the ideal recovery time necessary for a GBM to recover between pulses.It was found that a recovery time of 300 s allowed for a single GBM cell to be stimulated up to 11 times.
Fifthly, we highlight how a GBM cell stimulated by an optimal energy laser pulse could then also elicit Ca 2+ transients to its neighbouring cells and hence communicate to a network of GBM cells in vitro.It was found that Ca 2+ transients could be communicated to cells over 200 µm from the initial cell.
Finally, we determined the main initiation pathway for GBM UV laser stimulated Ca 2+ transients to be the ER as Tg abolished the ability for all three GBM cell-lines to produce Ca 2+ transients.
This work is the first of its kind and significant as it provides an effective modality of which to stimulate patient-derived GBM cells at the single-cell level in an accurate, repeatable, and reliable manner and is a first step toward Ca 2+ communication in GBM brain cancer cells and their networks being more effectively studied.

Figure 2 .
Figure 2. Ca 2+ transients after single and multiple nanosecond UV laser stimulation over a broad range of laser PEDs for FPW1 (red), RN1 (green) and RKI1 (blue).PEDs are logarithmically spaced due to the logarithmically spaced transmission steps of the UV laser's transmission attenuator.Error bars represent ± standard deviation.
, shows an example of this for the FPW1 cell-line.The cell shown in figures 3(a)-(d) was stimulated with a PED of 1.07 µJ µm −2 while the cell shown in figures 3(e)-(h) was stimulated with a PED of 3.4 µJ µm −2 .As a result of the laser pulse the cell in figures 3(a)-(d) maintains viability.This is shown by the lack of change in the fluorescent images and by brightfield images before, figures 3(a) and (b), and after, figures 3(c) and (d), laser stimulation.Whereas, the GBM cell shown in figures 3(e)-(h) does not maintain viability.This is demonstrated by the distinct change in the integrity of the cell-membrane from before laser stimulation, figures 3(e) and (f) and after laser stimulation, figures 3(g) and (h).

Figure 3 .
Figure 3. GBM cell membrane integrity is not lost at low laser PEDs but can be lost at high laser PEDs.(a) and (b) are fluorescent images of an FPW1 cell before and after being stimulated with a 1.58 µJ µm −2 laser pulse, with the corresponding brightfield images (c) and (d).(e) and (f) are fluorescent images of an FPW1 cell before and after being stimulated with a 3.40 µJ µm −2 laser pulse, with the corresponding fluorescent images (g) and (h).Cell membranes were stained in red with CMTPX and the nuclei in blue with NucBlue.

Figure 5 .
Figure 5. Average GBM Ca 2+ transient in response to an optimum UV laser pulse.(a)-(c) shows the fluorescent image sequences over a period of 25 s after the laser pulse was delivered for the FPW1, RN1, and RKI1 cell-lines respectively.The red dot represents the spatial position of the laser pulse that was delivered at t = 15 s from when imaging began.(d) shows the Ca 2+ transient temporal profile in response to a laser pulse at the optimal energy density (the red dot and black arrow indicate when the laser pulse was delivered) for FPW1 (red-solid), RN1 (green-dashed) and RKI1 (blue-dot/dash).Error bars represent ± standard deviation, n = 15.

Figure 6 .
Figure 6.Cross-correlation analysis of UV laser induced Ca 2+ transients and spontaneous Ca 2+ transients.(a)-(c) are the average Ca 2+ transients for the FPW1, RN1, and RKI1 cell-lines respectively with the UV laser induced Ca 2+ transients in red and spontaneous transients in blue.(d)-(f) is the cross-correlation for the FPW1, RN1, and RKI1 cell-lines respectively, red is the auto-correlation of the UV laser induced Ca 2+ transient, blue is the auto-correlation of the spontaneous Ca 2+ transient, and black is the cross-correlation of the UV laser induced and spontaneous Ca 2+ transients.All correlations were normalised to the UV laser induced Ca 2+ transient auto-correlation.

Figure 7 .
Figure 7. Consecutive laser pulses elicit successful Ca 2+ transients with similar profiles for each of the three cell-lines after a recovery period of 300 s.The average Ca 2+ transient displayed by each cell is shown in black with error bars corresponding to ± the standard deviation.
Ca 2+ transient displayed by each cell-line was calculated and shown in black at the bottom of figure 7.

Figure 8 .
Figure 8.A typical GBM cell response to ATP stimulation after UV laser stimulation.(a) Time-series showing laser pulse at optimal PED of 1.58 µJ µm −2 at 40 s generates a Ca 2+ transient.ATP perfusion begins at 275 s for 15 s until a subsequent Ca 2+ transient is observed at ∼290 s.(b) shows the fluorescent images corresponding to the laser stimulation and shows only the targeted cell responded.(c) shows the fluorescent images corresponding to ATP stimulation and shows that the same cell as in (b) responded as well as neighbouring cells.(d)-(f) shows the average ATP stimulated Ca 2+ transient for the FPW1, RN1, and RKI1 cell-lines respectively for cells which had previously been laser stimulated (red) and those which had not previously been laser stimulated (blue).n = 5 for each cell-line and each group.

Figure 9 .
Figure 9. GBM cells remain viable in the long term after UV laser stimulation at the optimal PED.(a) shows a square grid of FPW1 cells before (LHS) and 24 h after (RHS) targeting with a UV laser pulse with no PED.(b) shows a square grid of FPW1 cells before (LHS) and 24 h after (RHS) targeting with a UV laser pulse with optimal PED.(c) shows a square grid of FPW1 cells before (LHS) and 24 h after (RHS) targeting with a UV laser pulse with high PED.(d)-(f) are the equivalent images of (a)-(c) for the RN1 cell-line, and (g)-(i) are the equivalent images for the RKI1 cell-line.Live cell nuclei are stained blue and dead cell nuclei are stained red.The square grids are 500 µm × 500 µm.(j) shows the percentage change in the number of cells per square for the 3 GBM cell-lines at the 3 PEDs.FPW1 in red, RN1 in green, and RKI1 in blue (N = 9 for each cell-line and PED).Error bars are ±SEM.

Figure 10 .
Figure 10.GBM cells respond to laser stimulation eliciting Ca 2+ transients.(a)-(c) shows the fluorescent images of the network response of cells from the FPW1, RN1, and RKI1 cell-lines respectively within the field of view.Initially cell 1 (red circle) was stimulated by a laser pulse at the optimal energy and displayed an increase in Ca 2+ .This was then followed by similar Ca 2+ responses from the nearest neighbour cells and later by the next nearest neighbours.(d)-(f) shows the heat maps corresponding to (a)-(c) respectively.

Figure 11 .
Figure 11.Off target and on target responses of GBM cells to UV laser stimulation.(a) Timeseries of the fluorescent response in the GBM cells, FPW1 red, RN1 dash green, and RKI1 dash dot blue.(b)-(d) Brightfield images of GBM cells with the location of four consecutive laser pulses (red squares, 1-3, off target) and (4, on target) for the FPW1 cell-line (b), RN1 cell-line (c), and RKI1 cell-line (d).(e)-(g) Fluorescent images showing the cell after pulse 4 for the FPW1 cell-line (e), RN1 cell-line (f), and RKI1 cell-line (g).
(a) are shown in figures 12(b) and (c) for the FPW1, RN1, and RKI1 cell-lines respectively.For all three cell-lines and under Ca 2+ free treatment and Suramin treatment the average Ca 2+ transients displayed similar durations to the control.Under Tg treatment the average Ca 2+ transient was significantly shorter in

Figure 12 .
Figure 12.Source of Ca 2+ in UV laser pulse stimulated Ca 2+ transients.(a) shows the effect of different treatments which block or remove Ca 2+ sources on the three GBM cell-lines, FPW1 (red-square), RN1 (green-circle), RKI1 (blue-diamond).Tg and Ca 2+ free + Tg treatments both decrease max(∆F/F0) from the control and the difference is statistically significant.(b)-(d) shows the average Ca 2+ transients under the different treatments for the FPW1 cell-line, RN1 cell-line, and the RKI1 cell-line respectively.Control (black), Ca 2+ free (red-dashed), suramin (cyan), Tg (green-dot/dash) and Ca 2+ free + Tg (blue-dot).n = 10 for all cell-lines and treatments.Asterisks indicated statistical significance as calculated by Dunnett's multiple comparison test, p < 0.05.