International Journal of Molecular Sciences Article Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells Lucía Villamañan 1 , Laura Martínez-Escardó 2,3 , Carles Arús 1,4,5 , Victor J. Yuste 2,3 and Ana P. Candiota 1,4,5, * 1 Unitat de Bioquímica de Biociències, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; lucia_1303@hotmail.es (L.V.); Carles.Arus@uab.cat (C.A.) 2 Cell Death, Senescence and Survival Group, Department of Biochemistry and Molecular Biology and Institute of Neurosciences, Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; lauramescardo@gmail.com (L.M.-E.); Victor.Yuste@uab.cat (V.J.Y.) 3 Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (C.I.B.E.R.N.E.D.), Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 4 Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain 5 Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain * Correspondence: AnaPaula.Candiota@uab.cat Abstract: Background: The relevance of the cancer immune cycle in therapy response implies that  successful treatment may trigger the exposure or the release of immunogenic signals. Previous results  with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide Citation: Villamañan, L.; (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an Martínez-Escardó, L.; Arús, C.; Yuste, immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals V.J.; Candiota, A.P. Successful released in vitro by GB cells. Methods: GL261 GB cells were treated with TMZ and CX-4945 at Partnerships: Exploring the Potential different concentrations (25 µM–4 mM) and time frames (12–72 h). Cell viability was measured with of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, Treatment in GL261 Glioblastoma and ATP release was measured with bioluminescence. Results: TMZ showed cytostatic rather than Cells. Int. J. Mol. Sci. 2021, 22, 3453. cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. https://doi.org/10.3390/ijms22073453 Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at Academic Editor: Maria high concentrations, as well as with TMZ/CX-4945. Conclusions: combined treatment may produce Alexandra Brito the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able Received: 21 February 2021 to mimic pharmacokinetics observed in vivo fully. Accepted: 24 March 2021 Published: 26 March 2021 Keywords: preclinical glioblastoma; cancer immune cycle; immunogenic signals; calreticulin; ATP; protein kinase CK2; temozolomide Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Glioblastoma (GB) is the most prevalent malignant primary brain tumor in adults, and even after aggressive treatment, its prognosis is poor [1,2], leaving much room for Copyright: © 2021 by the authors. improvement. The current standard chemotherapeutic treatment for GB in clinical practice Licensee MDPI, Basel, Switzerland. is the alkylating agent temozolomide (TMZ), which is described as causing DNA damage This article is an open access article through the production of O6-methyl adducts in guanine residues [3]. TMZ was historically distributed under the terms and thought to display a damaging effect on GB tumors through massive cell death due to conditions of the Creative Commons DNA lesions and concomitant cell cycle arrest [4,5]. However, more recent work indicates Attribution (CC BY) license (https:// that the immune system may also be involved in GB response to TMZ [6–9]. creativecommons.org/licenses/by/ Immunotherapy is one of the most investigated new therapeutic approaches for 4.0/). treating cancer. It mostly works by either triggering the activation of the immune system Int. J. Mol. Sci. 2021, 22, 3453. https://doi.org/10.3390/ijms22073453 https://www.mdpi.com/journal/ijms avoiding its inactivation [10,11]. One modality of immunotherapy actually the use of certain drugs, called immunogenic, which are able to induce an immune response by the elicitation of immunogenic cell death (ICD) [12]. T Int. J. Mol. Sci. 2021, 22, 3453 2 of 18 non-tolerogenic form of cell death triggering an effective immune response ag cells through the activation of both the innate and adaptive host immune syst nogenic or avoiding drugs cause tumor its inactivation [10,11].cells to expose One modality and/or release of immunotherapy molecules actually profits fromcalled da ciated the use molecular patterns of certain drugs, called(DAMPs), immunogenic, which which may are ableinteract to inducewith immune syste an antitumoral immune response by the elicitation of immunogenic triggering an effective antitumor response. Two of the most accepted cell death (ICD) [12]. This ICD is DAMP a non-tolerogenic form of cell death triggering an effective immune response against intumor the literature cells through are thecalreticulin activation of both(CRT) exposure the innate and ATP and adaptive release.systems. host immune CRT exposu theImmunogenic first events drugsobserved cause tumor in cells the toICD cascade, expose beingmolecules and/or release a potent “eat-me” called damage- signal associated molecular patterns (DAMPs), which may interact the engulfment of tumor cells by antigen-presenting cells (APCs) and conseq with immune system elements, triggering an effective antitumor response. Two of the most accepted DAMPs described in ingtheto tumorareantigen literature presentation calreticulin (CRT) exposure and and specific ATP release.cytotoxic T lymphocyte CRT exposure is one of the (CTL [13,14]. Some first events studies observed also in the ICDsuggest that CRT cascade, being a potentcould be taken “eat-me” up by macrophag signal, facilitating the engulfment of tumor cells by antigen-presenting cells (APCs) M1-polarization [15], also being relevant in the general process of removing and consequently leading to da tumor antigen presentation and specific cytotoxic T lymphocyte (CTL) responses [13,14]. [16]. Some ATP release studies is a well-known also suggest that CRT could be “find-me” taken up bysignal released macrophages, fromM1- inducing apoptot cells, implicated polarization [15], alsoinbeing the relevant activation of theprocess in the general NLRP3 inflammasome of removing damaged cells and contrib [16]. ATP release is a well-known “find-me” signal released from CD8+ T cell polarization and dendritic cells (DCs) maturation [17,18]. See Fi apoptotic/damaged cells, implicated in the activation of the NLRP3 inflammasome and contributing to the CD8+ T schematic representation cell polarization of the and dendritic cells cancer (DCs) immune maturation cycle [17,18]. after1 an See Figure for aimmunogenic schematic d representation of the cancer immune cycle after an immunogenic drug hit. Figure 1. Scheme of the cycle for immune response elicitation against a preclinical glioblastoma (GB) Figure 1. Scheme of the cycle for immune response elicitation against a preclinical glio during a full therapy cycle. When treated with temozolomide (TMZ) (A), tumor cells release/expose (GB) during a full therapy cycle. When treated with temozolomide (TMZ) (A), tumor immunogenic signals, such as calreticulin (CRT), which attract dendritic cells (DCs) and macrophages lease/expose immunogenic (B). DCs migrate to proximal lymphsignals, such nodes and as calreticulin prime (CRT), naïve T-cells, which startwhich attract dendritic their proliferation [19]. ce macrophages (B). DCs migrate TMZ (or any antiproliferative agent) to proximal should lymph nodes not be administered and during prime this periodnaï veitT-cells, since would which proliferation [19]. TMZ (or any antiproliferative agent) should not be administered interfere with lymphocytes’ proliferation and hamper proper immune response (C). At days 5–6 of duri thesince riod cycle, effector it would T cells reach thewith interfere lesionlymphocytes’ site and attack tumor cells (D). and hamper proper immun proliferation (C). AtStill, daysregarding 5–6 of the cycle, cancer effector small treatment, T cellsmolecular reach theweight lesioninhibitors site and of attack tumor protein ki- cells ( nases have been a popular tool amongst targeted experimental therapies for GB [20,21]. Still,them, Among regarding cancer inhibitors treatment, of protein kinase CK2small havemolecular been provenweight inhibitors to be successful in of pro preclinical studies for cancer treatment, including GB [22,23]. CK2 is a pleiotropic serine– have been a popular tool amongst targeted experimental therapies for GB [20 them, inhibitors of protein kinase CK2 have been proven to be successful in studies for cancer treatment, including GB [22,23]. CK2 is a pleiotropic serin Int. J. Mol. Sci. 2021, 22, 3453 3 of 18 threonine kinase implicated in a multitude of prosurvival and anti-apoptotic pathways [24]. Its overexpression contributes to creating an anti-apoptotic environment; CK2 activity downregulation has been described as producing tumor cell destruction [25,26]. Sev- eral molecules have been described as CK2 inhibitors, among them, CX-4945 which is an ATP binding site competitive inhibitor, which has been evaluated in several clinical trials (https://clinicaltrials.gov/ct2/home, (accessed on 25 March 2021) NCT01199718, NCT02128282, NCT00891280). The use of animal models in oncological research is a key step in the search for new therapeutic strategies. GL261 cells grown in C57BL/6 mice is one of the most investigated immunocompetent models, widely used in GB preclinical studies [27–31]. Wu and Wax- man [32] described an immune friendly cyclophosphamide treatment triggering activation of the immune system against GL261 subcutaneous tumors, which also proved useful with TMZ treatment of an orthotopic GL261 GB model, improving mice survival [29,33] in comparison with previous protocols [34]. Our group has coined the expression Immune En- hanced metronomic Schedule (IMS) to refer to such an “immune friendly” administration protocol [35]. We have been working towards the improvement in preclinical GB outcome and non-invasive therapy response assessment, with special interest not only in TMZ but also in its combination with another non-mutagenic agent, such as CX-4945. Our previous study showed that the combined TMZ plus CX-4945 treatment, also in an IMS schedule, demonstrated improved survival rates in comparison with single IMS TMZ treatment [29] (54.7 ± 11.9 days in the combined treatment vs. 38.7 ± 2.7 days in single TMZ treatment). The IMS administration of CX-4945 itself only produced a slight, although significant, survival improvement (untreated: 22.5 ± 1.2 days and CX-4945: 24.5 ± 2 days [29]), which was not observed in continuous or alternate days treatment schedule. Overall, our previous results suggested that synergism between TMZ and CX-4945 actions was taking place, provided the IMS protocol was followed, producing better results than each agent alone. Based on those results, we decided to go back to in vitro experiments to unravel the mechanistic rationale for results obtained in vivo. We hypothesized, based on previous data described by Wu et al. [36] obtained with subcutaneously injected GL261 cells into C57BL/6J mice treated with cyclophosphamide, that a sustained antitumor immune response may be behind the survival increase when an IMS schedule using TMZ and CX-4945 is followed [29], since therapeutic antiproliferative administration is avoided during the amplification step shown in Figure 1. To further evaluate this possibility, we proceeded to study whether these drugs would produce additive/complementary immunogenic cell death, able to elicit the host immune system better, and, ultimately, improve the survival of treated mice. To assess this possibility, in vitro studies were carried out, and release/exposure of DAMPs was evaluated in GL261 cultured cells, as well as their potential for inducing cell death in tumor cells. Thus, the main goal of this work was to gain insight into TMZ and CX-4945 treatment, either single or combined treatment in GL261 cultured cells, having in mind the satisfactory in vivo results obtained with GL261 GB tumor-bearing mice. The characterization of their in vitro cytotoxic/cytostatic effect and immunogenic cell death signals profile should help to understand and explain the rationale of the combined treatment opening the door to future optimization. To the best of the author’s knowledge, this is the first study assessing the immunogenic potential of CX-4945 in GL261 GB cells. 2. Materials and Methods 2.1. GL261 Cell Culture GL261 murine glioma cells were obtained from the Tumor Bank Repository at the National Cancer Institute (Frederick, MD, USA) and cultured as previously described by our group in [37]. Mouse short tandem repeat (STR) profile and interspecies contamination were assessed, as well as PCR evaluation to discard Mycoplasma and virus contamination. Int. J. Mol. Sci. 2021, 22, 3453 4 of 18 2.2. Cell Treatments GL261 cells were treated with temozolomide (TMZ, T2577, Sigma–Aldrich, Darmstadt, Germany), CX-4945 (S2248, Selleckchem, Houston, TX, USA), and doxorubicin (Doxo 44583, Sigma–Aldrich, Darmstadt, Germany). Different concentrations and time point treatments were studied depending on the question being addressed (See Table 1 for ranges). GL261 cells (3500–500,000, depending on the test) were seeded in multiwell plates (6, 24, or 96 wells) or 60 mm dishes both from Sarstedt, and treatment was maintained during 12–72 h. Table 1. Drug concentrations and treatment time ranges used in this study. Range of Concentrations Range of Treatment Drug Assayed Time (h) TMZ 0.5–4 mM 24–72 CX-4945 25–150 µM 12–72 Doxorubicin 50 µM 24 2.3. Cell Viability Assays 2.3.1. Trypan Blue Exclusion Assay GL261 cells were seeded in a 24-well plate at a seeding density of 32,000 cells per well. After 24 h, the medium was removed, and new medium with the desired treatment was added. TMZ (0.5–4 mM) and CX-4945 (25–100 µM) treated cells were analyzed. After the treatment period (24–72 h), the medium was collected, cells washed with phosphate buffered saline (PBS), and trypsinized (0.5 mL of trypsin/EDTA). After 3 min, 1 mL of RPMI medium was used to resuspend cells, and they were centrifuged for 5 min at 900× g. The supernatant was discarded, and cells were finally resuspended in 1 mL of PBS. Then, 20 µL of the cell suspension was added to 20 µL of Trypan Blue (T8154, Sigma–Aldrich, Darmstadt, Germany), and 10 µL was loaded into the cell counting slide of the TC10 cell counter (Bio-Rad, Hercules, CA, USA). Cells excluding Trypan Blue were counted as living cells. 2.3.2. Flow Cytometry Analysis with Propidium Iodide Propidium iodide (PI, P4170, Sigma–Aldrich, Darmstadt, Germany) was used to stain dead cells since it cannot pass through intact cellular membranes, but it is able to enter cells with damaged plasma membranes. GL261 cells were seeded in a 24-well plate at 32,000 cells per well. Cells were treated with TMZ (500–2000 µM), CX-4945 (33–150 µM), and a combination of both (see Figure 2C for further details). After treatment (12–72 h depending on the case), cells were collected, washed with PBS, centrifuged at 300× g, and the supernatant was discarded. Cells were resuspended in 200 µL buffer containing PBS + 2% bovine serum albumin (BSA) + PBS with 1 µg/mL of PI. Each sample was analyzed with a FACSCalibur Cytometer (Becton Dickinson, San Jose, CA, USA) for 30 s at medium speed. Values were plot as size (forward scatter, FSC) vs. granularity (SSC, side scatter) and size (FSC, forward scatter) vs. FL-2 channel intensity. Positive PI cells were considered dead, regardless of the signal intensity. 2, 3453 5 of 18 Int. J. Mol. Sci. 2021, 22, 3453 5 of 18 Figure 2. Cell viability/cell death assessment in TMZ, CX-4945, and TMZ+CX-4945-treated GL261 cells. Living and dead Figure 2. Cell viability/cell death assessment in TMZ, CX-4945, and TMZ+CX-4945-treated GL261 cell counting, as well as the percentage of dead cells assessed by Trypan Blue exclusion assay after (A) TMZ and (B) CX-4945 cells. Living treatment (n = 2–3 and dead for every cell(C) case). counting, Living cellsasand well as the iodide propidium percentage of dead (PI) positive cells assessed cells percentage by cells) (i.e., dead Trypan evaluated by flow cytometry after 72 h of TMZ, CX-4945, and TMZ+CX-4945 treatment (n = 3 for every case). Mean ± SDLiving Blue exclusion assay after (A) TMZ and (B) CX-4945 treatment (n = 2–3 for every case). (C) arecells and shown. < 0.05), ∆ (p < iodide * (ppropidium (PI) 0.01), and δ (ppositive cells < 0.001) mark percentage statistical (i.e.,(Student’s significance dead cells)t-test)evaluated by flow comparing treated vs. cytom- control cells. etry after 72 h of TMZ, CX-4945, and TMZ+CX-4945 treatment (n = 3 for every case). Mean ± SD are shown. * (p < 0.05), 2.4. Δ (pDAMPs < 0.01),Release/Exposure and δ (p < 0.001) mark statistical significance (Student’s t-test) com- Assessment paring treated vs. control cells. DAMPs presence was assessed in GL261 cells untreated and treated with CX-4945, TMZ, or both. Drug concentrations and times of treatment will be described in detail in the 2.4. DAMPs corresponding Assessment Release/Exposure subsections. DAMPs presence was assessed inforGL261 2.4.1. Immunofluorescence cells untreated and treated with CX-4945, CRT Detection CRT exposure in the and TMZ, or both. Drug concentrations membrane of GL261 times cells was assessed of treatment will bewith immunofluorescence. described in detail in Cells were seeded in MatTek Glass Bottom Dishes (MatTek Corporation, Ashland, MA, the correspondingUSA). subsections. After 24h, the culture medium was replaced by medium containing treatment (TMZ 2 mM, CX-4945 25 µM and 100 µM, TMZ+CX-4945—both concentrations—and Doxo 2.4.1. Immunofluorescence 50 µM). Afterfor CRT 24 h Detection of treatment, cells were washed twice with 0.5 mL of PBS and fixed CRT exposure in the membrane of GL261 cells was assessed with immunofluores- cence. Cells were seeded in MatTek Glass Bottom Dishes (MatTek Corporation, Ashland, MA, USA). After 24h, the culture medium was replaced by medium containing treatment Int. J. Mol. Sci. 2021, 22, 3453 6 of 18 for 10 min with paraformaldehyde at 2%, pH 7.4. Further on, cells were washed twice with 2 mL of ice-cold PBS and incubated with blocking buffer (2 mL of 3% BSA in PBS) for 30 min. After that, cells were incubated overnight with primary anti-calreticulin antibody from Abcam (ab2907, Abcam, Cambridge, UK) at 1:100 dilution and 4 ◦ C and observed with a microscope Leica TCS SP5 (Leica Microsystems GmbH, Wetzlar, Germany) with a wavelength of 488 nm. The average number of cells counted was 150, depending on the treatment used in each case. 2.4.2. Bioluminescent Assay for ATP Measurement Extracellular ATP For these measurements, a specific medium containing inactivated fetal bovine serum (FBS) was used to avoid ATP degradation by ATPases present in the FBS used for sup- plementing the culture medium. FBS inactivation was achieved by incubation for 2 h at 65 ◦ C, as described in [38]. Cells were seeded in 24-well plates at 32,000 cells per well in a standard culture medium which was replaced after 24 h with new culture medium with inactivated FBS, and treatment was added (TMZ at 2 mM, CX-4945 at 25 µM and 100 µM, TMZ+CX-4945—both concentrations—and Doxo at 50 µM). To assess ATP degradation, the supernatant of cells growing in the medium with non-inactivated FBS was analyzed as an additional control. After the treatment period, 150 µL of cellular supernatant was collected and centrifuged at 900× g for 2 min to remove floating cells. Then, 100 µL of supernatant belonging to each sample was loaded in a 96-well plate, especially for luminescence assays. Victor3 Multilabel Counter (Perkin Elmer, Waltham, MA, USA) was used, and 100 µL of the ATP mix containing luciferin and luciferase was added with an automatic injection system. Immediately after addition, the generated light was measured during 10 s. Intracellular ATP For specific intracellular ATP measurement, cells were washed with PBS, trypsinized, and centrifuged. After that, the supernatant was removed, and cells were resuspended in 1 mL of Tris Borate buffer 40 mM pH 9.2 and incubated at 95 ◦ C for 10 min for cell lysis. Further on, samples were incubated on ice for 30 s and centrifuged at 13,000× g at 4 ◦ C for 5 min. The resulting cell lysate (100 µL) was used for ATP measurement as described for extracellular measurements. An estimation of the total ATP content per well was performed. As different treatments may decrease total cell number at wells, ATP content was calculated ‘per cell’. The number of living cells after each treatment was calculated from the Trypan Blue assay, and cellular water volume was estimated as ca. 1.5 pl, by comparison with C6 glioma cells volume in exponential phase [39]. 2.5. Statistical Analysis Sample distribution was assessed with Shapiro–Wilk or Kolmogorov–Smirnov tests, while variance homogeneity was assessed with Levene’s test. Parametric tests, such as Student’s t-test, were used for assessing differences in groups following normal distribution. On the other hand, for groups following non-normal distribution, nonparametric tests were performed (Mann–Whitney U test). For the extracellular ATP measurement, logarithm transformation of the data was used due to large variance differences among groups, as advised by our Statistical Facility (Servei d’Estadística UAB, http://sct.uab.cat/estadistica/, (accessed on 26 March 2021)). After transformation, the Kolmogorov–Smirnov test and Levene’s test were used for assessing sample distribution and variance homogeneity, respectively. The analysis of variance (ANOVA) test was applied with Tukey’s post-hoc correction for multiple comparisons. 3. Results In this work, the immunogenic signals triggered by TMZ, CX-4945, and the combina- tion of both drugs were studied in GL261 cells in vitro. First, the effects of these treatments on the GL261 cell viability were assessed. Int. J. Mol. Sci. 2021, 22, 3453 7 of 18 3.1. Effects over GL261 Cell Viability 3.1.1. TMZ Single Treatment To characterize the TMZ effect on GL261 cells and to elucidate whether a cytostatic or cytotoxic effect was taking place in those cells, treated cells were stained with Trypan Blue and analyzed by manual cell counting after 24, 48, and 72 h of treatment at 0.5, 2, and 4 mM (Figure 2A). The living cell counting decreased significantly after 48 and 72 h of treatment at every concentration studied. No significant changes were found in dead cell counting, except at 2 mM when a significantly lower number of dead cells was observed when compared to control cells. This apparent inconsistency may be caused by spontaneous cell death taking place in control cells since their total cell counting is clearly higher than treated cells. Thus, the total number of dead cells is not suitable to elucidate whether a drug is producing a cytotoxic effect. On the other hand, the percentage of the dead cells may be more useful for this purpose since it relates the dead cell counting to the total cell number. In this respect, no increase in cell death percentage was seen for 0.5 mM TMZ at any incubation time. Still, such percentage increased from 3 to 8% after 48 h at 2 and 4 mM, respectively. After 72 h, the dead cell percentage at 4 mM was 27%, thus indicating a possible cytotoxic effect of TMZ when used at 4 mM. However, we should also consider that the concentration described to have been reached following in vivo administration of TMZ at standard doses of 60 mg/Kg for mice (250 µM, according to [40], also in agreement with values described in [41]), is much lower than 4 mM, and below the 0.5 mM value at which no increase in cell death was detected. Propidium iodide (PI) staining was also used in TMZ-treated cells, which were ana- lyzed with flow cytometry after 72 h of treatment at 0.5, 1, 1.5, and 2 mM (Figure 2C). A decrease in living cell counting was observed at 1, 1.5, and 2 mM, while the percentage of dead cells increased from 6 up to 10% at 0.5 mM. Although this was statistically significant, in our opinion, it is not enough to identify TMZ as a cytotoxic agent for in vivo work because most cells remained alive, no significant cell death increase could be monitored for higher TMZ concentrations (1–2 mM) with respect to control values, and TMZ expo- sure in vivo is not continuous for 72 h (see also Section 4.4). In short, these results taken together suggest that TMZ could have a cytostatic rather than a cytotoxic effect in GL261 cells evaluated in vitro at the concentrations achieved during in vivo administration (ca. 250 µM [40]). This was further confirmed in other in vitro studies from our group (not shown) in which GL261 cell treatment with TMZ 298 µM during 72 h had only minor effects over cell viability (88% cell viability found with Trypan Blue). 3.1.2. CX-4945 Single Treatment GL261 cells treated with CX-4945 were collected, and a Trypan Blue exclusion assay was performed (Figure 2B). The living cell overall counting decreased significantly after 24 h of treatment, being higher at longer treatment times. No differences were found for dead cell counting between untreated and CX-4945 treated cells. With respect to the percentage of Trypan Blue positive dead cells, it was found to be significantly higher after 48 h of treatment and at the highest concentration analyzed (100 µM), with an average value of 27% of dead cells. After 72 h of treatment, the dead cell percentage was 21% at 50 µM and 50% at 100 µM, although significance was not reached in the latter in comparison to untreated cells due to high data values’ dispersion. Results showed that CX-4945 mainly produced a cytostatic effect reducing the number of living cells, especially at low concentrations. On the other hand, a higher cytotoxic effect than with TMZ (about 50% dead cells) was observed with the use of high concentrations and longer treatment times (Figure 2B). 3.1.3. CX-4945 and TMZ Combined Treatment In this work, the cytotoxic potential of the single and combined treatments was assessed through flow cytometry with PI-stained cells after treatment with TMZ, CX-4945, and a combination of both. The living cell counting was similar when comparing cells Int. J. Mol. Sci. 2021, 22, 3453 8 of 18 treated with CX-4945 and the combination TMZ+CX-4945 (the maximum difference found being 5.2%, non-significant). The dead cell percentage was 26.6% ± 9.9 for 100 µM CX-4945 and 29.6% ± 1.6 for the combination (same concentration of CX-4945 plus 1.5 mM TMZ), suggesting a discrete, although non-significant, synergistic effect between them, showing a coincident trend with [19]. Results suggest that the combined treatment has a slightly more pronounced effect, albeit non-significant, than the single CX-4945 treatment in cell viability, both being more cytotoxic than TMZ alone (Figure 2C). 3.2. Immunogenic Cell Death Signals Triggered In Vitro under TMZ and CX-4945 Treatment Alone and in Combination We assessed the ability of CX-4945, TMZ, and combined TMZ+CX-4945 in vitro treat- ments to produce immunogenic cell death and lead to the exposure/release of immuno- genic signals. This could help to explain the beneficial results obtained with IMS therapeu- tic schedule in GL261 tumor-bearing mice and to provide a better understanding of the combined effects in vivo already described by us [29]. 3.2.1. Calreticulin Exposure CRT exposure is an initial event in the ICD cascade acting as a potent “eat-me” signal [42]. This exposure has been analyzed by confocal microscopy in GL261 cells, both untreated and treated with TMZ (2 mM), CX-4945 (25 and 100 µM), and CX-4945+TMZ (all combinations) after 24 h of treatment. Cells treated with doxorubicin (Doxo) were used as a positive control since they have been described to trigger CRT exposure [43,44]. This can be seen with GL261 cells in Figure 3A (white brackets and arrows). At the assayed Doxo concentrations, most (75–90%) of GL261 cells evaluated by us should be dead/dying, and concentrations were far above the described EC50 [44]. For estimation and counting of confocal fluorescence microscopy observations, cells with an absent CRT signal in their membrane were considered negative (i.e., no exposure) (e.g., Figure 3B, most of untreated (UT) cells). It is worth noting that CRT exposed cells in Doxo and TMZ-treated samples presented large value dispersion, as can be seen in the violin plots (Figure 3D), and the registered values did not follow a normal distribution. Accordingly, nonparametric tests were used to compare untreated cells with the other groups. Untreated cells showed low percentages of CRT exposed cells (7.9 ± 13.5% on av- erage), which was considered as the GL261 cell line basal CRT exposure. Doxo-treated cells presented a different behavior: higher percentages of CRT-exposing cells were observed (average ca. 4-fold increase, 30.6 ± 32.4% of cells), although non-significant due to the dispersion of values. In this sense, Doxo-treated cells were considered a suitable positive control for the protocol used in this work. Regarding TMZ-treated cells, CRT positive cells were present in a higher percentage as compared to UT cells (ca. 3-fold change, 23.0 ± 25.0%). As for Doxo treated cells, due to the large data dispersion, no significant differences were found. Moreover, CX-4945 treated cells were also analyzed at two different concentrations, 25 µM (Figure 3B) and 100 µM (Figure 3C). At both concentrations, CX-4945 single treatment triggered CRT exposure in a small number of treated cells, with average values even lower than untreated cells, albeit not significantly different. The combined CX-4945+TMZ treatment (with CX-4945 at 25 µM) increased the percentage of CRT-exposing cells significantly when compared with single treatments (20-fold increase with respect to CX-4945 alone and ca. 2-fold with respect to TMZ alone). Regarding the combined treatment with the higher concentration of CX-4945 (100 µM), CRT exposure was not significantly different from the UT or CX-4945 treatment alone at this concentration (Figure 3C,D). Int. J. Mol. Sci. 2021, 22, 3453 Int. J. Mol. Sci. 2021, 22, 3453 9 of 18 Figure 3. CRT exposure in the GL261 cells plasma membrane after treatment. Nuclei were stained Figure with 3. CRT Hoechst exposure dye 33,342. in the GL261 White brackets and arrows cells plasma indicate linealmembrane or clusterized after treatment. Nucl CRT, respectively. with Hoechst dye 33,342. White brackets and arrows indicate lineal or clusterized Membranes were stained with Cell Mask and shown in red. (A) Example of CRT exposure in its C different conformations. Top: membranes are shown in red, tively. Membranes were stained with Cell Mask and shown in red. (A) Example ofnuclei in blue, and CRT in green, with the overlay CRT/membrane shown in yellow. Bottom: only nuclei (blue) and CRT in green in its different conformations. Top: membranes are shown in red, nuclei in blue, an are shown. On the left side, Doxo-treated cells (50 µM, 24 h of treatment) show CRT in a linear, with the overlay CRT/membrane shown in yellow. Bottom: only nuclei (blue) and elongated conformation (green). On the right side, TMZ-treated cells (2 mM, 24 h of treatment) show aare shown.focal clusterized, Onpattern the left side, of CRT Doxo-treated exposure. TMZ-treated cells cells(50 hadµnuclei M, 24 h of with stained treatment) Hoechst dye.show CR elongated In Doxo-treated conformation cells, Doxo prevents(green). Onofthe the union rightand Hoechst side, the TMZ-treated nuclear structures. cells (2 mM, Hence, blue 24 h o show acorresponds staining clusterized, focal patternofof to autofluorescence theCRT exposure. nuclei-bound DoxoTMZ-treated cells had nuclei sta itself. (B) Immunofluorescence staining for all conditions studied. All treatments were Hoechst dye. In Doxo-treated cells, Doxo prevents the union of Hoechst performed for 24 h: TMZ (2 mM), andCX- the nu 4945 (CX, 25 µM), TMZ+CX-4945 (2mM, 25 µM) and Doxo (50 µM). (C) Comparison between CRT tures. Hence, blue staining corresponds to autofluorescence of the nuclei-bound Do exposure staining in 24 h treatments of CX-4945 and CX-4945+TMZ at two different concentrations Immunofluorescence staining for all conditions studied. All treatments were perfo of CX-4945 (CX, 25 and 100 µM). (D) Violin plots indicating the quantitation of CRT-exposing GL261 TMZ cells (2treatment upon mM), CX-4945 (CX, 25explained with the conditions µ M), TMZ+CX-4945 (2mM, in (B,C). The percentage 25 µ M)cells of positive and Doxo (50 µ M is shown ison (n = 3–5between CRTMedian for each case). exposure is shown staining in dottedin 24 h lines, treatments while ofshown quartiles are CX-4945 and CX-4945+T with continuous ferent concentrations of CX-4945 (CX, 25 and 100 µ M). (D) Violin plots indicating t lines. UT, untreated cells. “*” states for p < 0.05 for the indicated comparisons. of CRT-exposing GL261 cells upon treatment with the conditions explained in (B,C age of positive cells is shown (n = 3–5 for each case). Median is shown in dotted lin tiles are shown with continuous lines. UT, untreated cells. “*” states for p < 0.05 for comparisons. Int. J. Mol. Sci. 2021, 22, 3453 10 of 18 3.2.2. Assessment of ATP Release Released ATP has been described as stimulating the immune response acting as a short-range “find-me” signal with a proinflammatory role by binding to P2Y2, P2X7, and P2 receptors [45] found in dendritic cells (DCs) and monocytes. The content of ATP in the supernatant of the GL261 cells was measured with a luciferin– luciferase based assay in TMZ, CX-4945, and TMZ+CX-4945 treated cells. Since TMZ needs more time to decrease cellular viability (Figure 2), we treated cells with TMZ for 24 h and, after this period, CX-4945 was added. Three different time points were studied: 12, 18, and 24 h after CX-4945 treatment. The TMZ concentration used was 2 mM, and CX-4945 was administered at two different concentrations, 25 and 100 µM, to be consistent with CRT exposure studied concentrations. Results showed that both CX-4945 and CX-4945+TMZ treated cells, at the highest concentration of CX-4945 (100 µM), released significantly more ATP compared to untreated or TMZ-treated cells (Figure 4A) for every time point studied. However, the maximum difference in ATP release when compared to untreated cells was found after 12 h of treatment (ATP content was found to be 37-fold higher for single CX-4945 treatment and ca 12-fold higher for the combined treatment). Moreover, single CX-4945 treatment was significantly different from combined CX-4945+TMZ at some time points, as follows: for the 100 µM concentration, differences were either significant (p < 0.05, 12 h) or tending to significance (0.05 < p ≤ 0.1, 18 and 24 h). For the 25 µM concentration, only the 24 h time point showed significance. The lower ATP release values observed after the combined treatment indicate that the combination of CX-4945 with TMZ triggered a 3-fold Int. J. Mol. Sci. 2021, 22, 3453 11 of 18 decrease in the overall amount of ATP released, although still significantly higher than in untreated cells. Figure 4. ATP quantification in GL261 treated cells (supernatant release and intracellular content), as well as cell viability assessment Figure 4. ATP after treatment. CX-4945 quantification in GL261(CX, 100 cells treated and 25 µM), TMZ release (supernatant (2 mM),and andintracellular combined CX-4945+TMZ content), as welltreatments were as cell viability evaluated. (A) ATP content released in the cell culture supernatant. Average ± SD values assessment after treatment. CX-4945 (CX, 100 and 25 µ M), TMZ (2 mM), and combined CX-4945+TMZ treatments were are shown for n = 3–6 in every condition. evaluated. δ(A) indicates p < 0.001 ATP content according released in thetocell ANOVA culture(Tukey’s post-hoc supernatant. comparisons) Average ± SD values vs. are TMZ and untreated shown for n = 3–6cells. No in every significant condition. differences δ indicates were found p < 0.001 between to according single ANOVA CX-4945 and joint (Tukey’s CX-4945+TMZ post-hoc treatments. comparisons) vs. TMZ Anand expansion is shown untreated on cells. No significant the differences left to appreciate werewith values found between small valuessingle betterCX-4945 and joint (higher values willCX-4945+TMZ be out of scale)treatments. (B) ContentAn of expansion is ATP intracellular shownin on the left to appreciate values with small values better (higher values will be out of scale) (B) Content GL261 treated cells after treatment, (n = 3). Average ± SD is shown, with no significant differences found. (C) Living cellsof intracellular ATP in GL261 after CX (100treated µM),cells TMZafter treatment, (2 mM), (n = 3). Average and combined treatment± CX+TMZ SD is shown, (100 with µM and no significant differences 2 mM), analyzed found. by flow (C) Living cytometry after cells after CX (100 µ M), TMZ (2 mM), and combined treatment CX+TMZ (100 µ M and 2 mM), analyzed by flow cytometry PI staining, (n = 3 for each condition). (D) Percentage of dead cells after treatments. Average ± SD is shown for all cases. *, after PI staining, (n = 3 for each condition). (D) Percentage of dead cells after treatments. Average ± SD is shown for all ∆ and δ indicate significance (p < 0.05, p < 0.01 and p < 0.001, respectively) according to Student’s t-test, for comparisons cases. *, Δ and δ indicate significance (p < 0.05, p < 0.01 and p < 0.001, respectively) according to Student’s t-test, for com- between parisons treated between and untreated treated cells. and untreated cells. Cytotoxicity Assessment after Short Treatment Times: Can It Affect ATP Release? ATP is presumably released either actively by pre/early apoptotic cells [46] or through passive release in necrotic cells [47]. One of the questions raised in this work was whether the ATP release measured after treatments originated in living or membrane- compromised cells. To elucidate this, CX-4945, TMZ, and CX-4945+TMZ treated cells were Int. J. Mol. Sci. 2021, 22, 3453 11 of 18 However, no differences were found at the lower concentration of CX-4945 (25 µM) in comparison with UT cells, indicating that this concentration was not enough to trigger ATP release in the concentrations and time points assayed. On the other hand, single TMZ treatment somewhat decreased the amount of ATP released (0.32 nM for UT vs. 0.17 nM for TMZ, non-significant, p > 0.3) at every time point studied. One possible reason could be the production of a different immunogenic signal related to ATP consumption or some interference with the ATP release in TMZ-treated GL261 cells. In addition, intracellular ATP and the estimated amount of ATP per cell were also measured (Figure 4B). Only the highest concentration of CX-4945 was evaluated since it was the only concentration that triggered significant ATP release. No significant differences were found among the different treatments, with no noticeable changes in the intracellular ATP content, as opposed to the extracellular released ATP. Cytotoxicity Assessment after Short Treatment Times: Can It Affect ATP Release? ATP is presumably released either actively by pre/early apoptotic cells [46] or through passive release in necrotic cells [47]. One of the questions raised in this work was whether the ATP release measured after treatments originated in living or membrane-compromised cells. To elucidate this, CX-4945, TMZ, and CX-4945+TMZ treated cells were analyzed by flow cytometry after incubation with PI in the same conditions, and results are shown in Figure 4C,D. CX-4945 treated cells were only analyzed at the highest concentration (100 µM) since it was the only one triggering significant ATP release. The maximum amount of ATP released was observed after 24 h of CX-4945 treat- ment in comparison with untreated cells. Flow cytometry results after CX-4945 and CX-4945+TMZ treatments showed 22.3% and 20.5% of dead cells, respectively, indicating that the remaining cells were still alive after 24 h. Thus, assuming the contribution of dying cells to extracellular ATP is similar in the two treatments, the larger ATP content in the supernatant must originate mostly from living cells (see also discussion in this regard). 4. Discussion 4.1. TMZ Is Not Enough: Pursuing Better Combinations/Schedules Despite the significant efforts made to improve therapeutic approaches and schedules for GB, its prognosis is still very poor in the clinical scenario. TMZ has been widely studied both in vitro and in vivo for GB treatment. Its beneficial effect is, in most studies, attributed to its effect as an alkylating agent able to activate the DNA repair mechanisms of the cells and, subsequently, the apoptotic cascade [4,48]. From our results, it is clear that TMZ alone, given to GL261 cultured cells in vitro, at concentrations similar to concentrations reaching the tumor when used in preclinical studies in vivo, has a cytostatic rather than a cytotoxic mechanism, in agreement with work by others [9,49,50]. The local concentration needed to produce some degree of cell death, about 2 mM, is definitely beyond the concentrations achieved in either preclinical or clinical settings (e.g., [9,51]). Thus, it is clear that a different mechanism is needed to explain the in vivo effects of TMZ, such as ICD/DAMP release. However, the potential and promising effects of TMZ as an immunogenic drug have not attracted much attention, although this is gaining prominence nowadays [9]. Moreover, although TMZ is currently the best chemotherapeutic option approved for GB, its beneficial effect is clearly insufficient. For instance, the median overall survival for GB with TMZ plus radiotherapy treatment did not really improve in terms of years (14–17 months) [52,53]. For this reason, combination schedules with drugs showing different mechanisms of action have been explored in preclinical models, such as the joint treatment with small molecular weight CK2 inhibitors [23,29] or the use of immune checkpoint inhibitors such as anti-PD1 [9,54–56]. Int. J. Mol. Sci. 2021, 22, 3453 12 of 18 4.2. Immunogenic Potential of Single and Combined Treatments and Possible Pitfalls While Interpreting Results In this work, we showed that both TMZ and, especially, joint TMZ+CX-4945 treatment of GL261 cells trigger CRT exposure on their surface. This CRT may act as a potent immunogenic “eat-me” signal for APCs and could be a key element in recruiting the immune system to produce the TMZ-related outcome in GB. In addition, having in mind that our results indicate that, at least in vitro, TMZ at the concentrations circulating in living mice models (about 0.3 mM [40,41]) mostly acts as a cytostatic agent, we need a different source of cytotoxicity to explain its therapeutic effect. Thus, in agreement with results in [9], it would be mostly TMZ-induced signaling recruited immune system cells (CTILs and macrophages), being the ones killing GL261 cells directly during the response to therapy, rather than TMZ direct activity over tumor cells. Furthermore, GL261 cells treated with CX-4945 (alone or in TMZ combination) are shown here to induce ATP release from living GL261 cells. This is a strong “find-me” DAMP that provides a chemotactic guide in the tumor surrounding tissue and within the tumor to facilitate APCs reaching the vicinity of damaged tumor cells exposing CRT “eat-me” signals. Thus, both signals, exposed CRT and released ATP, support the proposed role of TMZ and CX-4946 agents in attracting immune system cells to the vicinity of damaged/dying tumor cells to facilitate adequate priming and amplification of lymphocytes within the cancer immune cycle summarized in Figure 1. One could also hypothesize whether the increase in ATP content in the supernatant of cultured GL261 cells could be simply a consequence of the increased amount of dead cells due to different treatments. In this respect, apart from the considerations mentioned in Section 3.2.2, it is well known that under a certain ATP threshold (ca 15% of the basal con- centration [57]), cells may undergo necrosis, which in the end would release the remaining ATP content from the cell. Thus, taking as an example the comparison between UT cells and CX-4945 100 µM treatment, overall dead cell count only increased ca 1.4 fold, while the extracellular ATP content increased 37 fold. Hence, although a small part of the measured extracellular ATP can be indeed contributed by necrotic dying cells, most of the detected ATP in our conditions must have arisen from an active release from living cells. Several cellular mechanisms that could explain this release have been explained in detail in [58], and in this respect, it worth mentioning that the amounts of ATP measured were far below the threshold in which it could instead have immunosuppressive effects [59]. Additionally, the 12 h timing high mark agrees with the one reported for ATP release in C6 rat glioma cells after a targeted liposomal treatment [60]. Moreover, ATP released by glioma cells has been described to help in microglia/macrophage activation and production of macrophage inflammatory proteins [61]. Furthermore, it is worth noting that, as summarized in Figure 5, the release of im- munogenic signals by GL261 cells was both time and concentration-dependent. This is consistent with data from other authors. Thus, TMZ triggering CRT exposure in GL26 mouse GB cultured cells was already described by Kim et al. [6], although using longer treatment times than in our case (72 h). Others have gone a step beyond and proved that CRT exposure due to TMZ was observed in different GB cell lines, associated with endoplasmic reticulum stress [9]. In their case, the point of the maximum percentage of CRT-positive cells varied with the cell line (48–72 h) with average values similar to the ones described in this work. TMZ was also shown by them able to promote GB cell phagocytosis by bone marrow-derived phagocytes, reinforcing the key role of the innate immune system in response to TMZ therapy [9]. The CRT exposed in the plasma membrane was described as assembling in clusters hence facilitating the binding to the APCs receptor CD91 [62], triggering subsequent presentation of tumor-associated antigens to T lymphocytes [63]. First, CRT would be transported through the ER-Golgi pathway [46] towards the plasma membrane, first being organized in a linear shape and then further clusterized. This type of organization can be seen in Figure 3B (for TMZ treatment). Such CRT exposure was absent in our hands with CX-4945 treatment alone, but it was maximal in the combined gering subsequent presentation of tumor-associated antigens to T lymphocy CRT would be transported through the ER-Golgi pathway [46] towards the brane, first being organized in a linear shape and then further clusterized Int. J. Mol. Sci. 2021, 22, 3453 organization can be seen in Figure 3B (for TMZ treatment). Such CRT 13 of 18exposu in our hands with CX-4945 treatment alone, but it was maximal in the combi 4945 treatment (Figure 3B,D). This indicates that the joint treatment allow TMX/CX-4945 exposure, treatment could (Figure 3B,D). contribute, atThis indicates least that theto partially, joint treatment allows improved immune better system CRT exposure, could contribute, at least partially, to improved immune system recruitment into tumors, and explain a better survival of mice under the joint treatment into tumors, and explain a better survival of mice under the joint treatment compared to single IMS single IMS TMZTMZ in [29]. in vivo vivo [29]. Figure 5. Heatmap for comparison of (A) CRT exposure and (B) ATP extracellular release in the different 5. Figure conditions Heatmap studied. for CRT is expressedof comparison in(A) the same CRTunits as in Figure exposure and 3D,(B) andATP ATP release in extracellular r the same units as in Figure 4A. Hence, they are shown using different scales (columns at right). For different conditions studied. CRT is expressed in the same units as in Figure 3D, and Figure 5B, TMZ was also added 24 h before CX-4945, and the overall time passed is thus different for in the same units as in Figure 4A. Hence, they are shown using different scales (colu TMZ and CX-4945, as already stated in the Figure 4 legend. For Figure 5B, TMZ was also added 24 h before CX-4945, and the overall time passe Regarding ferent for TMZ ATP andrelease, and considering CX-4945, as alreadythestated discouraging in theresults Figureobtained with in vivo 4 legend. CX-4945 treatment of GL261 GB tumor-bearing mice [29], it seems clear that released ATP alone cannot be the major determinant immunogenic signal involved in GL261 GB Regarding treatment, ATP needing the release,ofand participation otherconsidering the discouraging DAMPs for an improved outcome. results obt vivo CX-4945 treatment of GL261 GB tumor-bearing mice [29], it seems clear 4.3. Word of Caution While Interpreting the Additive Effect of Immunogenic Signals In Vitro ATP alone cannot be the major determinant immunogenic signal involved In this work, we have shown that treatment of GL261 cells with 100 µM CX-4945 treatment, needingmore triggered significantly the ATP participation release whenof other DAMPs compared forcells to untreated anorimproved at 25 µM, outco either alone or in combination with TMZ. On the other hand, regarding CRT exposure, cells treated 4.3. with of Word TMZ plus thewhile Caution lowestInterpreting concentration of CX-4945 the (25 µM) Additive did of Effect show optimal CRT Signa Immunogenic exposure while combining TMZ with high CX-4945 (100 µM) did not expose CRT. Thus, In this we seem work, to need we have high CX-4945 (100shown that treatment µM) to induce high ATP “findof me” GL261signalcells with 100 optimal release, 25 µM CX-4945 is not enough for that. The added TMZ seems triggered significantly more ATP release when compared to untreated cells to partially inhibit ATP release induced by 100 µM CX-4945. On the other hand, combined TMZ and low either alone or 25 µM CX-4945 wasin combination optimal with TMZ. for CRT exposure, On the other while combined TMZ and hand, regarding high 100 µM C cells CX-4945treated with TMZ fully inhibited plus the CRT induced lowest exposure overconcentration untreated GL261 of CX-4945 cells. (25 µ M) d So, treatment conditions which seem optimal for “find me” immunogenic mal CRT exposure while combining TMZ with high CX-4945 (100 µ M) d signaling displayed through ATP release (100 µM CX-4945 alone or with TMZ) will make the “eat me” CRT signaling CRT. Thus, disappear, weconditions while seem tooptimal need forhigh “eatCX-4945 (100byμM) me” signaling CRT to induce exposure (25high µM CX-ATP “fin optimal 4945 with release, TMZ) will25 µM lose theCX-4945 “find me” is not enough signaling throughfor that.ATP. released TheThis added TMZ seem apparent discrepancy between in vivo CX-4945 and TMZ synergy [29] and in vitro results can have different explanations. In the first place, the concentrations of TMZ and CX-4945 reaching GL261 tumors in vivo and their pharmacokinetics in [26] are not known. Accordingly, the Int. J. Mol. Sci. 2021, 22, 3453 14 of 18 concentrations used in our in vitro experiments may not adequately mimic the in vivo situation (see also Section 4.3). In the second place, different sensitivity intrinsic to the detection methods used can hamper the adequate interpretation of some of the results described in this study. Thus, ATP measurement by luminescence may be less sensitive than the evaluation of CRT exposure by confocal microscopy. Moreover, confocal microscopy allowed studying individual glioma cells, while in the ATP released measurements, a pool of cells was being studied, and some heterogeneity can take place (i.e., some cells releasing ATP and others not, making the overall result an average). It is known that the combined action of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production is a prerequisite for the exposure and release of DAMPs to happen [42]. In this respect, Intemann et al. [64] described that CX-4945 treatment in several cell lines triggered ER stress, which would reinforce CX-4945 as an immunogenic drug in GB therapy, although, in our hands, it did not induce CRT exposure when used alone. A heatmap summarizing the main immunogenic signals observed in the different conditions studied is shown in Figure 5. The fact that the joint treatment did not prevent the appearance of both immunogenic signals (see also Section 4.3 for comments about the actual concentration of both agents in in vivo experiments) could possibly explain the reason for the synergistic action described in [29]. Acknowledgment of immune system participation in cancer therapy response is nowadays an accepted issue [65–67]. For proper immune system elicitation, the correct appearance of immunogenic signals should be triggered, and, in addition, no antiprolifera- tive treatment during the amplification stage of the cancer immune cycle (Figure 1C) must be performed, for example, using the IMS schedule [35]. 4.4. In Vitro Studies Do Not Properly Mimic the In Vivo Environment In previous work provided by us [29], the combined in vitro CX-4945+TMZ treatment showed better results than single treatments, using MTT assay approaches. Namely, the combined treatment proved to decrease 4.5 fold cell viability in comparison with untreated cells, whereas treatment with CX-4945 alone caused a 3-fold decrease, and TMZ alone caused only 1.7 fold-decrease. We were interested in elucidating the effect of TMZ in C57BL/6 mice harboring GL261 GB since this is the standard, and so far, best, chemotherapeutic treatment for human GB. However, it is worth noting that the preclinical model treatment did not fully replicate the therapeutic scheme performed in clinical settings. In human GB, TMZ is usually administered after tumor resection and/or in combination with radiotherapy, conditions that are not usually fully simulated, neither in GL261 GB nor, of course, in in vitro studies. Moreover, it is relevant to have in mind that in vitro studies hardly reproduce actual in vivo conditions, even preclinical ones. During in vivo therapy, drugs are administered and absorbed with different rates and half-lives, exposing the cells to determined temporary concentrations followed by washout, while in vitro studies provide constant concentrations of therapeutic compounds. If we compare CX-4945 hypothetical in vivo concentrations expected to be reached after oral administration (such as the one used in [29]) and also taking into account the pharmacokinetic profile [68], the CX-4945 plasma concentration in mice would have been 6.16 mM, far beyond concentrations assayed in our experiments (25 and 100 µM). Even though the hypothetical concentration in plasma of CX-4945 would be very high according to such previous literature, it is important to take into account that these authors also found out that almost all CX-4945 (98.8 ± 1%) was in a bound form in the plasma, which could finally prevent most drug reaching the tumor site. Nevertheless, even if only a small fraction of the administered drug reached the tumor, we could still hypothesize that tumor concentration would probably be a cytotoxic one for CX-4945 (i.e., >100 µM). The opposite should take place for TMZ. Concentration values expected after an in vivo administration Int. J. Mol. Sci. 2021, 22, 3453 15 of 18 (250 µM, according to [40], also in agreement with [41]) would be somewhat lower than the lowest concentrations studied in vitro in this work. Additional factors may affect drug bioavailability. For instance, drugs should face heterogeneous environments, with pH gradients and the presence of normal, non-tumoral cells and cells from the immune system infiltrating the tumor and competing for the drug. Thus, we can probably assume that the immunogenic signals detected in in vitro work will also be produced in vivo, but, having in mind different timing related to drugs half- life and bioavailability, this may mean that the drug and the induced DAMPs are not necessarily going to be present at the same time in the tumor environment. Finally, the introduction of different environmental factors, such as the “enriched environment” for animal housing [69], described to improve the performance of immune system cells, has had a clear impact on mice survival upon treatment [35], but it is hardly reproduced in in vitro work. 5. Conclusions In conclusion, the in vitro study performed with cultured GL261 GB cells indicated that TMZ treatment is able to trigger CRT exposure, while CX-4945 produces ATP release. A combined treatment using the two drugs may trigger both ICD signals, being a possible explanation for the beneficial effects detected in preclinical GL261 GB survival [29]. The effect of TMZ at the concentrations assumed to reach the tumor in vivo, would be essentially cytostatic, but immunogenic through CRT exposure, attracting APCs and macrophages. On the other hand, ATP release following CX-4945 treatment could reinforce the attraction of APCs to the CRT exposure site. To the best of our knowledge, this is the first study reporting production of classical immunogenic signals upon CX-4945 administration, although probably ATP alone is not enough to elicit a potent immune system response since only modest survival increases were obtained even with an IMS administration [26]. Translational studies that do not take into account the required synchronicity with the cancer immune cycle could fail, discarding an a priori promising drug that could present satisfactory results provided the correct timing in therapeutic schedules is respected. Finally, the mounting of suitable host immune response against GB tumors in immunocompetent models may benefit from combined TMZ and CX-4945 use. Author Contributions: Conceptualization, A.P.C., C.A., V.J.Y.; Investigation and experiment conduc- tion, L.V., L.M.-E.; Writing—original draft preparation, L.V.; Writing—review and editing, A.P.C., C.A.; Funding acquisition, C.A., A.P.C. All authors have read and agreed to the published version of the manuscript. Funding: ATTRACT project (H2020), Grant/Award Number: 777222 (A.P.C., C.A.); Centro de In- vestigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Grant/Award Number: CB06/01/0010 (C.A.); Universitat Autònoma de Barcelona, Grant/Award Numbers: 13th PIF Call (“Personal Investigador en Formació” (L.V., L.M.E.)). APC and CA acknowledge 2018 XARDI 00016 / IU68-013944 (XarTEC SALUT) for support. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data will be made available through UAB Digital repository (DDD UAB https://ddd.uab.cat/, (accessed on 25 March 2021)). Conflicts of Interest: The authors declare no conflict of interest. References 1. Louis, D.N.; Perry, A.; Reifenberger, G.; Von Deimling, A.; Figarella, D.; Webster, B.; Hiroko, K.C.; Wiestler, O.D.; Kleihues, P.; Ellison, D.W. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: A summary. Acta Neuropathol. 2016, 131, 803–820. [CrossRef] Int. J. Mol. Sci. 2021, 22, 3453 16 of 18 2. Gilbert, M.R.; Wang, M.; Aldape, K.D.; Stupp, R.; Hegi, M.E.; Jaeckle, K.A.; Armstrong, T.S.; Wefel, J.S.; Won, M.; Blumenthal, D.T.; et al. Dose-dense temozolomide for newly diagnosed glioblastoma: A randomized phase III clinical trial. J. Clin. Oncol. 2013, 31, 4085–4091. [CrossRef] 3. Zhang, J.; Stevens, M.F.G.; Bradshaw, T.D. Temozolomide: Mechanisms of Action, Repair and Resistance. Curr. Mol. Pharmacol. 2011, 5, 102–114. [CrossRef] 4. Roos, W.P.; Batista, L.F.Z.; Naumann, S.C.; Wick, W.; Weller, M.; Menck, C.F.M.; Kaina, B. Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine. Oncogene 2007, 26, 186–197. [CrossRef] [PubMed] 5. Kanzawa, T.; Germano, I.M.; Komata, T.; Ito, H.; Kondo, Y.; Kondo, S. Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ. 2004, 11, 448–457. [CrossRef] [PubMed] 6. Kim, T.-G.; Kim, C.-H.; Park, J.-S.; Park, S.-D.; Kim, C.K.; Chung, D.-S.; Hong, Y.-K. Immunological Factors Relating to the Antitumor Effect of Temozolomide Chemoimmunotherapy in a Murine Glioma Model. Clin. Vaccine Immunol. 2010, 17, 143–153. [CrossRef] [PubMed] 7. Fritzell, S.; Sandén, E.; Eberstål, S.; Visse, E.; Darabi, A.; Siesjö, P. Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion. Cancer Immunol. Immunother. 2013, 62, 1463–1474. [CrossRef] 8. Liikanen, I.; Ahtiainen, L.; Hirvinen, M.L.M.; Bramante, S.; Cerullo, V.; Nokisalmi, P.; Hemminki, O.; Diaconu, I.; Pesonen, S.; Koski, A.; et al. Oncolytic adenovirus with temozolomide induces autophagy and antitumor immune responses in cancer patients. Mol. Ther. 2013, 21, 1212–1223. [CrossRef] 9. Von Roemeling, C.A.; Wang, Y.; Qie, Y.; Yuan, H.; Zhao, H.; Liu, X.; Yang, Z.; Yang, M.; Deng, W.; Bruno, K.A.; et al. Therapeutic modulation of phagocytosis in glioblastoma can activate both innate and adaptive antitumour immunity. Nat. Commun. 2020, 11, 1508. [CrossRef] 10. Galluzzi, L.; Vacchelli, E.; Bravo-San Pedro, J.-M.; Buqué, A.; Senovilla, L.; Baracco, E.E.; Bloy, N.; Castoldi, F.; Abastado, J.-P.; Agostinis, P.; et al. Classification of current anticancer immunotherapies. Oncotarget 2014, 5, 12472–12508. [CrossRef] 11. Waldman, A.D.; Fritz, J.M.; Lenardo, M.J. A guide to cancer immunotherapy: From T cell basic science to clinical practice. Nat. Rev. Immunol. 2020, 20, 651–668. [CrossRef] [PubMed] 12. Kroemer, G.; Galluzzi, L.; Kepp, O.; Zitvogel, L. Immunogenic Cell Death in Cancer Therapy. Annu. Rev. Immunol. 2013, 31, 51–72. [CrossRef] 13. Li, Y.; Zeng, X.; He, L.; Yuan, H. Dendritic cell activation and maturation induced by recombinant calreticulin fragment 39-272. Int. J. Clin. Exp. Med. 2015, 8, 7288–7296. [PubMed] 14. Fucikova, J.; Spisek, R.; Kroemer, G.; Galluzzi, L. Calreticulin and cancer. Cell Res. 2021, 31, 5–16. [CrossRef] 15. Osman, R.; Tacnet-Delorme, P.; Kleman, J.P.; Millet, A.; Frachet, P. Calreticulin release at an early stage of death modulates the clearance by macrophages of apoptotic cells. Front. Immunol. 2017, 8, 1034. [CrossRef] [PubMed] 16. Chao, M.P.; Majeti, R.; Weissman, I.L. Programmed cell removal: A new obstacle in the road to developing cancer. Nat. Rev. Cancer 2012, 12, 58–67. [CrossRef] [PubMed] 17. Ghiringhelli, F.; Apetoh, L.; Tesniere, A.; Aymeric, L.; Ma, Y.; Ortiz, C.; Vermaelen, K.; Panaretakis, T.; Mignot, G.; Ullrich, E.; et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors. Nat. Med. 2009, 15, 1170–1178. [CrossRef] [PubMed] 18. Muenz, C.; Burgdorf, S.; Macleod, A.S.; Mahnke, K.; Silva-Vilches, C.; Ring, S. ATP and Its Metabolite Adenosine as Regulators of Dendritic Cell Activity. Front. Immunol. 2018, 9, 2581. [CrossRef] 19. Karman, J.; Ling, C.; Sandor, M.; Fabry, Z. Initiation of Immune Responses in Brain Is Promoted by Local Dendritic Cells. J. Immunol. 2004, 173, 2353–2361. [CrossRef] 20. An, Z.; Aksoy, O.; Zheng, T.; Fan, Q.W.; Weiss, W.A. Epidermal growth factor receptor and EGFRvIII in glioblastoma: Signaling pathways and targeted therapies. Oncogene 2018, 37, 1561–1575. [CrossRef] [PubMed] 21. Uhl, M.; Aulwurm, S.; Wischhusen, J.; Weiler, M.; Ma, J.Y.; Almirez, R.; Mangadu, R.; Liu, Y.-W.; Platten, M.; Herrlinger, U.; et al. SD-208, a Novel Transforming Growth Factor Receptor I Kinase Inhibitor, Inhibits Growth and Invasiveness and Enhances Immunogenicity of Murine and Human Glioma Cells In Vitro and In Vivo. Cancer Res. 2004, 64, 7954–7961. [CrossRef] 22. Zheng, Y.; McFarland, B.C.; Drygin, D.; Yu, H.; Bellis, S.L.; Kim, H.; Bredel, M.; Benveniste, E.N. Targeting protein kinase CK2 suppresses prosurvival signaling pathways and growth of glioblastoma. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2013, 19, 6484–6494. [CrossRef] 23. Liu, X.; Chen, J.; Li, W.; Hang, C.; Dai, Y. Inhibition of Casein Kinase II by CX-4945, but Not Yes-associated protein (YAP) by Verteporfin, Enhances the Antitumor Efficacy of Temozolomide in Glioblastoma. Transl. Oncol. 2020, 13, 70–78. [CrossRef] [PubMed] 24. Ruzzene, M.; Pinna, L.A. Addiction to protein kinase CK2: A common denominator of diverse cancer cells? Biochim. Biophys. Acta 2010, 1804, 499–504. [CrossRef] [PubMed] 25. Trembley, J.H.; Wang, G.; Unger, G.; Slaton, J.; Ahmed, K. Protein kinase CK2 in health and disease: CK2: A key player in cancer biology. Cell. Mol. Life Sci. C 2009, 66, 1858–1867. [CrossRef] [PubMed] 26. Silva-Pavez, E.; Tapia, J. Protein Kinase CK2 in Cancer Energetics. Front. Oncol. 2020, 10, 893. [CrossRef] 27. Reardon, D.A.; Gokhale, P.C.; Klein, S.R.; Ligon, K.L.; Rodig, S.J.; Ramkissoon, S.H.; Jones, K.L.; Conway, A.S.; Liao, X.; Zhou, J.; et al. Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model. Cancer Immunol. Res. 2016, 4, 124–135. [CrossRef] [PubMed] Int. J. Mol. Sci. 2021, 22, 3453 17 of 18 28. Feldman, L.A.; Fabre, M.-S.; Grasso, C.; Reid, D.; Broaddus, W.C.; Lanza, G.M.; Spiess, B.D.; Garbow, J.R.; McConnell, M.J.; Herst, P.M. Perfluorocarbon emulsions radiosensitise brain tumors in carbogen breathing mice with orthotopic GL261 gliomas. PLoS ONE 2017, 12, e0184250. [CrossRef] 29. Ferrer-Font, L.; Villamañan, L.; Arias-Ramos, N.; Vilardell, J.; Plana, M.; Ruzzene, M.; Pinna, L.A.; Itarte, E.; Arús, C.; Candiota, A.P. Targeting Protein Kinase CK2: Evaluating CX-4945 Potential for GL261 Glioblastoma Therapy in Immunocompetent Mice. Pharmaceuticals 2017, 10, 24. [CrossRef] 30. Towner, R.A.; Ihnat, M.; Saunders, D.; Bastian, A.; Smith, N.; Pavana, R.K. A new anti-glioma therapy, AG119: Pre-clinical assessment in a mouse GL261 glioma model. BMC Cancer 2015, 15, 522. [CrossRef] 31. Enríquez Pérez, J.; Kopecky, J.; Visse, E.; Darabi, A.; Siesjö, P. Convection-enhanced delivery of temozolomide and whole cell tumor immunizations in GL261 and KR158 experimental mouse gliomas. BMC Cancer 2020, 20, 7. [CrossRef] 32. Wu, J.; Waxman, D.J. Metronomic cyclophosphamide eradicates large implanted GL261 gliomas by activating antitumor Cd8+ T-cell responses and immune memory. Oncoimmunology 2015, 4, e1005521. [CrossRef] [PubMed] 33. Ferrer-Font, L.; Arias-Ramos, N.; Lope-Piedrafita, S.; Julià-Sapé, M.; Pumarola, M.; Arús, C.; Candiota, A.P. Metronomic treatment in immunocompetent preclinical GL261 glioblastoma: Effects of cyclophosphamide and temozolomide. NMR Biomed. 2017, 30, e3748. [CrossRef] 34. Delgado-Goñi, T.; Ortega-Martorell, S.; Ciezka, M.; Olier, I.; Candiota, A.P.; Julià-Sapé, M.; Fernández, F.; Pumarola, M.; Lisboa, P.J.; Arús, C. MRSI-based molecular imaging of therapy response to temozolomide in preclinical glioblastoma using source analysis. NMR Biomed. 2016, 29, 732–743. [CrossRef] 35. Wu, S.; Calero-Perez, P.; Villamañan, L.; Arias-Ramos, N.; Pumarola, M.; Ortega-Martorell, S.; Julia-Sapé, M.; Arús, C.; Candiota, A.P. Anti-tumour immune response in GL261 glioblastoma generated by Temozolamide Immune-Enhancing Metronomic Schedule monitored with MRSI-based nosological images. NMR Biomed. 2020, 33, e4229. [CrossRef] 36. Wu, J.; Jordan, M.; Waxman, D.J. Metronomic cyclophosphamide activation of anti-tumor immunity: Tumor model, mouse host, and drug schedule dependence of gene responses and their upstream regulators. BMC Cancer 2016, 16, 623. [CrossRef] 37. Simões, R.V.; Delgado-Goñi, T.; Lope-Piedrafita, S.; Arús, C. 1H-MRSI pattern perturbation in a mouse glioma: The effects of acute hyperglycemia and moderate hypothermia. NMR Biomed. 2010, 23, 23–33. [CrossRef] 38. Boyd-Tressler, A.; Penuela, S.; Laird, D.W.; Dubyak, G.R. Chemotherapeutic drugs induce ATP release via caspase-gated pannexin-1 channels and a caspase/pannexin-1-independent mechanism. J. Biol. Chem. 2014, 289, 27246–27263. [CrossRef] [PubMed] 39. Valverde, D.; Quintero, M.R.; Candiota, A.P.; Badiella, L.; Cabañas, M.E.; Arús, C. Analysis of the changes in the 1H NMR spectral pattern of perchloric acid extracts of C6 cells with growth. NMR Biomed. 2006, 19, 223–230. [CrossRef] [PubMed] 40. Liu, H.-L.; Huang, C.-Y.; Chen, J.-Y.; Wang, H.-Y.J.; Chen, P.-Y.; Wei, K.-C. Pharmacodynamic and therapeutic investigation of focused ultrasound-induced blood-brain barrier opening for enhanced temozolomide delivery in glioma treatment. PLoS ONE 2014, 9, e114311. [CrossRef] 41. Zhou, Q.; Guo, P.; Wang, X.; Nuthalapati, S.; Gallo, J.M. Preclinical pharmacokinetic and pharmacodynamic evaluation of metronomic and conventional temozolomide dosing regimens. J. Pharmacol. Exp. Ther. 2007, 321, 265–275. [CrossRef] [PubMed] 42. Garg, A.D.; Martin, S.; Golab, J.; Agostinis, P. Danger signalling during cancer cell death: Origins, plasticity and regulation. Cell Death Differ. 2014, 21, 26–38. [CrossRef] 43. Obeid, M.; Tesniere, A.; Ghiringhelli, F.; Fimia, G.M.; Apetoh, L.; Perfettini, J.L.; Castedo, M.; Mignot, G.; Panaretakis, T.; Casares, N.; et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 2007, 13, 54–61. [CrossRef] 44. Tavener, A.M.; Phelps, M.C.; Daniels, R.L. Anthracycline-induced cytotoxicity in the GL261 glioma model system. Mol. Biol. Rep. 2021, 48, 1017–1023. [CrossRef] [PubMed] 45. Di Virgilio, F.; Sarti, A.C.; Falzoni, S.; De Marchi, E.; Adinolfi, E. Extracellular ATP and P2 purinergic signalling in the tumour microenvironment. Nat. Rev. Cancer 2018, 18, 601–618. [CrossRef] [PubMed] 46. Garg, A.D.; Krysko, D.V.; Verfaillie, T.; Kaczmarek, A.; Ferreira, G.B.; Marysael, T.; Rubio, N.; Firczuk, M.; Mathieu, C.; Roebroek, A.J.M.; et al. A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J. 2012, 31, 1062–1079. [CrossRef] 47. Vénéreau, E.; Ceriotti, C.; Bianchi, M.E. DAMPs from Cell Death to New Life. Front. Immunol. 2015, 6, 422. [CrossRef] 48. De Salvo, M.; Maresca, G.; D’agnano, I.; Marchese, R.; Stigliano, A.; Gagliassi, R.; Brunetti, E.; Raza, G.H.; De Paula, U.; Bucci, B. Temozolomide induced c-Myc-mediated apoptosis via Akt signalling in MGMT expressing glioblastoma cells. Int. J. Radiat. Biol. 2011, 87, 518–533. [CrossRef] 49. Strobel, H.; Baisch, T.; Fitzel, R.; Schilberg, K.; Siegelin, M.D.; Karpel-Massler, G.; Debatin, K.-M.; Westhoff, M.-A. Temozolomide and Other Alkylating Agents in Glioblastoma Therapy. Biomedicines 2019, 7, 69. [CrossRef] 50. Günther, W.; Pawlak, E.; Damasceno, R.; Arnold, H.; Terzis, A.J. Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids. Br. J. Cancer 2003, 88, 463–469. [CrossRef] 51. Hori, Y.S.; Hosoda, R.; Akiyama, Y.; Sebori, R.; Wanibuchi, M.; Mikami, T.; Sugino, T.; Suzuki, K.; Maruyama, M.; Tsukamoto, M.; et al. Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells. J. Neurooncol. 2015, 122, 11–20. [CrossRef] [PubMed] Int. J. Mol. Sci. 2021, 22, 3453 18 of 18 52. Stupp, R.; Mason, W.P.; van den Bent, M.J.; Weller, M.; Fisher, B.; Taphoorn, M.J.B.; Belanger, K.; Brandes, A.A.; Marosi, C.; Bogdahn, U.; et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 2005, 352, 987–996. [CrossRef] 53. Syed, M.; Liermann, J.; Verma, V.; Bernhardt, D.; Bougatf, N.; Paul, A.; Rieken, S.; Debus, J.; Adeberg, S. Survival and recurrence patterns of multifocal glioblastoma after radiation therapy. Cancer Manag. Res. 2018, 10, 4229–4235. [CrossRef] [PubMed] 54. Park, J.; Kim, C.G.; Shim, J.-K.; Kim, J.H.; Lee, H.; Lee, J.E.; Kim, M.H.; Haam, K.; Jung, I.; Park, S.-H.; et al. Effect of combined anti-PD-1 and temozolomide therapy in glioblastoma. Oncoimmunology 2019, 8, e1525243. [CrossRef] [PubMed] 55. Wu, S.; Calero-Pérez, P.; Arús, C.; Candiota, A.P. Anti-pd-1 immunotherapy in preclinical gl261 glioblastoma: Influence of therapeutic parameters and non-invasive response biomarker assessment with mrsi-based approaches. Int. J. Mol. Sci. 2020, 21, 8775. [CrossRef] [PubMed] 56. Dai, B.; Qi, N.; Li, J.; Zhang, G. Temozolomide combined with PD-1 Antibody therapy for mouse orthotopic glioma model. Biochem. Biophys. Res. Commun. 2018, 501, 871–876. [CrossRef] 57. Grusch, M.; Polgar, D.; Gfatter, S.; Leuhuber, K.; Huettenbrenner, S.; Leisser, C.; Fuhrmann, G.; Kassie, F.; Steinkellner, H.; Smid, K.; et al. Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside. Cell Death Differ. 2002, 9, 169–178. [CrossRef] 58. Vultaggio-Poma, V.; Sarti, A.C.; Di Virgilio, F. Extracellular ATP: A Feasible Target for Cancer Therapy. Cells 2020, 9, 2496. [CrossRef] 59. Trabanelli, S.; Očadlíková, D.; Gulinelli, S.; Curti, A.; Salvestrini, V.; de Paula Vieira, R.; Idzko, M.; Di Virgilio, F.; Ferrari, D.; Lemoli, R.M. Extracellular ATP Exerts Opposite Effects on Activated and Regulatory CD4 + T Cells via Purinergic P2 Receptor Activation. J. Immunol. 2012, 189, 1303–1310. [CrossRef] 60. Zheng, Z.; Zhang, J.; Jiang, J.; He, Y.; Zhang, W.; Mo, X.; Kang, X.; Xu, Q.; Wang, B.; Huang, Y. Remodeling tumor immune microenvironment (TIME) for glioma therapy using multi-targeting liposomal codelivery. J. Immunother. Cancer 2020, 8, 207. [CrossRef] 61. Wu, S.Y.; Watabe, K. The roles of microglia/macrophages in tumor progression of brain cancer and metastatic disease. Front. Biosci. Landmark 2017, 22, 1805–1829. [CrossRef] 62. Colangelo, T.; Polcaro, G.; Ziccardi, P.; Muccillo, L.; Galgani, M.; Pucci, B.; Milone, M.R.; Budillon, A.; Santopaolo, M.; Mazzoccoli, G. The miR-27a-calreticulin axis affects drug-induced immunogenic cell death in human colorectal cancer cells. Cell Death Dis. 2016, 7, e2108. [CrossRef] [PubMed] 63. Sukkurwala, A.Q.; Martins, I.; Wang, Y.; Schlemmer, F.; Ruckenstuhl, C.; Durchschlag, M.; Michaud, M.; Senovilla, L.; Sistigu, A.; Ma, Y.; et al. Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8. Cell Death Differ. 2014, 21, 59–68. [CrossRef] [PubMed] 64. Intemann, J.; Saidu, N.E.B.; Schwind, L.; Montenarh, M. ER stress signaling in ARPE-19 cells after inhibition of protein kinase CK2 by CX-4945. Cell. Signal. 2014, 26, 1567–1575. [CrossRef] 65. Gebremeskel, S.; Johnston, B. Concepts and mechanisms underlying chemotherapy induced immunogenic cell death: Impact on clinical studies and considerations for combined therapies. Oncotarget 2015, 6, 41600–41619. [CrossRef] [PubMed] 66. Papaioannou, N.E.; Beniata, O.V.; Vitsos, P.; Tsitsilonis, O.; Samara, P. Harnessing the immune system to improve cancer therapy. Ann. Transl. Med. 2016, 4, 2. [CrossRef] 67. Esfahani, K.; Roudaia, L.; Buhlaiga, N.; Del Rincon, S.V.; Papneja, N.; Miller, W.H. A review of cancer immunotherapy: From the past, to the present, to the future. Curr. Oncol. 2020, 27, 87–97. [CrossRef] 68. Son, Y.H.; Song, J.S.; Kim, S.H.; Kim, J. Pharmacokinetic characterization of CK2 inhibitor CX-4945. Arch. Pharm. Res. 2013, 36, 840–845. [CrossRef] 69. Garofalo, S.; Porzia, A.; Mainiero, F.; Di Angelantonio, S.; Cortese, B.; Basilico, B.; Pagani, F.; Cignitti, G.; Chece, G.; Maggio, R.; et al. Environmental stimuli shape microglial plasticity in glioma. eLife 2017, 6, e33415. [CrossRef] [PubMed]