Proinflammatory macrophage-derived medium enhances temozolomide sensitivity in glioblastoma via pSTAT3-mediated downregulation of DNA repair enzymes

Proinflammatory macrophage-derived medium enhances temozolomide sensitivity in glioblastoma via pSTAT3-mediated downregulation of DNA repair enzymes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Proinflammatory macrophage-derived medium enhances temozolomide sensitivity in glioblastoma via pSTAT3-mediated downregulation of DNA repair enzymes Susana López-López, Beatriz Castro-Robles, María José M. Díaz‐Guerra, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5991999/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Chemoresistance to temozolomide (TMZ), the main agent in glioblastoma (GBM) treatment, negatively impacts prognosis due to efficient repair of TMZ-induced DNA lesions. Reduced expression of the DNA repair enzyme O6-methylguanine DNA-methyltransferase (MGMT) has been associated with improved chemotherapy response, though not consistently across patients. Our previous findings suggested that N-methylpurine-DNA-glycosylase (MPG), which repairs the most common TMZ-induced DNA adducts, would be implicated in glioblastoma resistance to TMZ, but its role in chemoresistance remains unclear. Tumor-associated macrophages (TAMs), a key component of the GBM microenvironment, impair chemotherapy efficacy, though mechanisms by which they contribute to drug resistance are not well defined. This study shows that reducing MPG expression with specific siRNAs sensitizes tumor cells to TMZ, and this effect is potentiated by simultaneously downregulating both MPG and MGMT. We also provide the first evidence that conditioned medium from proinflammatory macrophages (CM-M1), by suppressing STAT3 phosphorylation, reduces repair enzyme expression in glioblastoma cells, enhancing TMZ cytotoxicity. Molecular analysis of tumor samples from glioblastoma patients confirmed an association between high repair enzyme expression and STAT3 activation. Our findings uncover a novel mechanism by which TAMs contribute to chemoresistance, supporting strategies to reprogram TAMs towards an M1-phenotype to improve TMZ efficacy in GBM treatment. Health sciences/Oncology/Cancer/Cancer microenvironment Health sciences/Oncology/Cancer/Cns cancer Biological sciences/Cancer/Cancer therapy Biological sciences/Cell biology/Mechanisms of disease Glioblastoma Chemoresistance Proinflammatory macrophage DNA repair enzymes MGMT MPG Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Glioblastoma (GB) is the most common and aggressive primary malignant brain tumor in adults, with a poor prognosis for patients, with less than 5% survive beyond five years 1 . The current standard treatment for newly diagnosed glioblastoma is multimodal, consisting of maximum safe resection, followed by localized radiotherapy and concomitant and adjuvant temozolomide (TMZ) chemotherapy. Although chemotherapy after surgery has been proven to increase patient survival 2 , TMZ resistance remains a major obstacle to treatment efficacy in glioblastoma. This underscores the need to elucidate the molecular mechanisms underlying the resistant phenotype in order to improve the prognosis for glioblastoma patients. TMZ causes damage to cells through methylation at several nitrogenous bases in DNA, with the primary site of methylation occurring at the N7-guanine position (70%), followed by the N3-adenine position and at the O6-guanine residue (5%). However, the cytotoxic effect of TMZ in glioblastoma cells becomes limited by the action of cellular DNA damage repair systems, which represent one of the main factors contributing to chemoresistance. It is well-established in the literature that elevated levels of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme that removes the O6-methylguanine lesion, one of the most lethal lesions induced by TMZ, are a key factor driving TMZ resistance. Several studies conducted in patients treated with TMZ have reported that MGMT promoter methylation, leading to a lack of expression of the enzyme, is associated with improved overall survival compared to unmethylated glioblastomas 3 . Nevertheless, a considerable proportion of patients fail to respond to TMZ treatment and do not exhibit clinical improvement even when the tumor expresses low levels of MGMT, suggesting that this DNA repair enzyme is not the only predictor of response to TMZ. In this context, previous findings from our group and others have highlighted the role of N-methylpurine-DNA glycosylase (MPG), the initial enzyme in the base excision repair pathway (BER), in conferring TMZ chemoresistance in glioblastoma 4,5 . MPG removes the most frequent TMZ-induced damages (N7-methylguanine and N3-methyladenine), leading to the formation of different intermediate adducts that are subsequently removed by downstream enzymes such as apurinic/apyrimidinic (AP) sites endonuclease 1 (APE1) and poly(ADP-ribose) polymerase (PARP), with the repair process finalized by DNA polymerase, POL-β. Pharmacological inhibition of certain proteins in the BER pathway, such as APE1, PARP and poly-ADP-ribose glycohydrolase (PARG), has been demonstrated to enhance TMZ-induced cytotoxicity regardless of MGMT status 6-8 . However, MPG expression has been associated with both increased and decreased resistance to TMZ in glioblastoma 5,9 , thus the role of this enzyme in chemotherapy response remains undefined. In recent years, tumor-associated macrophages (TAMs), the most abundant cells in the glioblastoma microenvironment, have attracted considerable interest due to their role in promoting tumor growth, metastasis, and resistance to conventional therapies 10 . Macrophages exhibit two mutually polarizable phenotypes: M1, characterized by the secretion of a high amount of proinflammatory cytokines (e.g interleukin (IL)-1, CXCL10, TNF-α), and M2, which produces high levels of anti-inflammatory cytokines such as IL-10. TAMs usually display an M2-like phenotype that is associated with pro-tumoral features, whereas M1 macrophages exert antitumor functions 11 . Activated M2 macrophages have been reported to promote chemoresistance by enhancing anti-apoptotic signaling, such as STAT3 transcription factor and BCL2 pathways in tumor cells, while M1 macrophages activate the STAT1-mediated apoptotic pathway 12 . Notably, a study performed by Kohsaka et al. 13 revealed that STAT3 activation, frequently observed in tumors including glioblastoma, upregulates MGMT, suggesting this signaling pathway might be responsible for TMZ resistance. However, the underlying mechanisms involving chemoresistance-induced macrophages are still poorly understood. This study aimed to gain insight into the contribution of MPG to TMZ resistance, both independently and in combination with MGMT, through siRNA silencing in an in vitro model of glioblastoma. These analyses are intended to clarify the controversial findings of previous studies regarding the role of the BER molecular repair pathway in chemotherapy resistance. On the other hand, TAMs are known to hinder treatment effectiveness, but the mechanisms underlying macrophage-mediated chemoresistance are still not well understood. In addition, we examined the potential effect of conditioned medium from proinflammatory macrophage phenotype (CM-M1) on the expression of MPG and MGMT in glioblastoma cells and whether this effect is mediated by the STAT1 or STAT3 transcription factors. Gathering evidence of the interaction between the innate immune system and DNA repair pathways in glioblastoma could support strategies to reprogram M2 macrophages into an M1 phenotype, with the purpose of improving the chemotherapy response. Results TMZ resistance and the expression profile of DNA repair enzymes in an in vitro model of glioblastoma Viability assays performed on A172 and T98G glioblastoma cell lines incubated with increasing concentrations of TMZ for 72 hours demonstrated a dose-dependent reduction in cell survival following TMZ treatment (Fig. 1A). The results indicated that both cell lines exhibited resistance to the chemotherapeutic agent, as high doses (400 µM) reduced cell viability by less than 50% (40% in A172 and 20% in T98G). BER pathway enzymes (MPG, APE1, PARP, and POL-β) and the MMR enzyme MSH2 were highly expressed in both glioblastoma cell lines (Fig. 1B). While MGMT expression was absent in A172 cells, it was detected in T98G cells (Fig. 1B), potentially explaining the greater resistance observed in T98G cells (Fig. 1A). Furthermore, our findings align with previous studies reporting that A172 cells possess wild-type p53, whereas T98G cells harbor a mutated form of p53. This mutation disrupts the normal regulation of protein degradation, leading to increased protein accumulation (Fig. 1B). Such alterations may partly account for the elevated MGMT levels in T98G cells, as mutated p53 has been reported to upregulate MGMT expression. 14 Although the lack of active MGMT, along with high levels of MMR, is generally considered a sensitizing factor for TMZ treatment, our results suggest that the chemoresistance observed in A172 cells may be driven by other factors, such as enzymes involved in the BER pathway. In summary, the glioblastoma cell lines A172 and T98G used in this study exhibited similarly high chemoresistance but distinct profiles of DNA repair enzyme expression, allowing us to investigate the potential role of MPG in TMZ resistance, both in the presence and absence of MGMT. Increased glioblastoma chemosensitivity through MPG silencing, both alone and synergistically with MGMT downregulation To investigate the influence of DNA repair enzymes on TMZ efficacy, small interfering RNAs (siRNAs) were utilized to suppress MGMT and MPG expression. Glioblastoma cell lines were initially transfected with varying concentrations of MGMT- and MPG-targeting siRNAs, both individually and in combination, to determine the optimal conditions for achieving efficient reduction of enzyme expression at both the protein and mRNA levels. MPG expression was effectively inhibited in A172 cells using a siRNA concentration of 25 nM, whereas T98G cells required 40 nM for optimal inhibition (Figure s1), with maximum suppression observed 96 hours post-incubation in both glioblastoma cell lines (Figure s2, Fig. 2A). In T98G cells, which express both DNA repair enzymes, a 25 nM concentration of MGMT-targeted siRNA effectively reduced MGMT expression separately, and this peak effect was sustained when both enzymes were simultaneously knocked down 96 hours after exposure to the specific siRNAs (Figure s2, Fig. 2A). The expression of enzymes involved in the subsequent steps of the MPG-mediated base excision repair (BER) pathway, as well as the MMR enzyme MSH2, was unaffected in the absence of MPG and MGMT (Fig. 2A). Additionally, siRNAs targeting these DNA repair enzymes remained effective even in the presence of TMZ (Fig. 2A). It is worth mentioning that the optimal incubation time for gene silencing, set at 96 hours, enabled the viability studies with TMZ-treated glioblastoma cells, as it aligned with the experimental conditions. SiRNA typically requires approximately 48 hours to take effect, while TMZ induces cell death over extended periods (see experimental protocol scheme, Fig. 2B). As shown in Figure 2B, MPG siRNA significantly enhanced TMZ sensitivity in A172 cells, which do not express MGMT. Moreover, the combined inhibition of MPG and MGMT in TMZ-treated T98G cells resulted in a synergistic decrease in cell viability, with statistically significant differences compared to each siRNA treatment alone. Collectively, our results suggest that MPG activity, either alone or in combination with MGMT, plays a significant role in contributing to TMZ resistance in glioblastoma. Conditioned medium from M1 macrophages inhibits MGMT and MPG expression in glioblastoma cells through blocking pSTAT3 signaling The M2 phenotype of tumor-associated macrophages has been reported to potentiate resistance to TMZ. In an attempt to elucidate the mechanisms behind macrophage-mediated chemoresistance in glioblastoma, which are still not fully understood, we sought to explored whether media from polarized M1 and/or M2 macrophages are involved in regulating the expression of the DNA repair enzymes MPG and MGMT. First, macrophages underwent a differentiation process, as described in Methods, to collect conditioned media during M1 and M2 activation, which would subsequently be used in cell cultures to evaluate the regulation of DNA repair enzyme expression. M1 differentiation was confirmed by measuring STAT1 phosphorylation, a key transcription factor in pro-inflammatory activation, and by detecting increased expression of the pro-inflammatory genes TNFα and CXCL-10 compared to M0 or M2 macrophages (Fig. 3A). However, M2 markers such as Arg1 and IL-10 were only modestly induced, consistent with previous studies showing that THP-1-macrophage cells do not fully differentiate into the M2 phenotype (Fig. 3A). As a result, subsequent experiments were conducted using only the conditioned medium from M1 macrophages (CM-M1), which, despite this, allowed us to explore the interplay between glioblastoma immunology and the molecular mechanisms underlying chemoresistance. In addition, the total and activated (phosphorylated form) levels of STAT1, as well as STAT3, a marker of M2 macrophage signaling, were analyzed in glioblastoma cells. Both glioblastoma cell lines showed elevated levels of phosphorylated Tyr705-STAT3, with T98G exhibiting the highest expression, while the activated form of STAT1 was found to be lower in T98G compared to A172 (Fig. 3B). Then, Western blotting was performed to examine the levels of MPG and MGMT, as well as STAT3, STAT1, and their phosphorylated forms, in T98G and A172 cells after incubation with CM-M1 and control medium from non-activated macrophages (CM-M0). CM-M1-treated glioblastoma cells exhibited reduced levels of the activated (phosphorylated) form of STAT3, which exerts the function of a transcription factor, compared to CM-M0-treated cells, while the amounts of total STAT3 protein remained largely unchanged. In contrast, CM-M1 exposure triggered both increased STAT1 activation (phosphorylation) and expression (total protein levels) (Fig. 3C). Additionally, we observed a reduction in the expression of the DNA repair enzymes MPG and MGMT after incubation with M1 proinflammatory medium, compared to cells treated with CM-M0 (Fig. 3C). To gain insight into the transcription factor signaling responsible for M1-macrophage medium-mediated regulation of DNA repair enzymes, glioblastoma cells were transfected with overexpression vectors for STAT3 or STAT1 to determine whether the observed reduction in MPG and MGMT expression was due to decreased pSTAT3 or increased pSTAT1. Forced STAT1 expression and increased pSTAT1 did not lower MPG or MGMT levels compared to control cells, while overexpression of STAT3, which resulted in a rise in its phosphorylated form, significantly increased the expression of these enzymes (Fig. 3D). Next, we used a selective STAT3 inhibitor that completely abolished STAT3 phosphorylation 15 , leading to reduced MGMT and MPG expression (Fig. 3E). Finally, to confirm the role of STAT3 in the CM-M1-induced decrease in MGMT and MPG expression, glioblastoma cells, either transfected with an empty vector or overexpressing STAT3, were incubated with the proinflammatory medium. As shown in Fig. 3F, STAT3 overexpression and increased pSTAT3 attenuated the CM-M1-induced suppression of DNA repair enzyme expression. Our findings demonstrate that pro-inflammatory conditioned medium suppresses MGMT and MPG expression in glioblastoma cells by inhibiting pSTAT3 signaling, emphasizing the role of STAT3-mediated innate immunity in regulating DNA repair mechanisms in glioblastoma. Association between MGMT and MPG expression and STAT3 activation in patient-derived glioblastoma We investigated the potential correlation between MGMT and MPG expression, and pSTAT3 in glioblastoma patient samples. Western blot analysis was performed on 15 tumor tissue samples obtained from patients diagnosed with glioblastoma who underwent tumor resection surgery. The patients had a median age of 55 years (range: 40–78 years), with 40% female representation. The time between diagnosis and surgery ranged from 0 to 45 days, with an average of 15.6 days. The majority of patients received a combination of radiation therapy and temozolomide (RT-TMZ + TMZ) as their first-line treatment, although some were treated with temozolomide alone or did not receive treatment. Glioblastoma recurrence (assessed through imaging studies, such as MRI, showing new tumor growth or changes 4 to 6 months after the completion of the first therapeutic regimen) was observed in 10 (67%) patients (GB1, 2, 6, 7, 8, 9, 10, 12, 13, 15) The molecular results showed that 53% of patients (n=8) expressed pSTAT3 (Fig. 4). Among these, 87.5% (n=7) exhibited MGMT expression, and 87.5% (n=7) had detectable MPG protein levels. Notably, 75% of the pSTAT3-positive patients (n=6) expressed both MGMT and MPG (Fig. 4). Although the sample size is small and requires confirmation in a larger patient cohort, these findings suggest that activated STAT3 may regulate the expression of both DNA repair enzymes in patient tumors, consistent with the observations from our in vitro glioblastoma model. We did not find a significant relationship between enzyme expression, transcription factors and tumor relapsing after the treatment. However, it is worth noting that MPG expression was detected in a higher proportion of patients with recurrence compared to those without recurrence (80% vs. 60%, respectively) (Fig. 4). Proinflammatory medium potentiates TMZ efficacy by inducing apoptotic cell death through the downregulation of anti-apoptotic protein and DNA repair enzymes We finally analyzed the effect of CM-M1 on the viability of glioblastoma cell lines under TMZ treatment to determine whether this proinflammatory medium enhances chemosensitivity as a result of downregulating the expression of MGMT and MPG (as shown in the previous results section). Cell viability was assessed after incubating the cells with CM-M1 or non-activated macrophage medium (M0) for 24 hours, followed by an additional 72 hours in the presence or absence of TMZ. Our results showed that exposure to CM-M1 significantly inhibited cell viability in both cell lines (Fig. 5A), which could be partially due to the downregulation of BCL2 (Fig. 5B), an anti-apoptotic gene, along with strong activation of STAT1(Fig. 3C), a transcription factor identified as a negative regulator of cell proliferation and a promoter of apoptotic signaling. More interestingly, CM-M1 incubation combined with low-dose TMZ treatment (100 µM in A172 and 300 µM in T98G) led to a significantly greater reduction in cell survival compared to treatments with the inflammatory medium and chemotherapy agent separately (Fig. 5A). Cell death in glioblastoma induced by exposure to CM-M1 alone or in combination with TMZ was analyzed by flow cytometry. We found that apoptosis was synergistically increased by the combination of proinflammatory macrophage medium and TMZ, with a higher percentage (5.4±0.3% and 17.2±1%) of A172 cells undergoing early and late apoptosis, respectively, compared to 4.5±0.1% and 12.7±2%, or 4.4±0.2% and 14.7±1.5%, in the individual CM-M1 and TMZ treatment groups (Fig. 5C). Similar results were observed in T98G cells, with 40.3±2.7% total apoptotic cells, in comparison to 35.2±1.8% and 17.3±4% after separate incubation with M1 medium and chemotherapy agent (Fig. 5C). These findings were further supported by Western blot analysis, which showed increased levels of the pro-apoptotic protein cleaved Caspase-3 in the combined treatment group (Fig. 5D). Taken together, these results suggest that CM-M1 potentiates the deleterious effects of TMZ by triggering apoptosis, which, in synergy with chemotherapy, leads to an increased number of cells undergoing cell death via apoptosis. This increase in apoptotic processes could result from the simultaneous action of the proinflammatory medium on glioblastoma cells, downregulating the expression of both the anti-apoptotic protein BCL2 and, as previously shown, the DNA repair enzymes MPG and MGMT, in a p-STAT3-dependent manner. Discussion Chemoresistance in glioblastoma develops frequently, underscoring the need for further investigation of the molecular mechanisms driving GBM resistance. Our results, derived from an in vitro glioblastoma resistance model, demonstrate that MPG, which repairs the most common TMZ-induced lesions, significantly contributes to TMZ resistance in the absence of MGMT. Moreover, MPG acts synergistically with MGMT, a well-established mediator of chemoresistance, to limit TMZ efficacy. Importantly, we report for the first time that proinflammatory medium derived from M1 macrophages (CM-M1) leads to a pSTAT3-mediated reduction in the expression of DNA repair enzymes MGMT and MPG, thereby enhancing glioblastoma cell sensitivity to TMZ, as illustrated in Fig. 6. For more than a decade, chemoresistance in glioblastoma has been largely attributed to MGMT, which repairs the most cytotoxic TMZ-induced DNA lesions. Indeed, the methylation status of the MGMT promoter, leading to low MGMT expression, was the first molecular marker used to predict a better response to TMZ in clinical trials 16 . As chemotherapy-induced cytotoxicity, driven by MGMT deficiency, also requires subsequent repair via the MMR pathway, and its activity has been reported to decrease with TMZ treatment 17,18 , the MPG-initiated BER pathway, responsible for repairing the most frequent N-methylpurine lesions in DNA induced by TMZ, has emerged as a potential complementary factor in chemoresistance. However, in recent years, there has been controversy regarding its role in chemoresistance, as this DNA repair pathway has been associated with both contributing to chemoresistance and increasing TMZ efficacy. MPG expression has been correlated with worse prognosis in glioma patients 4,19 , and a similar pattern was observed in patients whose glioblastoma tumor analysis revealed that MGMT methylation alone could not explain TMZ resistance 20 . In this context, other studies have demonstrated that MPG and POL-β expression predict sensitivity to chemotherapeutic agents 21 , and the reduced expression of BER proteins, such as APE1 and PARP, by chemical inhibitors sensitizes glioblastoma cells to TMZ 6,7 . In contrast, Fosmark et al. 9 showed that high MPG expression correlated with improved survival in glioblastoma patients with MGMT promoter methylation. Growing evidence of TMZ efficacy potentiation by MPG in glioblastoma and other cancers comes from experiments combining MPG overexpression with the inhibition of downstream BER pathway enzymes, including AP1 and PARP 22,23 . The increased sensitivity resulted from enhanced repair of N-methylpurine lesions, saturating the BER rate-limiting enzyme POL-β and leading to the accumulation of cytotoxic 5’dRP repair intermediates. Notably, overexpression of POL-β in cells with high levels of MPG incubated with BER enzyme inhibitors abrogates the MPG-dependent enhancement of TMZ sensitivity 23 . Thus, according to these results, inhibiting downstream BER enzymes would only potentiate the effect of TMZ under conditions of high MPG levels and low POL-β levels. However, the results of our study revealed that downregulation of the first enzyme of BER, MPG, is capable of sensitizing glioblastoma cells to TMZ in both MGMT-expressing cells (T98G) and MGMT-negative cells (A172), regardless of elevated POL-β levels. These findings strongly support the role of this DNA repair enzyme in chemoresistance in glioblastoma, aligning with previous data from our group that demonstrated an association between elevated MPG expression and the TMZ-resistant phenotype in tumor cells derived from patients 5 . This suggests that inactivating these DNA repair enzymes could be an effective therapeutic strategy to overcome chemotherapy resistance. However, the development of effective MPG inhibitors has so far been unsuccessful due to the complexity of its active site. Similarly, pharmacological approaches targeting MGMT have not significantly reduced chemoresistance in clinical trials, as they required reduced TMZ doses to avoid the severe myelosuppressive toxicity caused by inhibitors such as Lomeguatrib or PaTrin-2 24,25 . On the other hand, research in recent years has demonstrated that TAMs dominate the cellular composition of the glioblastoma microenvironment, with the presence of the M2 phenotype contributing to TMZ resistance in this type of tumor 26-29 , although the underlying molecular mechanisms driving such chemoresistance remain unclear. The results of our study demonstrated that the proinflammatory medium derived from M1 macrophages triggered reduced cell proliferation and apoptotic induction, comparable to the effects seen in breast cancer cell cultures 30 and renal clear cell carcinoma 31 treated with CM-M1. Furthermore, we found that CM-M1 led to an increase in the phosphorylated form of STAT1, which has been previously described as an adaptor required for the formation of the death-inducing signaling complex involved in apoptosis induction, as well as a downregulation of the anti-apoptotic protein BCL-2. Therefore, the apoptosis induced by the proinflammatory medium could be due to an increase in STAT1 activity combined with the reduction of BCL-2 levels. More interestingly, the simultaneous treatment of glioblastoma cells with CM-M1 and TMZ synergistically increased apoptotic cell death in glioblastoma cultures, significantly enhancing sensitivity to chemotherapy compared to the individual CM-M1 and TMZ treatments. This cytotoxic potentiation effect of TMZ may result from the observed inhibition of MPG and MGMT expression, along with the reduced expression of BCL-2 and the elevated levels of pSTAT1. Using various technical approaches in this study, we found that the decreased expression of both DNA repair enzymes induced by the proinflammatory macrophage medium was not mediated by the upregulation of STAT1 and pSTAT1, but rather by the inhibition of STAT3 activation, as evidenced by the reduced protein levels of pSTAT3, while the total amount of STAT3 remained unaffected. Consistent with our findings, previous research has demonstrated that blocking STAT3 phosphorylation decreases the IC50 of TMZ in glioblastoma cells, enhancing TMZ-induced apoptosis, partly through reduced BCL-2 expression, a downstream target of p-STAT3 32-35 . The activated-phosphorylated form of STAT3 has also been reported to upregulate MGMT expression, contributing to TMZ desensitization 13 . In this context, our study demonstrates for the first time that pSTAT3 modulates the expression of both MPG and MGMT. This finding aligns with tumor analyses from patients, which show higher expression of both enzymes in pSTAT3-positive samples (75%) compared to those lacking activated STAT3. Therefore, our study provides new insights into how glioblastoma-associated macrophages may contribute to chemoresistance by upregulating DNA repair enzymes that diminish the cytotoxic effect of TMZ. Based on our results, we propose that converting macrophages from the M2 to the M1 phenotype represents a promising therapeutic strategy to overcome TMZ resistance. This approach could also serve as an alternative to current immunotherapies focused on enhancing adaptive immunity 36 , which have shown limited efficacy in glioblastoma 37 . Indeed, various compounds, including corosolic 38 and inhibitors of placental growth factor, C/EBPβ (CCAAT/enhancer-binding protein beta), and the hyaluronic acid pathway, have demonstrated efficacy in reprogramming macrophages from M2 to M1 in glioblastoma, leading to tumor suppression through STAT3 inhibition 39,40 . Although our study sheds light on the roles of MPG and MGMT in TMZ resistance in glioblastoma, as well as the effect of the proinflammatory medium from M1 macrophages on the expression of these DNA repair enzymes, leading to reduced levels and, consequently, increased chemotherapy sensitivity (see Fig. 6 for illustration), we acknowledge certain limitations. The in vitro glioblastoma cell models used in this study do not fully replicate the complex tumor microenvironment observed in glioblastoma patients. Therefore, further research utilizing patient-derived cells and animal models is necessary to confirm and validate our findings, and also to identify the proinflammatory molecules secreted that are responsible for the pSTAT3-mediated downregulation of MPG and MGMT. Methods Cell cultures, Conditioned media from differentiated macrophages, and Patient-derived glioblastoma samples A172 and T98G glioblastoma cell lines (ATCC# CRL-1620, CRL-1690) were maintained in DMEM and MEM, respectively, with 10% FBS and 1% penicillin-streptomycin, while the culture medium of THP-1 monocytes (ATCC# TIB-202) was RPMI 1640 with 10% FBS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, and 1% penicillin-streptomycin. All cell lines were cultured at 37°C in a 5% CO2 atmosphere. Suppression of STAT3 activation-phosphorylation was achieved using Stattic (ab120962, Abcam) according to the manufacturer's instructions. This selective STAT3 inhibitor blocks activation-phosphorylation, as well as subsequent dimerization and nuclear translocation of STAT3, by interacting with the SH2 domain. For macrophage differentiation, THP-1 cells were treated with 160 nM PMA (Sigma-Aldrich) for 48 hours, followed by culture in RPMI with 5% FBS for 24 hours. Macrophages were then polarized into M1 phenotype using IFN-γ (10 U/ml) and LPS (100 ng/ml), and into M2 using IL-13 (20 ng/ml) and IL-4 (20 ng/ml). After 24hours differentiation conditioned media (CM) was collected, centrifuged, and stored at -20°C until use. Tumor tissue samples were collected from 15 patients diagnosed with glioblastoma by the Neurology, Neurosurgery, and Oncology departments of the General University Hospital of Albacete (Spain). Written informed consent was obtained from all patients. All methods were performed in accordance with the 1964 Declaration of Helsinki and its later amendments, and all procedures were approved by the Human Ethics Committee of this hospital (Ethical approval number: 03/2014). Sociodemographic and clinical information was obtained from the hospital information system, including the date of diagnosis, surgery, cause of death (if applicable), type of antitumor therapy, and disease-free period (DFP). After collection, fresh tissue samples were homogenized in detergent lysis buffer (RIPA, Merck) for Western blot analysis. Gene silencing and Cell transfections A172 and T98G cells were transfected with increasing concentrations of siRNAs (25, 40,80 nM) targeting MPG and MGMT (Santa Cruz), using siRNA Transfection Reagent (sc-29527). Briefly, cells were seeded one day prior to transfection in 6-well plates for Western blot and qRT-PCR analysis, or in 24-well plates for cell viability assessment, with 10% FBS in their respective media. The siRNA-transfection reagent complex was prepared in transfection medium (sc-36868, Santa Cruz) according to the manufacturer’s instructions. The medium was replaced with fresh medium 8 hours after transfection. After a minimum of 48 hours, protein extracts were prepared for Western blot and qRT-PCR analysis. Cell viability was assessed after an additional 72-hour TMZ treatment. For plasmid transfections, A172 and T98G cells were seeded in 6-well plates and transfected 24 hours later with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, using OPTI-MEM medium (Gibco) and 2.5 µg of total plasmid DNA (pCMV-Sport6, pCMV-Sport6-STAT1, or pCMV-Sport6-STAT3) per well. Cells were analyzed 24 hours post-transfection. Western Blot analysis Proteins from cell culture and tumor tissue homogenates were extracted using RIPA buffer, and protein concentration was determined by the BCA assay. Protein extracts (30-40 µg) were separated on denaturing SDD-PAGE polyacrylamide gels and transferred to a PVDF membrane (Hybond-C Extra, Amersham Biosciences). Spectra TM Multicolor Broad Range Protein ladder (Thermo Scientific #26634) was used as a molecular weight marker for protein size estimation. The following primary antibodies were used: anti-MGMT (2739S), anti-pSTAT3 (D3A7), anti-pSTAT1 (58D6), anti-STAT3 (124H6), and anti-STAT1 (9172S) from Cell Signaling, anti-MPG (sc-101237), anti-MSH2 (sc-376384), anti-APE1 (sc-17774), anti-PARP-1 (sc-8007), anti-BLC2 (sc-7382), anti-caspase-3 (sc-56053) and anti-β-ACTIN (sc-81178) from Santa Cruz, and anti-DNA polymerase (ab26343) from Abcam. Chemiluminescence detection was performed using the SuperSignal™ West Dura (Thermo Scientific) on a Luminescent Image Analyzer LAS-mini 4000 system (Fujifilm, Tokyo, Japan) RNA isolation and qRT-PCR analysis Total RNA was extracted using the RNA Total Isolation Kit (Nzytech) and converted into cDNA using RevertAid™ Minus First Strand (Thermo Scientific). Gene expression was analyzed using SYBR™ Green PCR Master Mix (Applied Biosystems). Primers used included the following sequences: MGMT: Forward: 5′-GTCGTTCACCAGACAGGTGTTA-3′; Reverse: 5′-ACAGGATTGCCTCTCATTGCTC-3′ MPG: Forward:5′-TTGGAGTTCTTCGACCAGCC-3′; Reverse: 5′-CATGAACATGCCTCGGTTGC-3′ β-actin: Forward:5′-AAGATCATTGCTCCTCCTG-3′; Reverse: 5′-CGTCATACTCCTGCTTGCTG-3′ Cell viability and Apoptosis assays The MTT assay was used to assess cell viability according to the manufacturer’s protocol. A172 and T98G cells were plated in 24-well plates and cultured in 500 µL of medium containing the specified concentration of TMZ. After 72 hours, MTT solution (5 mg/mL) was added and incubated for 1 hour at 37°C. The medium was then removed, and a solubilizing solution was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate reader (SPECTROstar Omega, BMG LABTECH), with each sample analyzed in triplicate. Apoptosis was assessed by flow cytometry after 24 hours of CM-M1 or CM-M0 treatment followed by 72 hours of TMZ exposure or DMSO-vehicle control. Cells were resuspended in 450 µL of 1X Annexin V binding buffer (Immunostep) and stained with 10 µL of Annexin V conjugated to the fluorochrome Dy-634 (Immunostep), together with 40 µL of the vital dye propidium iodide (PI) (10 mg/mL, Invitrogen). Staining was performed for 1 hour in the dark and analyzed using a FACS Canto II flow cytometer. Early apoptotic cells (Annexin V-positive and PI-negative events), late apoptotic cells (Annexin V-positive and PI-positive events) and necrotic cells (Annexin V-negative and PI-positive events) were quantified. Additionally, caspase-3 cleavage was analyzed by Western blot as a complementary measure of apoptosis. To evaluate the effect of macrophage-conditioned medium (CM) on glioblastoma cells, A172 and T98G cells were treated with CM for 24 hours, followed by 72 hours with or without TMZ. Viability (MTT assay) and apoptosis (flow cytometry and Western blot) were measured. Statistical analysis Data were analyzed using IBM SPSS Statistics 22. Statistical significance was evaluated with Student’s t-test, Mann-Whitney U test for two-group comparisons, and one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. Declarations Acknowledgments We would like to express our deepest gratitude to the patients who participated in this study and generously donated tumor samples. The authors also wish to acknowledge Dr. Jaime Gallego Pérez de Larraya, Coordinator of the Neuro-Oncology Area at Clínica Universidad de Navarra, Spain, for his valuable advice and collaboration in this study. Author contributions S.L., MJM.D., T.S and G.S provided conception. S.L., B.C., MJM.D., L.A., RA.B. and G.S. conducted experiments and curated data. S.L., B.C., MJM.D., L.A., H.S., D.G., CJ.K., RA.B., T.S. and G.S. analyzed the results. H.S., D.G. and CJ.K. performed patient inclusion and collected human samples. S.L., T.S. and G.S. wrote the manuscript draft. T.S. and G.S. acquired funding and supervised the study. All authors reviewed the manuscript Funding information Susana López-López was the recipient of a contract funded by Fundación Hospital Nacional de Parapléjicos- Institute of Health Research of Castilla-La Mancha (IDISCAM), Castilla-La Mancha, Spain, during the years 2021 and 2022. Gemma Serrano-Heras is the recipient of a contract funded by the grant awarded to the project ´Reinforcement of the Research Activity of Castilla-La Mancha (EMER)´ (Fundación Hospital Nacional de Parapléjicos- Institute of Health Research of Castilla-La Mancha (IDISCAM), Castilla-La Mancha, Spain). Competing interests The author(s) declare no competing interests. Data availability Data is provided within the manuscript or supplementary information files. Ethics declarations Written informed consent was obtained from all patients. All methods were performed in accordance with the 1964 Declaration of Helsinki and its later amendments, and all procedures were approved by the Human Ethics Committee of this hospital (Ethical approval number: 03/2014). References Grochans, S. et al. Epidemiology of Glioblastoma Multiforme–Literature Review. Cancers . 14 , 2412; doi: 10.3390/cancers14102412 (2022). Stupp, R. et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. 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Cell Death Discov . 8 , 1–13; doi:10.1038/s41420-022-00973-y (2022). Additional Declarations No competing interests reported. Supplementary Files LpezLpezetalSupplementaryfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 06 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 Jun, 2025 Reviews received at journal 12 Jun, 2025 Reviewers agreed at journal 22 May, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers agreed at journal 26 Feb, 2025 Reviewers invited by journal 21 Feb, 2025 Editor assigned by journal 21 Feb, 2025 Editor invited by journal 14 Feb, 2025 Submission checks completed at journal 13 Feb, 2025 First submitted to journal 09 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5991999","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":415740986,"identity":"193d208f-6f59-4703-93d7-7c391d8adbfe","order_by":0,"name":"Susana López-López","email":"","orcid":"","institution":"General University Hospital of Albacete","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"","lastName":"López-López","suffix":""},{"id":415740988,"identity":"b64b78cb-6ba9-4c36-9824-785d123379dd","order_by":1,"name":"Beatriz Castro-Robles","email":"","orcid":"","institution":"General University Hospital of Albacete","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"","lastName":"Castro-Robles","suffix":""},{"id":415740989,"identity":"a6dc6042-24ae-417b-8928-18127d5ea114","order_by":2,"name":"María José M. 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(\u003cstrong\u003ea\u003c/strong\u003e) A172 and T98G cells were cultured for 5 days with different TMZ concentrations and chemoresistance was evaluated by MTT assay. Cell viability was normalized to untreated cells (mean ± SD from three independent experiments). Mann-Whitney U or Student’s t-test: *p \u0026lt; 0.05 or **p \u0026lt; 0.01 vs. untreated. (\u003cstrong\u003eb\u003c/strong\u003e) The grouped blots showing proteins of the BER, MGMT, and MMR repair pathways, as well as p53, with β-actin as the loading control, were cropped from different Western blot experiments. The original blots are presented in the Supplementary Material: \u003cstrong\u003eFigure s3\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/edfab7775c5bb599004e930d.png"},{"id":76575773,"identity":"3f129c11-bcbe-4735-b326-5eedd19b19a7","added_by":"auto","created_at":"2025-02-18 14:09:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":422911,"visible":true,"origin":"","legend":"\u003cp\u003esiRNA targeting of MPG, alone or with MGMT knockdown, potentiates TMZ efficacy. (a) Glioblastoma cells were transfected with scrambled (siC) or double siMGMT and siMPG for 48 hours, followed by TMZ treatment for 72 hours, as shown in the schematic protocol. Expression of repair enzymes was detected by Western blot analysis, with β-actin as the loading control. In this figure, the grouped blots were cropped from different Western blot experiments. The original blots are presented in the Supplementary Material: \u003cstrong\u003eFigure s4\u003c/strong\u003e. (b) TMZ cytotoxic effect assessed by MTT assay in siRNAs-transfected A172 and T98G after exposure to TMZ or vehicle. Cell viability was normalized to siC-untreated cells (100%) (mean ± SD, n=3). Kruskal-Wallis test: *p \u0026lt; 0.05 or **p \u0026lt; 0.01 vs. siC-untreated; #p \u0026lt; 0.05 or ##p \u0026lt; 0.01 vs. siC/TMZ-treated.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/ad15cefb09da43b94eb4878a.png"},{"id":76573965,"identity":"9c7a1c89-91d7-47a2-95e4-d961cba0919c","added_by":"auto","created_at":"2025-02-18 14:01:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":875780,"visible":true,"origin":"","legend":"\u003cp\u003eM1 macrophage-derived medium reduces MGMT and MPG expression in glioblastoma cells by inhibiting STAT3 activation. (\u003cstrong\u003ea)\u003c/strong\u003e mRNA expression of M1 (CXCL-10, TNF-alpha) and M2 (Arg1, IL-10) markers was analyzed by qRT-PCR and normalized to GAPDH mRNA following the differentiation of THP-1 macrophages (mean ± SD from three independent experiments). Student’s t-test: *p \u0026lt; 0.05 or **p \u0026lt; 0.01 compared to M0 (non-polarized macrophages). Western blot analysis was used to assess STAT1 expression and activation in M1 macrophages. (\u003cstrong\u003eb\u003c/strong\u003e) STAT3, STAT1 expression and phosphorylated forms were assessed in A172 and T98G glioblastoma cells. \u003cstrong\u003e(c\u003c/strong\u003e) Effect of M1 macrophage medium (24-hour incubation) on STAT1/STAT3 activation and DNA repair enzymes (MGMT, MPG) was evaluated by Western blot. (\u003cstrong\u003ed)\u003c/strong\u003e Protein levels of DNA repair enzymes and transcription factors were analyzed in glioblastoma cells transiently transfected with STAT1/STAT3 overexpression or empty vectors. (\u003cstrong\u003ee)\u003c/strong\u003e MGMT, MPG, p-STAT3, and STAT3 expression were analyzed in T98G cells treated with a STAT3 activation inhibitor. (\u003cstrong\u003ef\u003c/strong\u003e) Western blot analysis of MGMT and MPG in T98G cells transiently transfected with STAT3 or empty vectors, followed by treatment with conditioned medium from M1-differentiated or M0 control macrophages. In this figure, the grouped blots were cropped from different Western blot experiments, with β-actin as a loading control. The original blots are presented in the Supplementary Material: \u003cstrong\u003eFigures s5\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/bd264b7eaf5b3be5fece0c5f.png"},{"id":76575774,"identity":"2095dfbf-1e90-48af-97e0-2d4dc3a0846b","added_by":"auto","created_at":"2025-02-18 14:09:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":381387,"visible":true,"origin":"","legend":"\u003cp\u003eMGMT and MPG are preferentially expressed in glioblastoma patient samples with STAT3 activation. The grouped blots showing MGMT and MPG DNA repair enzymes, as well as STAT3/STAT1 phosphorylation in A172 and T98G cells and 15 patient-derived tumor samples, with β-actin as the loading control, were cropped from different Western blot experiments. The original blots are presented in the Supplementary Material: \u003cstrong\u003eFigure s6\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/47e5e6a9c041b7b55018cf9c.png"},{"id":76573969,"identity":"c8c9d8c2-42c1-478c-9552-1eec32c691f2","added_by":"auto","created_at":"2025-02-18 14:01:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":835944,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced TMZ cytotoxicity by M1 medium through the induction of apoptosis and suppression of BCL-2. (\u003cstrong\u003ea)\u003c/strong\u003e Glioblastoma cell viability was assessed in cells cultured in CM-M1 or CM-M0, followed by incubation with or without TMZ, using MTT assay. Cell viability was normalized to untreated cells (mean ± SD from three independent experiments). one-way ANOVA test: * or ** vs. not CM-incubated and TMZ-untreated; # or ## compared to the same medium without TMZ; $$ vs. CM-M0-incubated and TMZ treated (p \u0026lt; 0.05 or p \u0026lt;0.01). (\u003cstrong\u003eb\u003c/strong\u003e) BCL2 anti-apoptotic protein expression was evaluated by Western blot analysis in A172 and T98G cells incubated with M0 or M1 conditioned medium. (\u003cstrong\u003ec)\u003c/strong\u003e Representative flow cytometry dot plot (n=2) showing cell death by early (brown dots) and late (blue dots) apoptosis, and necrosis (orange dots) in cells treated under different conditions, using Annexin V-Dy634 and PI staining. (\u003cstrong\u003ed\u003c/strong\u003e) After treatment with M0/M1 0 medium and/or TMZ, cleaved caspase-3 levels were analyzed by Western blot. In this figure, the grouped blots were cropped from different Western blot experiments, with β-actin as a loading control. The original blots are presented in the Supplementary Material: \u003cstrong\u003eFigure s7\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/41f536d5d3865afdfda3886b.png"},{"id":76573966,"identity":"8a30d415-984b-42ed-b227-5715ad2e22eb","added_by":"auto","created_at":"2025-02-18 14:01:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":252845,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the novel mechanism by which M1 macrophage-derived medium, through suppression of STAT3 activation, inhibits DNA repair enzyme expression, reducing TMZ chemoresistance.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/0613dbb6ae5987b3a73a244f.png"},{"id":95564041,"identity":"5a9e8aa1-198e-4167-86a7-539ab9ed494a","added_by":"auto","created_at":"2025-11-10 16:06:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4268761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/51b40f96-d2e3-4dc6-b7ed-e7358a35cf85.pdf"},{"id":76576468,"identity":"7fc26616-0e3a-4f42-b17f-130c427f3e4e","added_by":"auto","created_at":"2025-02-18 14:17:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2628695,"visible":true,"origin":"","legend":"","description":"","filename":"LpezLpezetalSupplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5991999/v1/5807d135e491b38a071f338b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Proinflammatory macrophage-derived medium enhances temozolomide sensitivity in glioblastoma via pSTAT3-mediated downregulation of DNA repair enzymes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioblastoma (GB) is the most common and aggressive primary malignant brain tumor in adults, with a poor prognosis for patients, with less than 5% survive beyond five years\u003csup\u003e1\u003c/sup\u003e. The current standard treatment for newly diagnosed glioblastoma is multimodal, consisting of maximum safe resection, followed by localized radiotherapy and concomitant and adjuvant temozolomide (TMZ) chemotherapy. Although chemotherapy after surgery has been proven to increase patient survival\u003csup\u003e2\u003c/sup\u003e, TMZ resistance remains a major obstacle to treatment efficacy in glioblastoma. This underscores the need to elucidate the molecular mechanisms underlying the resistant phenotype in order to improve the prognosis for glioblastoma patients.\u003c/p\u003e\n\u003cp\u003eTMZ causes damage to cells through methylation at several nitrogenous bases in DNA, with the primary site of methylation occurring at the N7-guanine position (70%), followed by the N3-adenine position and at the O6-guanine residue (5%). However, the cytotoxic effect of TMZ in glioblastoma cells becomes limited by the action of cellular DNA damage repair systems, which represent one of the main factors contributing to chemoresistance. It is well-established in the literature that elevated levels of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme that removes the O6-methylguanine lesion, one of the most lethal lesions induced by TMZ, are a key factor driving TMZ resistance. Several studies conducted in patients treated with TMZ have reported that MGMT promoter methylation, leading to a lack of expression of the enzyme, is associated with improved overall survival compared to unmethylated glioblastomas\u003csup\u003e3\u003c/sup\u003e. Nevertheless, a considerable proportion of patients fail to respond to TMZ treatment and do not exhibit clinical improvement even when the tumor expresses low levels of MGMT, suggesting that this DNA repair enzyme is not the only predictor of response to TMZ. In this context, previous findings from our group and others have highlighted the role of N-methylpurine-DNA glycosylase (MPG), the initial enzyme in the base excision repair pathway (BER), in conferring TMZ chemoresistance in glioblastoma\u003csup\u003e4,5\u003c/sup\u003e. MPG removes the most frequent TMZ-induced damages (N7-methylguanine and N3-methyladenine), leading to the formation of different intermediate adducts that are subsequently removed by downstream enzymes such as apurinic/apyrimidinic (AP) sites endonuclease 1 (APE1) and poly(ADP-ribose) polymerase (PARP), with the repair process finalized by DNA polymerase, POL-\u0026beta;. Pharmacological inhibition of certain proteins in the BER pathway, such as APE1, PARP and poly-ADP-ribose glycohydrolase (PARG), has been demonstrated to enhance TMZ-induced cytotoxicity regardless of MGMT status\u003csup\u003e6-8\u003c/sup\u003e. However, MPG expression has been associated with both increased and decreased resistance to TMZ in glioblastoma\u003csup\u003e5,9\u003c/sup\u003e, thus the role of this enzyme in chemotherapy response remains undefined.\u003c/p\u003e\n\u003cp\u003eIn recent years, tumor-associated macrophages (TAMs), the most abundant cells in the glioblastoma microenvironment, have attracted considerable interest due to their role in promoting tumor growth, metastasis, and resistance to conventional therapies\u003csup\u003e10\u003c/sup\u003e. Macrophages exhibit two mutually polarizable phenotypes: M1, characterized by the secretion of a high amount of proinflammatory cytokines (e.g interleukin (IL)-1, CXCL10, TNF-\u0026alpha;), and M2, which produces high levels of anti-inflammatory cytokines such as IL-10. TAMs usually display an M2-like phenotype that is associated with pro-tumoral features, whereas M1 macrophages exert antitumor functions\u003csup\u003e11\u003c/sup\u003e. Activated M2 macrophages have been reported to promote chemoresistance by enhancing anti-apoptotic signaling, such as STAT3 transcription factor and BCL2 pathways in tumor cells, while M1 macrophages activate the STAT1-mediated apoptotic pathway\u003csup\u003e12\u003c/sup\u003e. Notably, a study performed by Kohsaka et al.\u003csup\u003e13\u003c/sup\u003e revealed that STAT3 activation, frequently observed in tumors including glioblastoma, upregulates MGMT, suggesting this signaling pathway might be responsible for TMZ resistance. However, the underlying mechanisms involving chemoresistance-induced macrophages are still poorly understood.\u003c/p\u003e\n\u003cp\u003eThis study aimed to gain insight into the contribution of MPG to TMZ resistance, both independently and in combination with MGMT, through siRNA silencing in an \u003cem\u003ein vitro\u003c/em\u003e model of glioblastoma. These analyses are intended to clarify the controversial findings of previous studies regarding the role of the BER molecular repair pathway in chemotherapy resistance. On the other hand, TAMs are known to hinder treatment effectiveness, but the mechanisms underlying macrophage-mediated chemoresistance are still not well understood. In addition, we examined the potential effect of conditioned medium from proinflammatory macrophage phenotype (CM-M1) on the expression of MPG and MGMT in glioblastoma cells and whether this effect is mediated by the STAT1 or STAT3 transcription factors. Gathering evidence of the interaction between the innate immune system and DNA repair pathways in glioblastoma could support strategies to reprogram M2 macrophages into an M1 phenotype, with the purpose of improving the chemotherapy response.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTMZ resistance and the expression profile of DNA repair enzymes in an \u003cem\u003ein vitro\u003c/em\u003e model of glioblastoma\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViability assays performed on A172 and T98G glioblastoma cell lines incubated with increasing concentrations of TMZ for 72 hours demonstrated a dose-dependent reduction in cell survival following TMZ treatment (Fig. 1A). The results indicated that both cell lines exhibited resistance to the chemotherapeutic agent, as high doses (400 \u0026micro;M) reduced cell viability by less than 50% (40% in A172 and 20% in T98G). BER pathway enzymes (MPG, APE1, PARP, and POL-\u0026beta;) and the MMR enzyme MSH2 were highly expressed in both glioblastoma cell lines (Fig. 1B). While MGMT expression was absent in A172 cells, it was detected in T98G cells (Fig. 1B), potentially explaining the greater resistance observed in T98G cells (Fig. 1A). Furthermore, our findings align with previous studies reporting that A172 cells possess wild-type p53, whereas T98G cells harbor a mutated form of p53. This mutation disrupts the normal regulation of protein degradation, leading to increased protein accumulation (Fig. 1B). Such alterations may partly account for the elevated MGMT levels in T98G cells, as mutated p53 has been reported to upregulate MGMT expression.\u003csup\u003e14\u003c/sup\u003e Although the lack of active MGMT, along with high levels of MMR, is generally considered a sensitizing factor for TMZ treatment, our results suggest that the chemoresistance observed in A172 cells may be driven by other factors, such as enzymes involved in the BER pathway.\u003c/p\u003e\n\u003cp\u003eIn summary, the glioblastoma cell lines A172 and T98G used in this study exhibited similarly high chemoresistance but distinct profiles of DNA repair enzyme expression, allowing us to investigate the potential role of MPG in TMZ resistance, both in the presence and absence of MGMT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncreased glioblastoma chemosensitivity through MPG silencing, both alone and synergistically with MGMT downregulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the influence of DNA repair enzymes on TMZ efficacy, small interfering RNAs (siRNAs) were utilized to suppress MGMT and MPG expression. Glioblastoma cell lines were initially transfected with varying concentrations of MGMT- and MPG-targeting siRNAs, both individually and in combination, to determine the optimal conditions for achieving efficient reduction of enzyme expression at both the protein and mRNA levels. MPG expression was effectively inhibited in A172 cells using a siRNA concentration of 25 nM, whereas T98G cells required 40 nM for optimal inhibition (Figure s1), with maximum suppression observed 96 hours post-incubation in both glioblastoma cell lines (Figure s2, Fig. 2A). In T98G cells, which express both DNA repair enzymes, a 25 nM concentration of MGMT-targeted siRNA effectively reduced MGMT expression separately, and this peak effect was sustained when both enzymes were simultaneously knocked down 96 hours after exposure to the specific siRNAs (Figure s2, Fig. 2A). The expression of enzymes involved in the subsequent steps of the MPG-mediated base excision repair (BER) pathway, as well as the MMR enzyme MSH2, was unaffected in the absence of MPG and MGMT (Fig. 2A). Additionally, siRNAs targeting these DNA repair enzymes remained effective even in the presence of TMZ (Fig. 2A).\u003c/p\u003e\n\u003cp\u003eIt is worth mentioning that the optimal incubation time for gene silencing, set at 96 hours, enabled the viability studies with TMZ-treated glioblastoma cells, as it aligned with the experimental conditions. SiRNA typically requires approximately 48 hours to take effect, while TMZ induces cell death over extended periods (see experimental protocol scheme, Fig. 2B). As shown in Figure 2B, MPG siRNA significantly enhanced TMZ sensitivity in A172 cells, which do not express MGMT. Moreover, the combined inhibition of MPG and MGMT in TMZ-treated T98G cells resulted in a synergistic decrease in cell viability, with statistically significant differences compared to each siRNA treatment alone. Collectively, our results suggest that MPG activity, either alone or in combination with MGMT, plays a significant role in contributing to TMZ resistance in glioblastoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConditioned medium from M1 macrophages inhibits MGMT and MPG expression in glioblastoma cells through blocking pSTAT3 signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe M2 phenotype of tumor-associated macrophages has been reported to potentiate resistance to TMZ. In an attempt to elucidate the mechanisms behind macrophage-mediated chemoresistance in glioblastoma, which are still not fully understood, we sought to explored whether media from polarized M1 and/or M2 macrophages are involved in regulating the expression of the DNA repair enzymes MPG and MGMT.\u003c/p\u003e\n\u003cp\u003eFirst, macrophages underwent a differentiation process, as described in Methods, to collect conditioned media during M1 and M2 activation, which would subsequently be used in cell cultures to evaluate the regulation of DNA repair enzyme expression. M1 differentiation was confirmed by measuring STAT1 phosphorylation, a key transcription factor in pro-inflammatory activation, and by detecting increased expression of the pro-inflammatory genes TNF\u0026alpha; and CXCL-10 compared to M0 or M2 macrophages (Fig. 3A). However, M2 markers such as Arg1 and IL-10 were only modestly induced, consistent with previous studies showing that THP-1-macrophage cells do not fully differentiate into the M2 phenotype (Fig. 3A). As a result, subsequent experiments were conducted using only the conditioned medium from M1 macrophages (CM-M1), which, despite this, allowed us to explore the interplay between glioblastoma immunology and the molecular mechanisms underlying chemoresistance. In addition, the total and activated (phosphorylated form) levels of STAT1, as well as STAT3, a marker of M2 macrophage signaling, were analyzed in glioblastoma cells. Both glioblastoma cell lines showed elevated levels of phosphorylated Tyr705-STAT3, with T98G exhibiting the highest expression, while the activated form of STAT1 was found to be lower in T98G compared to A172 (Fig. 3B). Then, Western blotting was performed to examine the levels of MPG and MGMT, as well as STAT3, STAT1, and their phosphorylated forms, in T98G and A172 cells after incubation with CM-M1 and control medium from non-activated macrophages (CM-M0). CM-M1-treated glioblastoma cells exhibited reduced levels of the activated (phosphorylated) form of STAT3, which exerts the function of a transcription factor, compared to CM-M0-treated cells, while the amounts of total STAT3 protein remained largely unchanged. In contrast, CM-M1 exposure triggered both increased STAT1 activation (phosphorylation) and expression (total protein levels) (Fig. 3C). Additionally, we observed a reduction in the expression of the DNA repair enzymes MPG and MGMT after incubation with M1 proinflammatory medium, compared to cells treated with CM-M0 (Fig. 3C).\u003c/p\u003e\n\u003cp\u003eTo gain insight into the transcription factor signaling responsible for M1-macrophage medium-mediated regulation of DNA repair enzymes, glioblastoma cells were transfected with overexpression vectors for STAT3 or STAT1 to determine whether the observed reduction in MPG and MGMT expression was due to decreased pSTAT3 or increased pSTAT1. Forced STAT1 expression and increased pSTAT1 did not lower MPG or MGMT levels compared to control cells, while overexpression of STAT3, which resulted in a rise in its phosphorylated form, significantly increased the expression of these enzymes (Fig. 3D). Next, we used a selective STAT3 inhibitor that completely abolished STAT3 phosphorylation\u003csup\u003e15\u003c/sup\u003e, leading to reduced MGMT and MPG expression (Fig. 3E). Finally, to confirm the role of STAT3 in the CM-M1-induced decrease in MGMT and MPG expression, glioblastoma cells, either transfected with an empty vector or overexpressing STAT3, were incubated with the proinflammatory medium. As shown in Fig. 3F, STAT3 overexpression and increased pSTAT3 attenuated the CM-M1-induced suppression of DNA repair enzyme expression.\u003c/p\u003e\n\u003cp\u003eOur findings demonstrate that pro-inflammatory conditioned medium suppresses MGMT and MPG expression in glioblastoma cells by inhibiting pSTAT3 signaling, emphasizing the role of STAT3-mediated innate immunity in regulating DNA repair mechanisms in glioblastoma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssociation between MGMT and MPG expression and STAT3 activation in patient-derived glioblastoma\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; We investigated the potential correlation between MGMT and MPG expression, and pSTAT3 in glioblastoma patient samples. Western blot analysis was performed on 15 tumor tissue samples obtained from patients diagnosed with glioblastoma who underwent tumor resection surgery. The patients had a median age of 55 years (range: 40\u0026ndash;78 years), with 40% female representation. The time between diagnosis and surgery ranged from 0 to 45 days, with an average of 15.6 days. The majority of patients received a combination of radiation therapy and temozolomide (RT-TMZ + TMZ) as their first-line treatment, although some were treated with temozolomide alone or did not receive treatment. Glioblastoma recurrence (assessed through imaging studies, such as MRI, showing new tumor growth or changes 4 to 6 months after the completion of the first therapeutic regimen) was observed in 10 (67%) patients (GB1, 2, 6, 7, 8, 9, 10, 12, 13, 15)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe molecular results showed that 53% of patients (n=8) expressed pSTAT3 (Fig. 4). Among these, 87.5% (n=7) exhibited MGMT expression, and 87.5% (n=7) had detectable MPG protein levels. Notably, 75% of the pSTAT3-positive patients (n=6) expressed both MGMT and MPG (Fig. 4). Although the sample size is small and requires confirmation in a larger patient cohort, these findings suggest that activated STAT3 may regulate the expression of both DNA repair enzymes in patient tumors, consistent with the observations from our \u003cem\u003ein vitro\u003c/em\u003e glioblastoma model. We did not find a significant relationship between enzyme expression, transcription factors and tumor relapsing after the treatment. However, it is worth noting that MPG expression was detected in a higher proportion of patients with recurrence compared to those without recurrence (80% vs. 60%, respectively) (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProinflammatory medium potentiates TMZ efficacy by inducing apoptotic cell death through the downregulation of anti-apoptotic protein and DNA repair enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe finally analyzed the effect of CM-M1 on the viability of glioblastoma cell lines under TMZ treatment to determine whether this proinflammatory medium enhances chemosensitivity as a result of downregulating the expression of MGMT and MPG (as shown in the previous results section). Cell viability was assessed after incubating the cells with CM-M1 or non-activated macrophage medium (M0) for 24 hours, followed by an additional 72 hours in the presence or absence of TMZ. Our results showed that exposure to CM-M1 significantly inhibited cell viability in both cell lines (Fig. 5A), which could be partially due to the downregulation of BCL2 (Fig. 5B), an anti-apoptotic gene, along with strong activation of STAT1(Fig. 3C), a transcription factor identified as a negative regulator of cell proliferation and a promoter of apoptotic signaling. More interestingly, CM-M1 incubation combined with low-dose TMZ treatment (100 \u0026micro;M in A172 and 300 \u0026micro;M in T98G) led to a significantly greater reduction in cell survival compared to treatments with the inflammatory medium and chemotherapy agent separately (Fig. 5A).\u003c/p\u003e\n\u003cp\u003eCell death in glioblastoma induced by exposure to CM-M1 alone or in combination with TMZ was analyzed by flow cytometry. We found that apoptosis was synergistically increased by the combination of proinflammatory macrophage medium and TMZ, with a higher percentage (5.4\u0026plusmn;0.3% and 17.2\u0026plusmn;1%) of A172 cells undergoing early and late apoptosis, respectively, compared to 4.5\u0026plusmn;0.1% and 12.7\u0026plusmn;2%, or 4.4\u0026plusmn;0.2% and 14.7\u0026plusmn;1.5%, in the individual CM-M1 and TMZ treatment groups (Fig. 5C). Similar results were observed in T98G cells, with 40.3\u0026plusmn;2.7% total apoptotic cells, in comparison to 35.2\u0026plusmn;1.8% and 17.3\u0026plusmn;4% after separate incubation with M1 medium and chemotherapy agent (Fig. 5C). These findings were further supported by Western blot analysis, which showed increased levels of the pro-apoptotic protein cleaved Caspase-3 in the combined treatment group (Fig. 5D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, these results suggest that CM-M1 potentiates the deleterious effects of TMZ by triggering apoptosis, which, in synergy with chemotherapy, leads to an increased number of cells undergoing cell death via apoptosis. This increase in apoptotic processes could result from the simultaneous action of the proinflammatory medium on glioblastoma cells, downregulating the expression of both the anti-apoptotic protein BCL2 and, as previously shown, the DNA repair enzymes MPG and MGMT, in a p-STAT3-dependent manner.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChemoresistance in glioblastoma develops frequently, underscoring the need for further investigation of the molecular mechanisms driving GBM resistance. Our results, derived from an \u003cem\u003ein vitro\u003c/em\u003e glioblastoma resistance model, demonstrate that MPG, which repairs the most common TMZ-induced lesions, significantly contributes to TMZ resistance in the absence of MGMT. Moreover, MPG acts synergistically with MGMT, a well-established mediator of chemoresistance, to limit TMZ efficacy. Importantly, we report for the first time that proinflammatory medium derived from M1 macrophages (CM-M1) leads to a pSTAT3-mediated reduction in the expression of DNA repair enzymes MGMT and MPG, thereby enhancing glioblastoma cell sensitivity to TMZ, as illustrated in Fig. 6.\u003c/p\u003e\n\u003cp\u003eFor more than a decade, chemoresistance in glioblastoma has been largely attributed to MGMT, which repairs the most cytotoxic TMZ-induced DNA lesions. Indeed, the methylation status of the MGMT promoter, leading to low MGMT expression, was the first molecular marker used to predict a better response to TMZ in clinical trials\u003csup\u003e16\u003c/sup\u003e. As chemotherapy-induced cytotoxicity, driven by MGMT deficiency, also requires subsequent repair via the MMR pathway, and its activity has been reported to decrease with TMZ treatment\u003csup\u003e17,18\u003c/sup\u003e, the MPG-initiated BER pathway, responsible for repairing the most frequent N-methylpurine lesions in DNA induced by TMZ, has emerged as a potential complementary factor in chemoresistance. However, in recent years, there has been controversy regarding its role in chemoresistance, as this DNA repair pathway has been associated with both contributing to chemoresistance and increasing TMZ efficacy. MPG expression has been correlated with worse prognosis in glioma patients\u003csup\u003e4,19\u003c/sup\u003e, and a similar pattern was observed in patients whose glioblastoma tumor analysis revealed that MGMT methylation alone could not explain TMZ resistance\u003csup\u003e20\u003c/sup\u003e. In this context, other studies have demonstrated that MPG and POL-\u0026beta; expression predict sensitivity to chemotherapeutic agents\u003csup\u003e21\u003c/sup\u003e, and the reduced expression of BER proteins, such as APE1 and PARP, by chemical inhibitors sensitizes glioblastoma cells to TMZ\u003csup\u003e6,7\u003c/sup\u003e. In contrast, Fosmark et al. \u003csup\u003e9\u003c/sup\u003e showed that high MPG expression correlated with improved survival in glioblastoma patients with MGMT promoter methylation. Growing evidence of TMZ efficacy potentiation by MPG in glioblastoma and other cancers comes from experiments combining MPG overexpression with the inhibition of downstream BER pathway enzymes, including AP1 and PARP\u003csup\u003e22,23\u003c/sup\u003e. The increased sensitivity resulted from enhanced repair of N-methylpurine lesions, saturating the BER rate-limiting enzyme POL-\u0026beta; and leading to the accumulation of cytotoxic 5\u0026rsquo;dRP repair intermediates. Notably, overexpression of POL-\u0026beta; in cells with high levels of MPG incubated with BER enzyme inhibitors abrogates the MPG-dependent enhancement of TMZ sensitivity\u003csup\u003e23\u003c/sup\u003e. Thus, according to these results, inhibiting downstream BER enzymes would only potentiate the effect of TMZ under conditions of high MPG levels and low POL-\u0026beta; levels. However, the results of our study revealed that downregulation of the first enzyme of BER, MPG, is capable of sensitizing glioblastoma cells to TMZ in both MGMT-expressing cells (T98G) and MGMT-negative cells (A172), regardless of elevated POL-\u0026beta; levels. These findings strongly support the role of this DNA repair enzyme in chemoresistance in glioblastoma, aligning with previous data from our group that demonstrated an association between elevated MPG expression and the TMZ-resistant phenotype in tumor cells derived from patients\u003csup\u003e5\u003c/sup\u003e. This suggests that inactivating these DNA repair enzymes could be an effective therapeutic strategy to overcome chemotherapy resistance. However, the development of effective MPG inhibitors has so far been unsuccessful due to the complexity of its active site. Similarly, pharmacological approaches targeting MGMT have not significantly reduced chemoresistance in clinical trials, as they required reduced TMZ doses to avoid the severe myelosuppressive toxicity caused by inhibitors such as Lomeguatrib or PaTrin-2\u003csup\u003e24,25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOn the other hand, research in recent years has demonstrated that TAMs dominate the cellular composition of the glioblastoma microenvironment, with the presence of the M2 phenotype contributing to TMZ resistance in this type of tumor\u003csup\u003e26-29\u003c/sup\u003e, although the underlying molecular mechanisms driving such chemoresistance remain unclear. The results of our study demonstrated that the proinflammatory medium derived from M1 macrophages triggered reduced cell proliferation and apoptotic induction, comparable to the effects seen in breast cancer cell cultures\u003csup\u003e30\u003c/sup\u003e and renal clear cell carcinoma\u003csup\u003e31\u003c/sup\u003e treated with CM-M1. Furthermore, we found that CM-M1 led to an increase in the phosphorylated form of STAT1, which has been previously described as an adaptor required for the formation of the death-inducing signaling complex involved in apoptosis induction, as well as a downregulation of the anti-apoptotic protein BCL-2. Therefore, the apoptosis induced by the proinflammatory medium could be due to an increase in STAT1 activity combined with the reduction of BCL-2 levels. More interestingly, the simultaneous treatment of glioblastoma cells with CM-M1 and TMZ synergistically increased apoptotic cell death in glioblastoma cultures, significantly enhancing sensitivity to chemotherapy compared to the individual CM-M1 and TMZ treatments. This cytotoxic potentiation effect of TMZ may result from the observed inhibition of MPG and MGMT expression, along with the reduced expression of BCL-2 and the elevated levels of pSTAT1. Using various technical approaches in this study, we found that the decreased expression of both DNA repair enzymes induced by the proinflammatory macrophage medium was not mediated by the upregulation of STAT1 and pSTAT1, but rather by the inhibition of STAT3 activation, as evidenced by the reduced protein levels of pSTAT3, while the total amount of STAT3 remained unaffected. Consistent with our findings, previous research has demonstrated that blocking STAT3 phosphorylation decreases the IC50 of TMZ in glioblastoma cells, enhancing TMZ-induced apoptosis, partly through reduced BCL-2 expression, a downstream target of p-STAT3\u003csup\u003e32-35\u003c/sup\u003e. The activated-phosphorylated form of STAT3 has also been reported to upregulate MGMT expression, contributing to TMZ desensitization\u003csup\u003e13\u003c/sup\u003e. In this context, our study demonstrates for the first time that pSTAT3 modulates the expression of both MPG and MGMT. This finding aligns with tumor analyses from patients, which show higher expression of both enzymes in pSTAT3-positive samples (75%) compared to those lacking activated STAT3. Therefore, our study provides new insights into how glioblastoma-associated macrophages may contribute to chemoresistance by upregulating DNA repair enzymes that diminish the cytotoxic effect of TMZ. Based on our results, we propose that converting macrophages from the M2 to the M1 phenotype represents a promising therapeutic strategy to overcome TMZ resistance. This approach could also serve as an alternative to current immunotherapies focused on enhancing adaptive immunity\u003csup\u003e36\u003c/sup\u003e, which have shown limited efficacy in glioblastoma\u003csup\u003e37\u003c/sup\u003e. Indeed, various compounds, including corosolic\u003csup\u003e38\u003c/sup\u003e and inhibitors of placental growth factor, C/EBP\u0026beta; (CCAAT/enhancer-binding protein beta), and the hyaluronic acid pathway, have demonstrated efficacy in reprogramming macrophages from M2 to M1 in glioblastoma, leading to tumor suppression through STAT3 inhibition\u003csup\u003e39,40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAlthough our study sheds light on the roles of MPG and MGMT in TMZ resistance in glioblastoma, as well as the effect of the proinflammatory medium from M1 macrophages on the expression of these DNA repair enzymes, leading to reduced levels and, consequently, increased chemotherapy sensitivity (see Fig. 6 for illustration), we acknowledge certain limitations. The \u003cem\u003ein vitro\u003c/em\u003e glioblastoma cell models used in this study do not fully replicate the complex tumor microenvironment observed in glioblastoma patients. Therefore, further research utilizing patient-derived cells and animal models is necessary to confirm and validate our findings, and also to identify the proinflammatory molecules secreted that are responsible for the pSTAT3-mediated downregulation of MPG and MGMT.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell cultures, Conditioned media from differentiated macrophages, and Patient-derived glioblastoma samples\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA172 and T98G glioblastoma cell lines (ATCC# CRL-1620, CRL-1690) were maintained in DMEM and MEM, respectively, with 10% FBS and 1% penicillin-streptomycin, while the culture medium of THP-1 monocytes (ATCC# TIB-202) was RPMI 1640 with 10% FBS, 2 mM L-glutamine, 50 \u0026micro;M 2-mercaptoethanol, and 1% penicillin-streptomycin. All cell lines were cultured at 37\u0026deg;C in a 5% CO2 atmosphere. Suppression of STAT3 activation-phosphorylation was achieved using Stattic (ab120962, Abcam) according to the manufacturer\u0026apos;s instructions. This selective STAT3 inhibitor blocks activation-phosphorylation, as well as subsequent dimerization and nuclear translocation of STAT3, by interacting with the SH2 domain. For macrophage differentiation, THP-1 cells were treated with 160 nM PMA (Sigma-Aldrich) for 48 hours, followed by culture in RPMI with 5% FBS for 24 hours. Macrophages were then polarized into M1 phenotype using IFN-\u0026gamma; (10 U/ml) and LPS (100 ng/ml), and into M2 using IL-13 (20 ng/ml) and IL-4 (20 ng/ml). After 24hours differentiation\u0026nbsp;conditioned media (CM) was collected, centrifuged, and stored at -20\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003eTumor tissue samples were collected from 15 patients diagnosed with glioblastoma by the Neurology, Neurosurgery, and Oncology departments of the General University Hospital of Albacete (Spain). Written informed consent was obtained from all patients. All methods were performed in accordance with the 1964 Declaration of Helsinki and its later amendments, and all procedures were approved by the Human Ethics Committee of this hospital (Ethical approval number: 03/2014). Sociodemographic and clinical information was obtained from the hospital information system, including the date of diagnosis, surgery, cause of death (if applicable), type of antitumor therapy, and disease-free period (DFP). After collection, fresh tissue samples were homogenized in detergent lysis buffer (RIPA, Merck) for Western blot analysis.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene silencing and Cell transfections\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA172 and T98G cells were transfected with increasing concentrations of siRNAs (25, 40,80 nM) targeting MPG and MGMT (Santa Cruz), using siRNA Transfection Reagent (sc-29527). Briefly, cells were seeded one day prior to transfection in 6-well plates for Western blot and qRT-PCR analysis, or in 24-well plates for cell viability assessment, with 10% FBS in their respective media. The siRNA-transfection reagent complex was prepared in transfection medium (sc-36868, Santa Cruz) according to the manufacturer\u0026rsquo;s instructions. The medium was replaced with fresh medium 8 hours after transfection. After a minimum of 48 hours, protein extracts were prepared for Western blot and qRT-PCR analysis. Cell viability was assessed after an additional 72-hour TMZ treatment.\u003c/p\u003e\n\u003cp\u003eFor plasmid transfections, A172 and T98G cells were seeded in 6-well plates and transfected 24 hours later with Lipofectamine 2000 (Invitrogen) according to the manufacturer\u0026rsquo;s instructions, using OPTI-MEM medium (Gibco) and 2.5 \u0026micro;g of total plasmid DNA (pCMV-Sport6, pCMV-Sport6-STAT1, or pCMV-Sport6-STAT3) per well. Cells were analyzed 24 hours post-transfection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot analysis\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins from cell culture and tumor tissue homogenates were extracted using RIPA buffer, and protein concentration was determined by the BCA assay. Protein extracts (30-40 \u0026micro;g) were separated on denaturing SDD-PAGE polyacrylamide gels and transferred to a PVDF membrane (Hybond-C Extra, Amersham Biosciences). Spectra\u003csup\u003eTM\u003c/sup\u003e Multicolor Broad Range Protein ladder (Thermo Scientific #26634) was used as a molecular weight marker for protein size estimation. The following primary antibodies were used: anti-MGMT (2739S), anti-pSTAT3 (D3A7), anti-pSTAT1 (58D6), anti-STAT3 (124H6), \u0026nbsp;and anti-STAT1 (9172S) from Cell Signaling, anti-MPG (sc-101237), anti-MSH2 (sc-376384), anti-APE1 (sc-17774), anti-PARP-1 (sc-8007), anti-BLC2 (sc-7382), anti-caspase-3 (sc-56053) and anti-\u0026beta;-ACTIN (sc-81178) from Santa Cruz, and anti-DNA polymerase (ab26343) from Abcam. Chemiluminescence detection was performed using the SuperSignal\u0026trade; West Dura (Thermo Scientific) on a Luminescent Image Analyzer LAS-mini 4000 system (Fujifilm, Tokyo, Japan)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and qRT-PCR analysis\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the RNA Total Isolation Kit (Nzytech) and converted into cDNA using RevertAid\u0026trade; Minus First Strand (Thermo Scientific). Gene expression was analyzed using SYBR\u0026trade; Green PCR Master Mix (Applied Biosystems). Primers used included the following sequences:\u003c/p\u003e\n\u003cp\u003eMGMT:\u003c/p\u003e\n\u003cp\u003eForward: 5\u0026prime;-GTCGTTCACCAGACAGGTGTTA-3\u0026prime;;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReverse: 5\u0026prime;-ACAGGATTGCCTCTCATTGCTC-3\u0026prime;\u003c/p\u003e\n\u003cp\u003eMPG:\u003c/p\u003e\n\u003cp\u003eForward:5\u0026prime;-TTGGAGTTCTTCGACCAGCC-3\u0026prime;;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReverse: 5\u0026prime;-CATGAACATGCCTCGGTTGC-3\u0026prime;\u003c/p\u003e\n\u003cp\u003e\u0026beta;-actin:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eForward:5\u0026prime;-AAGATCATTGCTCCTCCTG-3\u0026prime;;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReverse: 5\u0026prime;-CGTCATACTCCTGCTTGCTG-3\u0026prime;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability and Apoptosis assays\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MTT assay was used to assess cell viability according to the manufacturer\u0026rsquo;s protocol. A172 and T98G cells were plated in 24-well plates and cultured in 500 \u0026micro;L of medium containing the specified concentration of TMZ. After 72 hours, MTT solution (5 mg/mL) was added and incubated for 1 hour at 37\u0026deg;C. The medium was then removed, and a solubilizing solution was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate reader (SPECTROstar Omega, BMG LABTECH), with each sample analyzed in triplicate.\u003c/p\u003e\n\u003cp\u003eApoptosis was assessed by flow cytometry after 24 hours of CM-M1 or CM-M0 treatment followed by 72 hours of TMZ exposure or DMSO-vehicle control. Cells were resuspended in 450 \u0026micro;L of 1X Annexin V binding buffer (Immunostep) and stained with 10 \u0026micro;L of Annexin V conjugated to the fluorochrome Dy-634 (Immunostep), together with 40 \u0026micro;L of the vital dye propidium iodide (PI) (10 mg/mL, Invitrogen). Staining was performed for 1 hour in the dark and analyzed using a FACS Canto II flow cytometer. Early apoptotic cells (Annexin V-positive and PI-negative events), late apoptotic cells (Annexin V-positive and PI-positive events) and necrotic cells (Annexin V-negative and PI-positive events) were quantified. Additionally, caspase-3 cleavage was analyzed by Western blot as a complementary measure of apoptosis.\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of macrophage-conditioned medium (CM) on glioblastoma cells, A172 and T98G cells were treated with CM for 24 hours, followed by 72 hours with or without TMZ. Viability (MTT assay) and apoptosis (flow cytometry and Western blot) were measured.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using IBM SPSS Statistics 22. Statistical significance was evaluated with Student\u0026rsquo;s t-test, Mann-Whitney U test for two-group comparisons, and one-way ANOVA with Dunnett\u0026rsquo;s post-hoc test for multiple comparisons.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our deepest gratitude to the patients who participated in this study and generously donated tumor samples. The authors also wish to acknowledge Dr. Jaime Gallego P\u0026eacute;rez de Larraya, Coordinator of the Neuro-Oncology Area at Cl\u0026iacute;nica Universidad de Navarra, Spain, for his valuable advice and collaboration in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS.L., MJM.D., T.S and G.S\u0026nbsp;\u003c/strong\u003eprovided conception. S.L., B.C., MJM.D., L.A., RA.B. and G.S. conducted experiments and curated data. \u0026nbsp;S.L., B.C., MJM.D., L.A., H.S., D.G., CJ.K., RA.B., T.S. and G.S. analyzed the results. H.S., D.G. and CJ.K. performed patient inclusion and collected human samples. S.L., T.S. and G.S. wrote the manuscript draft. T.S. and G.S. acquired funding and supervised the study. \u003cstrong\u003eAll authors reviewed the manuscript\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSusana L\u0026oacute;pez-L\u0026oacute;pez was the recipient of a contract funded by Fundaci\u0026oacute;n Hospital Nacional de Parapl\u0026eacute;jicos-\u0026nbsp;Institute of Health Research of Castilla-La Mancha (IDISCAM), Castilla-La Mancha, Spain, during the years 2021 and 2022. Gemma Serrano-Heras is the recipient of a contract funded by the grant awarded to the project \u0026acute;Reinforcement of the Research Activity of Castilla-La Mancha (EMER)\u0026acute; (Fundaci\u0026oacute;n Hospital Nacional de Parapl\u0026eacute;jicos- Institute of Health Research of Castilla-La Mancha (IDISCAM), Castilla-La Mancha, Spain).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all patients. All methods were performed in accordance with the 1964 Declaration of Helsinki and its later amendments, and all procedures were approved by the Human Ethics Committee of this hospital (Ethical approval number: 03/2014).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGrochans, S. et al. Epidemiology of Glioblastoma Multiforme\u0026ndash;Literature Review. \u003cem\u003eCancers\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e14\u003c/strong\u003e, 2412; doi: 10.3390/cancers14102412 (2022).\u003c/li\u003e\n \u003cli\u003eStupp, R. et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. \u003cem\u003eN Eng J Med\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e352\u003c/strong\u003e, 987\u0026ndash;996; doi: 10.1056/NEJMoa043330 (2005).\u003c/li\u003e\n \u003cli\u003eBinabaj, M.M. et al. 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Absent in melanoma 2-mediating M1 macrophages facilitate tumor rejection in renal carcinoma. \u003cem\u003eTransl Oncol\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e14\u003c/strong\u003e, 101018; doi:10.1016/j.tranon.2021.101018 (2021).\u003c/li\u003e\n \u003cli\u003eLee, E.S., Ko, K.K., Joe, Y.A., Kang, S.G. \u0026amp; Hong, Y.K. Inhibition of STAT3 reverses drug resistance acquired in temozolomide-resistant human glioma cells. \u003cem\u003eOncol Lett\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e2\u003c/strong\u003e, 115\u0026ndash;121; doi: 10.3892/ol.2010.210 (2011).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWang, Y., et al. Inhibition of STAT3 reverses alkylator resistance through modulation of the AKT and \u0026beta;-catenin signaling pathways. \u003cem\u003eOncol Rep\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e26\u003c/strong\u003e, 1173\u0026ndash;1180; doi:\u0026nbsp;10.3892/or.2011.1396 (2011).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHan, T.J. et al. 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Reprogramming of Tumor-Associated Macrophages with Anticancer Therapies: Radiotherapy versus Chemo- and Immunotherapies. \u003cem\u003eFront Immunol\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e8\u003c/strong\u003e, 828;\u003cem\u003e\u0026nbsp;\u003c/em\u003edoi: 10.3389/fimmu.2017.00828 (2017).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYan, T. et al. Suppression of the hyaluronic acid pathway induces M1 macrophages polarization via STAT1 in glioblastoma. \u003cem\u003eCell Death Discov\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e8\u003c/strong\u003e, 1\u0026ndash;13; doi:10.1038/s41420-022-00973-y (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Glioblastoma, Chemoresistance, Proinflammatory macrophage, DNA repair enzymes, MGMT, MPG","lastPublishedDoi":"10.21203/rs.3.rs-5991999/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5991999/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Chemoresistance to temozolomide (TMZ), the main agent in glioblastoma (GBM) treatment, negatively impacts prognosis due to efficient repair of TMZ-induced DNA lesions. Reduced expression of the DNA repair enzyme O6-methylguanine DNA-methyltransferase (MGMT) has been associated with improved chemotherapy response, though not consistently across patients. Our previous findings suggested that N-methylpurine-DNA-glycosylase (MPG), which repairs the most common TMZ-induced DNA adducts, would be implicated in glioblastoma resistance to TMZ, but its role in chemoresistance remains unclear. Tumor-associated macrophages (TAMs), a key component of the GBM microenvironment, impair chemotherapy efficacy, though mechanisms by which they contribute to drug resistance are not well defined. This study shows that reducing MPG expression with specific siRNAs sensitizes tumor cells to TMZ, and this effect is potentiated by simultaneously downregulating both MPG and MGMT. We also provide the first evidence that conditioned medium from proinflammatory macrophages (CM-M1), by suppressing STAT3 phosphorylation, reduces repair enzyme expression in glioblastoma cells, enhancing TMZ cytotoxicity. Molecular analysis of tumor samples from glioblastoma patients confirmed an association between high repair enzyme expression and STAT3 activation. Our findings uncover a novel mechanism by which TAMs contribute to chemoresistance, supporting strategies to reprogram TAMs towards an M1-phenotype to improve TMZ efficacy in GBM treatment.","manuscriptTitle":"Proinflammatory macrophage-derived medium enhances temozolomide sensitivity in glioblastoma via pSTAT3-mediated downregulation of DNA repair enzymes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-18 14:01:30","doi":"10.21203/rs.3.rs-5991999/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-17T10:50:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T14:49:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147002154133957737234564861130030244778","date":"2025-05-22T13:12:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T20:52:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32441916240864918986488243246146512589","date":"2025-03-28T15:33:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301616789983599361792325578954993296843","date":"2025-02-27T03:44:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-21T18:51:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-21T18:45:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-14T09:27:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-13T12:17:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-09T11:02:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f358ecc0-b3bb-4932-adc7-3416e041c2f2","owner":[],"postedDate":"February 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44333513,"name":"Health sciences/Oncology/Cancer/Cancer microenvironment"},{"id":44333514,"name":"Health sciences/Oncology/Cancer/Cns cancer"},{"id":44333515,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":44333516,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2025-11-10T16:01:47+00:00","versionOfRecord":{"articleIdentity":"rs-5991999","link":"https://doi.org/10.1038/s41598-025-22674-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-06 15:57:08","publishedOnDateReadable":"November 6th, 2025"},"versionCreatedAt":"2025-02-18 14:01:30","video":"","vorDoi":"10.1038/s41598-025-22674-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-22674-z","workflowStages":[]},"version":"v1","identity":"rs-5991999","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5991999","identity":"rs-5991999","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}