Olaparib reverses prostate cancer resistance to Rapamycin by promoting macrophage polarization towards the M1 phenotype

Olaparib reverses prostate cancer resistance to Rapamycin by promoting macrophage polarization towards the M1 phenotype | 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 Research Article Olaparib reverses prostate cancer resistance to Rapamycin by promoting macrophage polarization towards the M1 phenotype Kai Ye, Gang Shi, Jian Xu, Kunyan Qiao, Qinghai Dai, Zhixiao Huo, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5399970/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2025 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract Prostate cancer (PCa) is the most common non-cutaneous malignancy and the second leading cause of cancer-related death in men. Despite its prevalence, treatment outcomes are often unsatisfactory, necessitating the search for more effective therapeutic approaches. mTOR inhibitor Rapamycin (RAPA) has shown promise in managing PCa, but the emergence of resistance often undermines its long-term effectiveness. Recent studies suggest that PARP inhibitor Olaparib (OLP) may overcome drug resistance in various tumor types. This study aims to assess the efficacy of OLP in treating RAPA-resistant PCa, with a specific focus on elucidating its underlying molecular mechanisms. This study utilized drug exposure and concentration escalation experiments to establish human RAPA-resistant PCa cell line (PC-3R) based on the human PCa cell line (PC-3). PC-3R cell lines were screened through a cloning assay. The efficacy of OLP in RAPA-resistant PCa, as well as its regulatory impact on tumor-associated macrophages (TAMs), was evaluated through a combination of real-time PCR, ELISA, immunohistochemistry, and fluorescence experiments. This study unveiled that the combination of OLP and RAPA effectively suppressed the proliferation, stemness, invasion, angiogenesis, apoptosis resistance, and anti-oxidative stress capacity of RAPA-resistant PCa. Additionally, it demonstrated the capacity of OLP to regulate macrophage polarization within the tumor microenvironment and reverse drug resistance to RAPA in PCa. The findings of this study lay a theoretical foundation for the potential utilization of OLP in the treatment of RAPA-resistant PCa, offering substantial academic significance and promising application prospects. Prostate cancer Rapamycin Olaparib Tumor microenvironment Macrophage polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous malignant tumor and ranks as the second leading cause of cancer-related mortality in men globally [ 1 ]. Despite its widespread occurrence, current treatment outcomes often fail to meet expectations, prompting the need for more effective therapeutic strategies. While the mTOR inhibitor Rapamycin (RAPA) has shown efficacy in managing PCa, the development of treatment resistance frequently compromises its long-term effectiveness, leading to disease progression. Recent studies have shed light on the potential of the PARP inhibitor Olaparib (OLP) in overcoming drug resistance observed across various tumor types [ 2 , 3 ]. Therefore, this study aims to assess the efficacy of OLP in treating RAPA-resistant PCa, with a specific focus on elucidating its underlying molecular mechanisms. Tumor-associated macrophages (TAMs) have emerged as pivotal players in facilitating tumor drug resistance [ 4 ]. Within the tumor microenvironment (TME), TAMs, among bone marrow-derived cells, are notably linked with the advancement and immune suppression observed in PCa [ 5 ]. It is widely acknowledged that macrophages can infiltrate tumor tissues. In one of the earliest investigations concerning macrophage presence in PCa, a significant disparity was noted in macrophage density between benign areas adjacent to prostate tumors and the tumors themselves [ 6 ]. Despite the established increase in TAMs within prostate cancers, the precise mechanisms through which TAMs regulate tumor growth remain largely elusive. TAMs serve as pivotal components within the tumor microenvironment (TME), exerting significant influence over tumor growth and disease progression [ 7 ]. Depending on their polarization state, TAMs can either impede or bolster tumor growth, adopting either a classically-activated macrophage (M1) or alternatively-activated macrophage (M2) phenotype, respectively [ 8 , 9 ]. The polarization phenotype of TAMs has been associated with prognosis across various cancer types [ 7 , 10 ]. Notably, a higher infiltrate of M1 macrophages correlates with a more favorable prognosis, whereas a higher M2 infiltrate is indicative of a poorer prognosis. M1 macrophages, characterized by their pro-inflammatory nature, counteract tumor growth through the activation of adaptive immune responses or direct tumor cell killing. Conversely, M2 macrophages, with their anti-inflammatory characteristics, sustain tumor cell growth by fostering angiogenesis, facilitating matrix remodeling, and promoting immune suppression [ 11 , 12 ]. Unfortunately, the predominant phenotype among TAMs often leans towards the M2-like state, consequently supporting tumor growth rather than facilitating tumor elimination. Therefore, the reprogramming of TAMs within the TME presents a promising therapeutic avenue to enhance anti-tumor activity. The effectiveness of OLP in RAPA-resistant PCa, along with its regulatory impact on TAMs, was evaluated through a combination of real-time PCR, ELISA, immunohistochemistry, and fluorescence experiments. Our study revealed that the combination of OLP and RAPA effectively suppressed the proliferation, stemness, invasion, angiogenesis, apoptosis resistance, and anti-oxidative stress capacity of PC-3R cells. In addition, it proved the ability of OLP to regulate macrophage polarization and reverse the drug resistance of PCa to RAPA in tumor microenvironment. Methods Animal and tumor cell lines Six-week-old male BALB/c nude mice were obtained from Charles River Laboratories. Human macrophage cell line (THP-1), human PCa cell line (PC-3) and human umbilical vein endothelial cells (HUVECs) were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin. F-THP-1 cells, F-PC-3 cells and F-HUVECs were transduced with a lentivirus encoding luciferase and green fluorescent protein (GFP). R-PC-3 was transduced with a lentivirus encoding luciferase and red fluorescent protein (RFP). The expression of GFP and RFP was monitored by using a flow cytometry analysis. All cells were incubated at 37℃ with 5% CO 2 . The construction of RAPA-resistant PCa cell line PC-3R First, we expose the parental PCa cell line (PC-3) to low concentrations of RAPA to initiate the resistance process. Then, we incrementally increase the concentration of RAPA over time, allowing the cells to adapt and develop resistance. Finally we isolate single-cell clones from the drug-exposed population and expand them. Cell cytotoxicity assessment For cell cytotoxicity assays, PC-3R cells were seeded in 96-well plates at a density of 1 × 10 3 per well, add different concentrations of RAPA and/or OLP for 24–72 h treatment, and the cell growth rate was evaluated by CCK-8 kit (C0038, Beyotime) in terms of the manufacturer's instructions. bioluminescence imaging In vitro bioluminescence imaging F-PC-3R cells (5 × 10 3 per well) or F-HUVECs (1 × 10 4 per well) were placed on 48-well plates. D-luciferin substrate (200µg/ml) was added to each well at room temperature. Cell bioluminescence signals were detected by the IVIS Spectrum Imaging System and analyzed by Carestream molecular imaging software (Caliper Life Sciences, Alameda, CA). Enzyme-linked immunosorbent assay (ELISA) The tumor tissue homogenates were collected and the concentration of multiple cytokines was measured by corresponding ELISA kit in terms of the manufacturer's instruction. Briefly, the primary antibody (provided in kit) was coated and incubated for 2 h at room temperature. Samples were added and incubated for 1 h, following which wells were washed and a biotinylated antibody (provided in kit) was added for 1 h. The plates were washed again and streptavidin conjugated to horseradish peroxidase was added for 10 min at room temperature. Plates were washed and tetramethylbenzidine was added for color development about 30 min at room temperature and the reaction was terminated with 1M H 2 SO 4 . Absorbance was measured at 450nm by using a microplate reader (Thermo Fisher Scientific, Inc.). Concentrations in the samples were calculated using a standard curve and values were expressed as pg/ml or ng/ml. All experiments were performed in terms of the manufacturer’s instructions. RNA extraction, RT-PCR and real-time RT-PCR In terms of the manufacturer's guideline, total RNA was drawn from cell lines and tissues by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized by using TransScript® First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China). Reverse transcription polymerase chain reaction (RT-PCR) was conducted by using TransScript® Two-Step RT-PCR SuperMix (TransGen, Beijing, China) in line with the manufacturer's instructions. The PCR products were analyzed on 1% agarose gel and visualized. Real-time RT-PCR was performed using TransStart® Top Green qPCR SuperMix (TransGen, Beijing, China). The primers (GENEWIZ, Tianjin, China) were listed in Table S1 . Lactate dehydrogenase (LDH) cytotoxicity assay LDH assay was performed to analyze cytotoxicity activity of RAPA and/or OLP. PC-3R cells were prepared in 96-well plates. Supernatants were collected and used for the LDH assay measurement, according to the LDH cytotoxicity assay kit (Beyotime institute of Biotechnology) manufacturer’s protocol. Cell apoptosis assay Annexin V-FITC/PI staining method was used to distinguish early and late stages of apoptosis cells and necrosis cells. PC-3R cells were seeded in a 6-well plate, then cells were collected and resuspended to 1 × 10 6 cells/ml in 1 × binding buffer, subsequently cells were washed with PBS. Approximately 1 × 10 5 cells (100 µl) were aliquoted into a flow cytometry tube. Annexin V-FITC (5 µl) was added into the tube and incubated for 10 min, at room temperature (RT) and in darkness. Propidium iodide (5 µl) was then incubated with the cells for 5 min, at RT and in darkness. The mixed solution was added to 500 µl PBS and blended gently. The prepared cell samples were immediately tested by flow cytometer (FACS Calibur, BD, US). Mitochondrial Membrane Potential (MMP) Assay MMP assay was performed as described previously [ 13 ]. PC-3R cells in each group were inoculated on 6-well plates, incubated for 24 h, and added with JC-1 (Beyotime, Beijing, China) at room temperature to avoid light reaction for 40 min. The intensity of red and green fluorescence was measured and recorded by flow cytometry. Determination of oxidative stress kit 20 mg of tumor blocks were minced and homogenized in ice-cold RIPA buffer (Sigma, C0278). Homogenates were subject to centrifuge at 13000g for 15 min at 4°C to obtain the supernatant as sample tissue total protein preparation. The protein concentration was measured with a BCA (bicinchoninic acid) protein assay kit (Beyotime Biotechnology, Shanghai, China). MDA, OFR, 4-HNE, GSH, PHD, SOD and GSH-Px activities were measured using the corresponding kits (Jiancheng Bioengineering Institute, Nanjing, China). All experiments were performed in terms of the manufacturer’s instructions. Immunofluorescence staining Immunofluorescence staining was performed using the paraffin-embedded sections. Antibodies were used at a dilution factor of 1:100. Staining signals were visualized with the Alexa Fluor 633 or 488 conjugated secondary antibodies (1:200; Invitrogen, Shanghai, China). The sections were counter-stained with DAPI (Southern Biotech, England) and examined using a laser confocal scanning microscopy (Imager Z2, Zeiss, Germany). At least 10 different stained random fields were assessed for each marker by a researcher that was blinded to the study. Caspase-3 activity detection The activity of caspase-3 was measured using caspase-3 activity assay kit (Beyotime Institute of Biotechnology). Briefly, PC-3R cells were cultured for 24 h. After addition of RAPA and/or OLP, cells were incubated for another 12 h. The extraction procedure of caspase-3 was the same as the instructions. Caspase-3 substrate (provided in the kit) was changed into a yellow formazan product in the presence of caspase-3. Caspase-3 activity was measured at 405 nm on a microplate reader, and the activity ratio was calculated according to the kit instructions. Tube formation experiment Add 220 µl of matrigel to each well of a 24-well plate and incubate at 37°C for 30 min; add 6×10 4 HUVECs and 600 µl of different groups of reagents (DMEM, PC-3R + RAPA, PC-3R + OLP or PC-3R + RAPA/OLP), placed in a cell incubator for 4 h. In vivo antitumor model BALB/c nude mice (male, 6 weeks age) were purchased from Charles River Laboratories (Beijing, China). All animal protocols were approved by the Institutional Animal Care and Use Committee. An in vivo subcutaneous tumor xenograft mouse model was established. Briefly, F-PC-3R cell suspension (1×10 6 cells) were injected into the right flank of mice. When tumors reached ~ 50 mm 3 after approximately one week, mice were subsequently randomly divided into the following four treatment group (PBS, RAPA, OLP and RAPA/OLP). Treatments were injected at the tumor site. The antitumor effect was quantified according to tumor weight and volume ((L×W 2 )/2). When the tumor grew to 1000mm 3 , mice were anesthetized by inhaling isoflurane through the nose cone. For euthanasia, the cervical dislocation was employed. All procedures involving anesthesia and euthanasia were carried out by trained personnel under the supervision of a veterinarian to ensure the utmost care and compliance with animal welfare regulations. Statement of ethics All animal experiments were performed in accordance with ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals (National Research Council, 8th edition, 2011) and approved by the Institutional Animal Care And Use Committee (IACUC) of Tianjin Second People's Hospital under Assurance Number LL-BG-032. Statistical Analyses Statistical analyses were determined by one-way ANOVA, followed by Tukey's multiple comparison test analysis. All statistical analyses were conducted using GraphPad Prism 7 (GraphPad). Results The optimum concentration of PC-3R cells to OLP and RAPA In this study, we employed the CCK-8 assay to investigate the cytotoxic effects of OLP on PC-3R cells, as well as to determine the tolerance threshold of these cells to RAPA. Treatment with low concentrations (0.1, 0.2, and 0.5 µM) of OLP resulted in only a slight decrease in the viability of PC-3R cells. However, higher concentrations (1, 2, and 5 µM) of OLP demonstrated significant inhibition of PC-3R cell growth at 24 hours (Fig. 1 A), as well as at 48 and 72 hours (Supplementary Fig. 1A). Importantly, all concentrations of RAPA showed no significant inhibition of growth in PC-3R cells at 24 hours (Fig. 1 B), as well as at 48 and 72 hours (Supplementary Fig. 1B). Based on the above-mentioned findings, RAPA or OLP at the concentration of 1 µM was used for subsequent experiments. OLP augments the inhibitory efficacy of RAPA on the proliferation and stemness of PC-3R cells Bioluminescent imaging unveiled a notably reduced proliferation of PC-3R cells upon treatment with the combination of RAPA and OLP, in comparison to the RAPA monotherapy group. This suggests that OLP enhances the sensitivity of RAPA in PC-3R cells (Fig. 1 C). Moreover, we conducted real-time PCR assay and observed that the combination of RAPA and OLP led to a reduction in the expression of pro-proliferative genes, including Igf1 , Hgf , Egf , Il6 , Il33 , and Csf3 in PC-3R cells (Fig. 1 D). Among these genes,the most significant down-regulation was observed in the expression of the Csf3 gene (Fig. 1 D). In parallel, the secretion of proliferation-related proteins, such as insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), epidermal growth factor (EGF), IL-6, IL-33 and granulocyte colony-stimulating factor (G-CSF), in PC-3R cells were down-regulated in combined treatment group compared to the monotherapy group (Fig. 1 E). Among these proteins, the most significant down-regulation was observed in the expression of G-CSF (Fig. 1 E). These results indicate that the inhibition of PC-3R cell proliferation by the combination of RAPA and OLP is primarily mediated through the suppression of G-CSF expression. Moreover, treatment with RAPA combined with OLP resulted in decreased expression levels of pluripotency-related genes, including Aldh1a1 , Nanog and Lin28a in PC-3R cells (Fig. 1 F). Among these genes, the most significant down-regulation was observed in the expression of Lin28a (Fig. 1 F). Consistent findings were obtained at the protein level (Fig. 1 G). These results suggest that the combination of RAPA and OLP could attenuate the stemness of PC-3R cells primarily by suppressing the expression of LIN28A. OLP augments the promoting efficacy of RAPA on oxidative stress and apoptosis of PC-3R cells Subsequently, we conducted in vitro assays to investigate whether OLP can potentiate the apoptosis induced by RAPA in PC-3R cells. The combination of RAPA and OLP triggered the release of lactate dehydrogenase (LDH, a necrotic marker) in PC-3R cells (Fig. 2 A), indicating strong cytotoxicity against RAPA-resistant PCa cells. This observation was supported by annexin V/PI assay, which demonstrated a higher percentage of apoptotic cells in the combination therapy group (Fig. 2 B). Activation of apoptosis frequently involves mitochondrial deregulation and oxidative stress [ 14 ]. Our data revealed that the combined treatment with RAPA and OLP resulted in mitochondrial damage, as evidenced by an increase in collapsed mitochondrial membrane potential in PC-3R cells (Fig. 2 C). In addition, we observed an elevation in oxygen free radical (OFR) (Fig. 2 D) and in lipid peroxidation markers, including 4-hydroxynonenal (4-HNE) and malonaldehyde (MDA) (Fig. 2 E) in PC-3R cells treated with RAPA and OLP. This suggests the potential involvement of oxidative stress in initiating apoptosis. Importantly, these changes occurred concurrently with a decrease in intracellular levels of antioxidants, such as superoxide dismutase (SOD), glutathione (GSH), and the activity of glutathione peroxidase (GSH-Px) (Fig. 2 F). Consistently, PC-3R cells treated with RAPA and OLP exhibited increased mRNA expression of pro-oxidant genes (Fig. 2 G) and reduced expression of anti-oxidative genes, as revealed by real-time RT-PCR (Supplementary Fig. 2). We also observed that the combined treatment with RAPA and OLP significantly upregulated the expression of p53, a tumor suppressor protein known to positively regulate apoptosis (Fig. 2 H). In addition, PC-3R cells subjected to the combined treatment of RAPA and OLP exhibited increased expression and activity of caspase-3 (Fig. 2 I and J), indicating the involvement of the intrinsic apoptotic pathway. Supporting this observation is the modulation of a broad spectrum of apoptotic genes, including cytochrome c ( Cycs ), Bax , Casp3 , Casp9 and poly (ADP-ribose) polymerase 1 ( Parp1 ) in the combined treatment group (Fig. 2 K). Importantly, the expression of Bax gene was significantly down-regulated (Fig. 2 K). The same conclusion is confirmed at the protein level (Fig. 2 L). Taken together, these findings suggest that the combination of RAPA and OLP can induce PC-3R cell apoptosis via activation of oxidative stress and a mitochondrial-mediated pathway. The combination of OLP and RAPA indirectly inhibits angiogenesis by modulating PCa cells in vitro Next, we aim to explore whether OLP can enhance the inhibitory effect of RAPA on angiogenesis. Our results showed that the combination of OLP and RAPA significantly down-regulated the expression of a series of angiogenesis-related genes compared with the RAPA group in HUVECs (Fig. 3 A). This was accompanied by enhanced PHD activity in PC-3R cells exposed to OLP and RAPA (Supplementary Fig. 3). Among them, Vegfa and Angpt1 genes were down-regulated most significantly. The same conclusion is confirmed at the protein level (Fig. 3 B). To further elucidate the mechanism underlying the anti-angiogenic property of the combined treatment, conditioned medium (CM) of PC-3R cells was harvested and used to treat HUVECs (Fig. 3 C). CM derived from RAPA-treated PC-3R cells had a weak impact on the viability and function of HUVECs. In contrast, CM from the combined treatment group markedly reduced cell proliferation (Fig. 3 D), increased LDH release (Fig. 3 E), impaired tubule formation (Fig. 3 F) and inhibited migratory capacity (Fig. 3 G) of HUVECs. In addition, exposure to CM derived from the combined treatment group significantly reduced the expression of VEGFA and Ang-1 (Fig. 3 H) and suppressed mRNA expression of Vegfa and Angpt1 in HUVECs (Fig. 3 I). Thus, our data indicate that OLP can indirectly inhibit tumor angiogenesis mainly by inhibiting the expression of VEGFA and Ang-1. OLP augments the inhibitory efficacy of RAPA on the mesenchymal transition (EMT) and migratory capacity of PC-3R cells Epithelial to mesenchymal transition (EMT) is a critical process involving phenotypic transition that confers invasive and migratory properties to PCa cells, ultimately leading to metastasis [ 15 ]. To investigate whether the combination of RAPA and OLP could reverse the metastatic phenotype of PC-3R cells, we examined the expression profiles of EMT-related genes in PC-3R cells. Our results demonstrated that RAPA combined with OLP significantly decreased the mRNA expression of a wide range of mesenchymal markers (e.g. Vim and Cdh2 ) and transcription factors (e.g. Snail1/2 and Twist1 ) (Supplementary Fig. 4), while restoring the expression of epithelial markers such as Cdh1 , Tjp1 and Claudin (Fig. 4 A) in PC-3R cells. Subsequently, these findings were further validated by ELISA experiments (Fig. 4 B). It is well established that matrix metalloproteinases (MMPs) promote tumor invasion and metastasis by degrading the extracellular matrix, and this effect can be inhibited by tissue inhibitors of metalloproteinases (TIMPs) [ 16 ]. Thus, we next investigated whether the combination of RAPA and OLP could suppress the invasion potential of PC-3R cells by regulating the expression of MMPs and TIMPs. We observed no change in mRNA expression of Mmp9 , Timp1 , Timp4 , or Kiss1 in PC-3R cells due to OLP and RAPA treatment (Fig. 4 C and E). Most importantly, the expression of Mmp2 was significantly down-regulated and Timp3 was markedly up-regulated after combined treatment (Fig. 4 C and E). The conclusions were further verified by ELISA experiments (Fig. 4 D and F). The above results indicate that RAPA combined with OLP could inhibit the migration and invasion of PC-3R cells mainly by regulating the expression of MMP2 and TIMP3. The combination of RAPA and OLP inhibit growth of PCa via promoting apoptosis We subsequently evaluated whether the combination of RAPA and OLP could effectively hinder tumor growth in a xenograft model. PC-3R cells expressing Fluc-GFP were implanted subcutaneously into the flanks of nude mice, which were then intratumorally injected with either saline, OLP, RAPA or a combination of OLP + RAPA (Fig. 5 A). Our findings indicated that RAPA alone exerted limited inhibitory effects on tumor growth, resulting in only a slight delay in tumor progression. However, the combination of RAPA and OLP induced a remarkable degree of tumor regression in all mice (Fig. 5 A and B). Furthermore, local administration of OLP and RAPA significantly enhanced the overall survival of xenograft mice compared to the OLP or RAPA groups alone (Fig. 5 C). In order to evaluate the inhibitory effect of the combination of RAPA and OLP on tumor growth, we conducted immunofluorescence experiments on tumor samples. Our results revealed a significant reduction in the number of ki-67 + proliferating cells in the group treated with both OLP and RAPA compared to those treated with OLP or RAPA alone (Fig. 5 D). Additionally, real-time RT-PCR analysis demonstrated that the combined treatment downregulated the expression of several anti-apoptosis genes, including Bcl2 , Bcl2l1 , Birc5 , Akt1 and Pik3ca , among which Bcl2 gene is the most significant (Fig. 5 E). Consistent with these findings, ELISA experiments confirmed the downregulation of anti-apoptosis protein expression (Fig. 5 F). These observations suggest that OLP primarily enhances the apoptotic effect of RAPA by potentiating its inhibition of Bcl2 gene expression. Furthermore, we found that the combined treatment upregulated the expression of several pro-apoptotic genes, such as Bax , Bad , Bcl2l11 , Cycs , Casp3 , Casp9 , and Parp1 , with Bax being the most significant (Fig. 5 G). Notably, the expression trends of these proteins were similar to those of their respective mRNAs (Fig. 5 H). Collectively, these findings indicate that OLP accelerates the apoptosis of PC-3R cells primarily by potentiating RAPA's promotion of Bax gene expression. The combination of RAPA and OLP inhibit tumor growth by blocking angiogenesis To assess whether OLP potentiates the anti-angiogenic effects of RAPA in PC-3R xenografts, we first performed real-time PCR analysis on tumor samples. Our findings revealed that the combined treatment of RAPA and OLP downregulated the expression of multiple pro-angiogenic genes, including Vegfa , Kdr , Fgf2 , Pigf , Pdgfa , Hif1a , Cxcl12 , Angpt1 , Angpt2 and Edn1 (Fig. 6 A). Consistent with these observations, ELISA results demonstrated a significant downregulation of pro-angiogenic protein expression, particularly VEGFA and Ang-1, in PC-3R xenografts treated with the combination of RAPA and OLP. Collectively, these results suggest that the anti-angiogenic activity of RAPA and OLP is primarily achieved by suppressing the expression of VEGFA and Ang-1. The combination of RAPA and OLP promote the differentiation of M2 macrophages into M1 in vivo It is well-established that macrophages can promote advanced tumor metastasis by releasing cytokines [ 17 ]. Therefore, to investigate the impact of RAPA combined with OLP on macrophage polarization, we evaluated the expression patterns of 17 genes related to M1 and M2 macrophages in PC-3R transplanted tumor following intratumoral injection of OLP and RAPA. Among these genes, six M1 macrophage gene expressions ( Il12a , Il6 , Il1b , Tnfa , Nos2 and Ccl2 ) were prominently augmented (Fig. 7 A). Similarly, the gene expressions of several M2 macrophages ( Il10 , Vegfa , Egf , Arg1 , Mrc1 and Tgfb1 ) were significantly inhibited (Fig. 7 B). Notably, there were no significant differences in the expression of other genes (M1: Il23a and Clec4e ; M2: Retnlb , Ym1 and Pdgfa ) before and after treatment with OLP and RAPA (Supplementary Fig. 5), indicating that the combination of RAPA and OLP does not regulate macrophage infiltration and polarization through these genes in vivo . The ELISA assay demonstrated that the combination of RAPA and OLP enhances the secretion of cytokines by M1 macrophages and suppresses the secretion of cytokines by M2 macrophages (Fig. 7 C and D). Among the six cytokines influenced in the M2 phenotype, IL-10 and TGF-β play a crucial role in regulating the function of TAMs [ 18 ]. Additionally, we evaluated the surface markers of M1 and M2 macrophages. The results revealed that the combined treatment of RAPA and OLP not only up-regulates the protein expression on the surface of anti-tumor M1 macrophages, but also downregulates the protein expression on the surface of tumor-promoting M2 macrophages (Fig. 7 E and F). These findings suggest that the combination of RAPA and OLP effectively polarizes macrophages in PC-3R transplanted tumors towards the M1 phenotype. The combination of RAPA and OLP inhibit tumor angiogenesis indirectly through macrophages in vitro Numerous studies have demonstrated that M1 macrophages could inhibit angiogenesis [ 19 , 20 ]. To investigate the direct impact of the combination of RAPA and OLP on M1 phenotype gene expression profiles, we analyzed THP-1 cells. Our results demonstrated that the combined treatment significantly upregulated the gene expressions of Il12a , Il6 , Il1b , Tnfa , Nos2 and Ccl2 compared to the RAPA group alone (Fig. 8 A). Notably, there were no significant differences in the expression of Il23a and Clec4e genes (Supplementary Fig. 6), indicating that the combination of RAPA and OLP does not stimulate M1 macrophages to produce cytokines encoded by these two genes. To further validate the findings from RT-PCR, we conducted ELISA assays to assess the expression levels of M1 markers in the combined treatment group. These assays confirmed the upregulation of IL-12α, IL-6, IL-1β, TNF-α, iNOS, and CCL2, thereby reinforcing the conclusion that the combination of RAPA and OLP directly promotes M1 macrophage expression (Fig. 8 B). There have already been several reports indicating that TAMs could vastly promote tumor angiogenesis [ 5 , 21 ]. To investigate the impact of RAPA combined with OLP on tumor angiogenesis by modulating macrophage in vitro , conditioned medium (CM) of THP-1 cells was harvested and used to treat HUVECs (Fig. 8 C). RT-qPCR was employed to measure the gene expression of angiogenesis markers ( Edn1 , Angpt1 , Vegfa , Pdgfb , Fgf2 , Egln1 and Hif1a ) in HUVECs. The results demonstrated a significantly reduced expression of these markers in the MΦ + OLP + RAPA group compared to the OLP + RAPA group (Fig. 8 D). The protein expression trend was consistent with the mRNA expression, indicating that the combination of RAPA and OLP indirectly suppresses angiogenesis by regulating macrophage secretion. The combination of RAPA and OLP affects macrophage polarization by regulating PC-3R cells in vitro Recent publications have unequivocally demonstrated the promising therapeutic potential of targeting TAMs for cancer immunotherapy [ 22 ]. Thus, to ascertain whether the regulation of PC-3R cells by OLP and RAPA can induce the transformation of M2 macrophages into M1 macrophages in vitro , we employed real-time PCR to assess the effect of the culture medium after treatment with RAPA combined with OLP on the polarization of TGF-β-treated M2 macrophages following PC-3R cell treatment. The findings revealed a significant upregulation of M1 macrophage markers in the TGF-β + PC-3R + OLP + RAPA group compared to the TGF-β + PC-3R + RAPA group (Fig. 9 A), while the expression levels of M2 phenotype markers were notably downregulated in the TGF-β + PC-3R + OLP + RAPA group compared to the TGF-β + PC-3R + RAPA group (Fig. 9 B). These results suggest that the treatment of PC-3R cells with OLP and RAPA could more effectively promote the polarization of M2 macrophages towards the M1 phenotype compared to RAPA alone. Additionally, we assessed the expression of M1 and M2-related proteins in macrophages treated with OLP and RAPA using ELISA, yielding consistent conclusions (Fig. 9 C and D). Discussion Prostate cancer (PCa) remains a significant health concern worldwide due to its high incidence and mortality rates. Despite advancements in treatment modalities, including surgical intervention, radiation therapy, and androgen deprivation therapy, the management of PCa remains challenging, especially in cases where resistance to conventional therapies develops. In this context, the exploration of alternative therapeutic strategies is imperative to improve patient outcomes and reduce mortality rates. The present study aimed to investigate the efficacy of the PARP inhibitor Olaparib (OLP) in treating RAPA-resistant PCa, with a specific focus on understanding its underlying molecular mechanisms. Our findings demonstrate that OLP effectively suppresses the proliferation, stemness, invasion, angiogenesis, apoptosis resistance, and anti-oxidative stress capacity of PC-3R cells. These results suggest that OLP holds promise as a potential therapeutic agent for overcoming drug resistance and inhibiting tumor progression in PCa patients who have failed RAPA treatment. One of the key findings of this study is the ability of OLP to regulate macrophage polarization within the tumor microenvironment. Tumor-associated macrophages (TAMs) play a crucial role in tumor progression and therapeutic resistance by promoting angiogenesis, immunosuppression, and tissue remodeling. Our results indicate that OLP treatment alters the phenotype of TAMs, potentially shifting them from a pro-tumorigenic M2 phenotype to an anti-tumorigenic M1 phenotype. This modulation of TAM polarization by OLP could contribute to its anti-tumor effects and reversal of drug resistance in PCa. The mechanisms underlying the efficacy of OLP in RAPA-resistant PCa are likely multifaceted. Previous studies have implicated the role of PARP inhibitors in inducing synthetic lethality in tumors with homologous recombination deficiency (HRD), leading to enhanced sensitivity to DNA damage [ 23 ]. Given the inherent genomic instability observed in PCa, particularly in advanced stages, OLP may exploit this vulnerability to induce tumor cell death. Moreover, OLP has been shown to inhibit the repair of DNA single-strand breaks (SSBs) and promote the accumulation of DNA double-strand breaks (DSBs), further sensitizing cancer cells to cytotoxic insults [ 24 ]. Additionally, the crosstalk between cancer cells and the tumor microenvironment (TME) is increasingly recognized as a determinant of therapeutic response and resistance [ 25 ]. In this context, our findings suggest that OLP-mediated modulation of TAMs may contribute to the restoration of sensitivity to RAPA in PC-3R cells. By reprogramming the immunosuppressive TME towards an anti-tumorigenic state, OLP may enhance the efficacy of RAPA and other anti-cancer therapies. However, it is essential to acknowledge the limitations of our study. While our findings provide compelling evidence for the potential therapeutic utility of OLP in RAPA-resistant PCa, further preclinical and clinical studies are warranted to validate these findings in a broader patient population. Additionally, the precise mechanisms underlying the crosstalk between OLP-treated cancer cells and TAMs require further elucidation. Future research efforts should focus on deciphering the signaling pathways involved in this interaction to identify novel therapeutic targets and optimize treatment strategies for PCa patients. Conclusion Our study highlights the therapeutic potential of OLP in overcoming drug resistance and inhibiting tumor progression in RAPA-resistant PCa. By elucidating the molecular mechanisms underlying its efficacy, we provide a rationale for the clinical evaluation of OLP as a novel therapeutic approach for PCa patients who have failed conventional treatments. Moreover, our findings underscore the importance of considering the dynamic interplay between cancer cells and the TME in the development of effective anti-cancer therapies. Ultimately, the translation of these research findings into clinical practice holds the promise of improving outcomes and quality of life for PCa patients. Declarations Availability of data and material All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author. Acknowledgements We are particularly grateful to all the people who have given us help on our article. Funding This work was supported by the National Science and Technology Major Project (No.2023ZD0508702), Natural Science Foundation of Tianjin (23JCYBJC00950), Tianjin Health Science and Technology Project key discipline special (TJWJ2022XK034), Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-059B), Research project in key areas of TCM in 2024 (2024022). Author information Kai Ye and Gang Shi have contributed equally to this work. Contributions Yuqiang Mi and Rui Su supervised the project; Kai Ye and Gang Shi drafted the manuscript; Kai Ye, Gang Shi, Kunyan Qiao, Jian Xu, Qinghai Dai, Zhixiao Huo, Yu Cao, Wei Liu, Yue Hu and Lihua Yan performed the experiments; Kai Ye and Gang Shi performed the statistical analyses; Ping Li, Liang Xu, Yu Zhu, Rui Su, Yuqiang Mi revised the article. All authors read and approved the final article. Corresponding authors Correspondence to Yuqiang Mi, Liang Xu, Rui Su or Ping Li. Ethics declarations Competing interests The authors declare that they have no competing interests. Ethics approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. Consent to participate: Not applicable. Consent for publication: Not applicable. Informed consent: Not applicable. References He J, Chen WQ, Li N, Cao W, Ye DW, Ma JH, Xing NZ, Peng J and Tian JH (2022) [China guideline for the screening and early detection of prostate cancer (2022, Beijing)]. Zhonghua Zhong Liu Za Zhi 44:29-53. doi: 10.3760/cma.j.cn112152-20211226-00975 Yang XD, Kong FE, Qi L, Lin JX, Yan Q, Loong JHC, Xi SY, Zhao Y, Zhang Y, Yuan YF, Ma NF, Ma S, Guan XY and Liu M (2021) PARP inhibitor Olaparib overcomes Sorafenib resistance through reshaping the pluripotent transcriptome in hepatocellular carcinoma. 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Basic Res Cardiol 115:22. doi: 10.1007/s00395-020-0781-7 Larionova I and Kazakova E (2021) New Angiogenic Regulators Produced by TAMs: Perspective for Targeting Tumor Angiogenesis. Cancers (Basel) 13. doi: 10.3390/cancers13133253 Wang YC, Wang X, Yu J and Ma F (2021) Targeting monoamine oxidase A-regulated tumor-associated macrophage polarization for cancer immunotherapy. Nat Commun 12:3530. doi: 10.1038/s41467-021-23164-2 Petropoulos M, Karamichali A and Rossetti GG (2024) Transcription-replication conflicts underlie sensitivity to PARP inhibitors. Nature 628:433-441. doi: 10.1038/s41586-024-07217-2 Kim DS, Camacho CV and Kraus WL (2021) Alternate therapeutic pathways for PARP inhibitors and potential mechanisms of resistance. Exp Mol Med 53:42-51. doi: 10.1038/s12276-021-00557-3 Badve SS and Gökmen-Polar Y (2023) Targeting the Tumor-Tumor Microenvironment Crosstalk. Expert Opin Ther Targets 27:447-457. doi: 10.1080/14728222.2023.2230362 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure.docx SupplementaryTable.docx Cite Share Download PDF Status: Published Journal Publication published 21 Feb, 2025 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Editorial decision: Revision requested 19 Jan, 2025 Reviews received at journal 17 Jan, 2025 Reviewers agreed at journal 24 Dec, 2024 Reviewers invited by journal 23 Nov, 2024 Editor assigned by journal 23 Nov, 2024 Submission checks completed at journal 07 Nov, 2024 First submitted to journal 06 Nov, 2024 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-5399970","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381775729,"identity":"849cfb09-c4af-4605-a8bf-bc00a3cabe3d","order_by":0,"name":"Kai Ye","email":"","orcid":"","institution":"Tianjin Institute of Hepatology, Tianjin Second People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Ye","suffix":""},{"id":381775730,"identity":"1436c5ca-4713-4db0-a381-0ee82a746c58","order_by":1,"name":"Gang Shi","email":"","orcid":"","institution":"Tianjin Anding Hospital, Mental Health 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06:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5399970/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5399970/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11010-025-05231-0","type":"published","date":"2025-02-21T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69937666,"identity":"df32b3c5-de2a-41a9-9aef-a21f3bbe5ff7","added_by":"auto","created_at":"2024-11-26 19:44:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2955331,"visible":true,"origin":"","legend":"\u003cp\u003eOLP enhances the inhibitory effect of RAPA on the proliferation of PC-3R cells \u003cem\u003ein vitro\u003c/em\u003e. (A) The effect of different concentrations of OLP on the proliferation of PC-3R cells after 24 hours of treatment. Data are presented as mean ± SD (n = 4). *(P \u0026lt; 0.05)significant difference comparable to the Control group. (B) The effect of different concentrations of RAPA on the proliferation of PC-3R cells after 24 hours of treatment. Data are presented as mean ± SD (n = 4). *(P \u0026lt; 0.05) significant difference comparable to the Control group. (C) The combination effect of OLP and RAPA on the proliferation of PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05)significant difference comparable to the RAPA group. (D) The combined effect of OLP and RAPA on the secretion of pro-proliferative factors in PC-3R cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05)significant difference comparable to the RAPA group. (E) The combined effect of OLP and RAPA on pro-proliferative gene expression in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05)significant difference comparable to the RAPA group. (F) The combination effect of OLP and RAPA on the expression of pluripotency-related genes in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05)significant difference comparable to RAPA group. (G) The combined effect of OLP and RAPA on the expression of pluripotency-related proteins in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05)significant difference comparable to the RAPA group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/f8ea816aafa1a2cd686513ca.png"},{"id":69937671,"identity":"8f105805-a6e2-49f5-8270-522a8d4ba0cd","added_by":"auto","created_at":"2024-11-26 19:44:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4113151,"visible":true,"origin":"","legend":"\u003cp\u003eOLP enhances the promoting efficacy of RAPA on oxidative stress and apoptosis of PC-3R cells \u003cem\u003ein vitro\u003c/em\u003e. (A) The combined effect of OLP and RAPA on LDH release in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (B) The combined effect of OLP and RAPA on PC-3R cell apoptosis detected by PI/Annexin-V kit. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (C) JC-1 kit detects the combined effect of OLP and RAPA on mitochondrial damage in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (D) The combined effect of OLP and RAPA on oxygen free radicals (OFR) in PC-3R cells. Data are presented as mean ± SD (n = 4). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (E) The combined effect of OLP and RAPA on endogenous lipid peroxidation products (4-HNE and MDA) in PC-3R cells. Data are presented as mean ± SD (n = 4). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (F) The combined effect of OLP and RAPA on superoxide dismutase (SOD) and glutathione peroxidase (GSH and GSH-Px) in PC-3R cells. Data are presented as mean ± SD (n = 4). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (G) The combined effect of OLP and RAPA on the expression of pro-oxidant-related genes in PC-3R cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (H) The combined effect of OLP and RAPA on gene expression of tumor suppressor protein P53 in PC-3R cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (I) Caspase-3 immunofluorescence staining to evaluate the combined effect of OLP and RAPA on apoptosis of PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (J) Caspase-3 enzyme activity assay. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (K) The combined effect of OLP and RAPA on the expression of apoptosis-related genes in PC-3R cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (L) The combined effect of OLP and RAPA on the expression of apoptosis-related proteins in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/1ae0ecf9d44b4a5d098a4331.png"},{"id":69937919,"identity":"256c2453-836f-4b8f-b3e0-0d0ffd7e9baf","added_by":"auto","created_at":"2024-11-26 19:52:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4535179,"visible":true,"origin":"","legend":"\u003cp\u003ePC-3R cells pretreated with OLP and RAPA indirectly inhibit the angiogenesis of vascular endothelial cells \u003cem\u003ein vitro\u003c/em\u003e. (A) The combined effect of OLP and RAPA on the expression of angiogenesis-related genes in HUVECs. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (B) The secretion of angiogenesis-related factors was detected by ELISA. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (C) Schematic of cell processing: PC-3R cells were treated with OLP and RAPA, and the supernatant was collected to treat HUVECs. (D) Indirect effect of OLP and RAPA on HUVECs proliferation. Data are presented as mean ± SD (n = 4). *(P \u0026lt; 0.05) significant difference comparable to the RAPA CM group. (E) Indirect effect of OLP and RAPA on LDH release from HUVECs. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA CM group. (F) Indirect effect of OLP and RAPA on tube formation in HUVECs. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA CM group. (G) Indirect effect of OLP and RAPA on HUVEC migration. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA CM group. (H) Indirect effect of OLP and RAPA on angiogenic factor secretion in HUVECs. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA CM group. (I) Indirect effect of OLP and RAPA on angiogenesis-related gene expression in HUVECs. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA CM group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/27644c3b003700f3158b9557.png"},{"id":69937921,"identity":"e2d93f6b-87d0-48ea-b910-a13ae9084820","added_by":"auto","created_at":"2024-11-26 19:52:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2696979,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of RAPA and OLP inhibits migration and invasion of PC-3R cells \u003cem\u003ein vitro\u003c/em\u003e. (A) The combined effect of OLP and RAPA on epithelial cell marker gene expression in PC-3R cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (B) The combined effect of OLP and RAPA on epithelial cell protein expression in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (C) The combined effect of OLP and RAPA on the expression of pro-metastasis-related genes in PC-3R cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (D) The combined effect of OLP and RAPA on the expression of pro-metastasis-related protein in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (E) The combined effect of OLP and RAPA on anti-metastasis-related gene expression in PC-3R cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (F) The combined effect of OLP and RAPA on anti-metastasis-related protein expression in PC-3R cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/1c92c8f726e2a3f7182a9dc7.png"},{"id":69937922,"identity":"c42d399e-4d7e-4cf8-9412-37b8ce548b23","added_by":"auto","created_at":"2024-11-26 19:52:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3851279,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of RAPA and OLP inhibits the growth of PC-3R xenografts \u003cem\u003ein vivo\u003c/em\u003e. (A) PC-3R cells were seeded on day 0, with daily intratumoral injections of OLP and/or RAPA on days 6-8, and tumor volume was measured with a vernier caliper on days 5, 9, 12, 15 and 19. (B) Growth curve of PC-3R transplanted tumors. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (C) The combined effect of OLP and RAPA on the survival of tumor-bearing mice. (p=0.0005) significant difference comparable to the RAPA group (n = 12). (D) Ki-67 immunofluorescence staining to evaluate cell proliferation in PC-3R xenografts. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (E) The combined effect of OLP and RAPA on anti-apoptotic gene expression in PC-3R xenograft tumors. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (F) The combined effect of OLP and RAPA on anti-apoptotic protein expression in PC-3R xenograft tumors. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (G) The combined effect of OLP and RAPA on pro-apoptotic gene expression in PC-3R xenografts. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to RAPA group. (H) The combined effect of OLP and RAPA on pro-apoptotic protein expression in PC-3R xenografts. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/011b24e0707b20cdba3b6ce0.png"},{"id":69937669,"identity":"b9adb1f1-ad73-4c8d-84e3-f3908bef9303","added_by":"auto","created_at":"2024-11-26 19:44:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1529649,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of RAPA and OLP inhibits angiogenesis in PC-3R xenografts \u003cem\u003ein vivo\u003c/em\u003e. (A) The combined effect of OLP and RAPA on the expression of angiogenesis-related genes in PC-3R xenograft tumors. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (B) The combined effect of OLP and RAPA on the secretion of angiogenesis-related proteins in PC-3R xenograft tumors. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/ba64620f83d9fcc10fa84581.png"},{"id":69937923,"identity":"4c7d91d2-8457-4f20-b019-cc74754c9f66","added_by":"auto","created_at":"2024-11-26 19:52:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3417999,"visible":true,"origin":"","legend":"\u003cp\u003eRAPA combined with OLP promotes the differentiation of M2 macrophages into M1 \u003cem\u003ein vivo\u003c/em\u003e. (A and B) Real-time RT-PCR analysis of M1 and M2 macrophage-related gene expression in PC-3R cell xenograft section. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (C and D) Analysis of M1 and M2 macrophage-related protein expression by ELISA. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (E) Quantitative analysis of F-PC-3R cell xenograft section stained for CD80, CD86 and MHCII (red) at day 21. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (F) Quantitative analysis of F-PC-3R cell xenograft section stained for CD163, CD206 and SLAM (red) at day 21. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/a96f85027c414fe40b74989e.png"},{"id":69937673,"identity":"cba8919a-c519-42c6-90c0-e8fdc7fd88f9","added_by":"auto","created_at":"2024-11-26 19:44:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":320429,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of RAPA and OLP inhibits tumor angiogenesis indirectly through macrophages \u003cem\u003ein vitro\u003c/em\u003e. (A) Real-time RT-PCR analysis of M1 macrophage expression in THP-1 cells. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (B) Analysis of M1 macrophage-related protein expression in THP-1 cells by ELISA. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the RAPA group. (C) Schematic diagram of cell processing. (D) Real-time RT-PCR analysis of angiogenesis-related protein expression in HUVECs. Data are presented as mean ± SD (n = 6). *(P \u0026lt; 0.05) significant difference comparable to the OLP+RAPA group. (E) Analysis of angiogenesis expression in HUVECs. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the OLP+RAPA group. Statistical analysis was determined by one-way ANOVA, followed by Tukey's multiple comparison test analysis.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/8dd85e006eb5b2a5dae929b2.png"},{"id":69937668,"identity":"38fc4f8a-112e-475a-8b39-47a0ddc6c9a8","added_by":"auto","created_at":"2024-11-26 19:44:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4016455,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of RAPA and OLP affects the polarization and infiltration capacity of macrophages by regulating PC-3R cells \u003cem\u003ein vitro\u003c/em\u003e. (A and B) Real-time RT-PCR analysis of M1 and M2 macrophage expression in THP-1 cells. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the TGF-β+PC-3R+RAPA group. (C and D) Analysis of M1 and M2 macrophage-related protein expression by ELISA. Data are presented as mean ± SD (n = 5). *(P \u0026lt; 0.05) significant difference comparable to the TGF-β+PC-3R+RAPA group.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/a7bfe04858e6234026779cba.png"},{"id":77052737,"identity":"b0f725b4-18ea-4106-867d-050208069ab3","added_by":"auto","created_at":"2025-02-24 16:24:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":38236155,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/65d846c6-cf76-4e88-9e1a-7479af2fc5a9.pdf"},{"id":69937920,"identity":"2fe6d9da-ebaf-45b1-b477-17d66bb58b33","added_by":"auto","created_at":"2024-11-26 19:52:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":80593,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/6be1904a61298b4c84e7d0aa.docx"},{"id":69937662,"identity":"03f0c718-fcf1-4974-ac7f-afefc8b84a4c","added_by":"auto","created_at":"2024-11-26 19:44:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24081,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-5399970/v1/a85b6fd6f001977fbbc9082a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Olaparib reverses prostate cancer resistance to Rapamycin by promoting macrophage polarization towards the M1 phenotype","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer (PCa) is the most commonly diagnosed non-cutaneous malignant tumor and ranks as the second leading cause of cancer-related mortality in men globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite its widespread occurrence, current treatment outcomes often fail to meet expectations, prompting the need for more effective therapeutic strategies. While the mTOR inhibitor Rapamycin (RAPA) has shown efficacy in managing PCa, the development of treatment resistance frequently compromises its long-term effectiveness, leading to disease progression. Recent studies have shed light on the potential of the PARP inhibitor Olaparib (OLP) in overcoming drug resistance observed across various tumor types [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, this study aims to assess the efficacy of OLP in treating RAPA-resistant PCa, with a specific focus on elucidating its underlying molecular mechanisms.\u003c/p\u003e \u003cp\u003eTumor-associated macrophages (TAMs) have emerged as pivotal players in facilitating tumor drug resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Within the tumor microenvironment (TME), TAMs, among bone marrow-derived cells, are notably linked with the advancement and immune suppression observed in PCa [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is widely acknowledged that macrophages can infiltrate tumor tissues. In one of the earliest investigations concerning macrophage presence in PCa, a significant disparity was noted in macrophage density between benign areas adjacent to prostate tumors and the tumors themselves [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite the established increase in TAMs within prostate cancers, the precise mechanisms through which TAMs regulate tumor growth remain largely elusive.\u003c/p\u003e \u003cp\u003eTAMs serve as pivotal components within the tumor microenvironment (TME), exerting significant influence over tumor growth and disease progression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Depending on their polarization state, TAMs can either impede or bolster tumor growth, adopting either a classically-activated macrophage (M1) or alternatively-activated macrophage (M2) phenotype, respectively [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The polarization phenotype of TAMs has been associated with prognosis across various cancer types [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, a higher infiltrate of M1 macrophages correlates with a more favorable prognosis, whereas a higher M2 infiltrate is indicative of a poorer prognosis. M1 macrophages, characterized by their pro-inflammatory nature, counteract tumor growth through the activation of adaptive immune responses or direct tumor cell killing. Conversely, M2 macrophages, with their anti-inflammatory characteristics, sustain tumor cell growth by fostering angiogenesis, facilitating matrix remodeling, and promoting immune suppression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Unfortunately, the predominant phenotype among TAMs often leans towards the M2-like state, consequently supporting tumor growth rather than facilitating tumor elimination. Therefore, the reprogramming of TAMs within the TME presents a promising therapeutic avenue to enhance anti-tumor activity.\u003c/p\u003e \u003cp\u003eThe effectiveness of OLP in RAPA-resistant PCa, along with its regulatory impact on TAMs, was evaluated through a combination of real-time PCR, ELISA, immunohistochemistry, and fluorescence experiments. Our study revealed that the combination of OLP and RAPA effectively suppressed the proliferation, stemness, invasion, angiogenesis, apoptosis resistance, and anti-oxidative stress capacity of PC-3R cells. In addition, it proved the ability of OLP to regulate macrophage polarization and reverse the drug resistance of PCa to RAPA in tumor microenvironment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal and tumor cell lines\u003c/h2\u003e \u003cp\u003eSix-week-old male BALB/c nude mice were obtained from Charles River Laboratories. Human macrophage cell line (THP-1), human PCa cell line (PC-3) and human umbilical vein endothelial cells (HUVECs) were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin. F-THP-1 cells, F-PC-3 cells and F-HUVECs were transduced with a lentivirus encoding luciferase and green fluorescent protein (GFP). R-PC-3 was transduced with a lentivirus encoding luciferase and red fluorescent protein (RFP). The expression of GFP and RFP was monitored by using a flow cytometry analysis. All cells were incubated at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe construction of RAPA-resistant PCa cell line PC-3R\u003c/h3\u003e\n\u003cp\u003eFirst, we expose the parental PCa cell line (PC-3) to low concentrations of RAPA to initiate the resistance process. Then, we incrementally increase the concentration of RAPA over time, allowing the cells to adapt and develop resistance. Finally we isolate single-cell clones from the drug-exposed population and expand them.\u003c/p\u003e\n\u003ch3\u003eCell cytotoxicity assessment\u003c/h3\u003e\n\u003cp\u003eFor cell cytotoxicity assays, PC-3R cells were seeded in 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e per well, add different concentrations of RAPA and/or OLP for 24\u0026ndash;72 h treatment, and the cell growth rate was evaluated by CCK-8 kit (C0038, Beyotime) in terms of the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003e bioluminescence imaging\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cem\u003eIn vitro\u003c/em\u003e bioluminescence imaging\u003c/div\u003e \u003cp\u003eF-PC-3R cells (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e per well) or F-HUVECs (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e per well) were placed on 48-well plates. D-luciferin substrate (200\u0026micro;g/ml) was added to each well at room temperature. Cell bioluminescence signals were detected by the IVIS Spectrum Imaging System and analyzed by Carestream molecular imaging software (Caliper Life Sciences, Alameda, CA).\u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eThe tumor tissue homogenates were collected and the concentration of multiple cytokines was measured by corresponding ELISA kit in terms of the manufacturer's instruction. Briefly, the primary antibody (provided in kit) was coated and incubated for 2 h at room temperature. Samples were added and incubated for 1 h, following which wells were washed and a biotinylated antibody (provided in kit) was added for 1 h. The plates were washed again and streptavidin conjugated to horseradish peroxidase was added for 10 min at room temperature. Plates were washed and tetramethylbenzidine was added for color development about 30 min at room temperature and the reaction was terminated with 1M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Absorbance was measured at 450nm by using a microplate reader (Thermo Fisher Scientific, Inc.). Concentrations in the samples were calculated using a standard curve and values were expressed as pg/ml or ng/ml. All experiments were performed in terms of the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, RT-PCR and real-time RT-PCR\u003c/h2\u003e \u003cp\u003eIn terms of the manufacturer's guideline, total RNA was drawn from cell lines and tissues by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized by using TransScript\u0026reg; First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China). Reverse transcription polymerase chain reaction (RT-PCR) was conducted by using TransScript\u0026reg; Two-Step RT-PCR SuperMix (TransGen, Beijing, China) in line with the manufacturer's instructions. The PCR products were analyzed on 1% agarose gel and visualized. Real-time RT-PCR was performed using TransStart\u0026reg; Top Green qPCR SuperMix (TransGen, Beijing, China). The primers (GENEWIZ, Tianjin, China) were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLactate dehydrogenase (LDH) cytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eLDH assay was performed to analyze cytotoxicity activity of RAPA and/or OLP. PC-3R cells were prepared in 96-well plates. Supernatants were collected and used for the LDH assay measurement, according to the LDH cytotoxicity assay kit (Beyotime institute of Biotechnology) manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eCell apoptosis assay\u003c/h3\u003e\n\u003cp\u003eAnnexin V-FITC/PI staining method was used to distinguish early and late stages of apoptosis cells and necrosis cells. PC-3R cells were seeded in a 6-well plate, then cells were collected and resuspended to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ml in 1 \u0026times; binding buffer, subsequently cells were washed with PBS. Approximately 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells (100 \u0026micro;l) were aliquoted into a flow cytometry tube. Annexin V-FITC (5 \u0026micro;l) was added into the tube and incubated for 10 min, at room temperature (RT) and in darkness. Propidium iodide (5 \u0026micro;l) was then incubated with the cells for 5 min, at RT and in darkness. The mixed solution was added to 500 \u0026micro;l PBS and blended gently. The prepared cell samples were immediately tested by flow cytometer (FACS Calibur, BD, US).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Membrane Potential (MMP) Assay\u003c/h2\u003e \u003cp\u003eMMP assay was performed as described previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PC-3R cells in each group were inoculated on 6-well plates, incubated for 24 h, and added with JC-1 (Beyotime, Beijing, China) at room temperature to avoid light reaction for 40 min. The intensity of red and green fluorescence was measured and recorded by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of oxidative stress kit\u003c/h2\u003e \u003cp\u003e20 mg of tumor blocks were minced and homogenized in ice-cold RIPA buffer (Sigma, C0278). Homogenates were subject to centrifuge at 13000g for 15 min at 4\u0026deg;C to obtain the supernatant as sample tissue total protein preparation. The protein concentration was measured with a BCA (bicinchoninic acid) protein assay kit (Beyotime Biotechnology, Shanghai, China). MDA, OFR, 4-HNE, GSH, PHD, SOD and GSH-Px activities were measured using the corresponding kits (Jiancheng Bioengineering Institute, Nanjing, China). All experiments were performed in terms of the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed using the paraffin-embedded sections. Antibodies were used at a dilution factor of 1:100. Staining signals were visualized with the Alexa Fluor 633 or 488 conjugated secondary antibodies (1:200; Invitrogen, Shanghai, China). The sections were counter-stained with DAPI (Southern Biotech, England) and examined using a laser confocal scanning microscopy (Imager Z2, Zeiss, Germany). At least 10 different stained random fields were assessed for each marker by a researcher that was blinded to the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-3 activity detection\u003c/h2\u003e \u003cp\u003eThe activity of caspase-3 was measured using caspase-3 activity assay kit (Beyotime Institute of Biotechnology). Briefly, PC-3R cells were cultured for 24 h. After addition of RAPA and/or OLP, cells were incubated for another 12 h. The extraction procedure of caspase-3 was the same as the instructions. Caspase-3 substrate (provided in the kit) was changed into a yellow formazan product in the presence of caspase-3. Caspase-3 activity was measured at 405 nm on a microplate reader, and the activity ratio was calculated according to the kit instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTube formation experiment\u003c/h2\u003e \u003cp\u003eAdd 220 \u0026micro;l of matrigel to each well of a 24-well plate and incubate at 37\u0026deg;C for 30 min; add 6\u0026times;10\u003csup\u003e4\u003c/sup\u003e HUVECs and 600 \u0026micro;l of different groups of reagents (DMEM, PC-3R\u0026thinsp;+\u0026thinsp;RAPA, PC-3R\u0026thinsp;+\u0026thinsp;OLP or PC-3R\u0026thinsp;+\u0026thinsp;RAPA/OLP), placed in a cell incubator for 4 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eantitumor model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBALB/c nude mice (male, 6 weeks age) were purchased from Charles River Laboratories (Beijing, China). All animal protocols were approved by the Institutional Animal Care and Use Committee. An \u003cem\u003ein vivo\u003c/em\u003e subcutaneous tumor xenograft mouse model was established. Briefly, F-PC-3R cell suspension (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells) were injected into the right flank of mice. When tumors reached\u0026thinsp;~\u0026thinsp;50 mm\u003csup\u003e3\u003c/sup\u003e after approximately one week, mice were subsequently randomly divided into the following four treatment group (PBS, RAPA, OLP and RAPA/OLP). Treatments were injected at the tumor site. The antitumor effect was quantified according to tumor weight and volume ((L\u0026times;W\u003csup\u003e2\u003c/sup\u003e)/2).\u003c/p\u003e \u003cp\u003eWhen the tumor grew to 1000mm\u003csup\u003e3\u003c/sup\u003e, mice were anesthetized by inhaling isoflurane through the nose cone. For euthanasia, the cervical dislocation was employed. All procedures involving anesthesia and euthanasia were carried out by trained personnel under the supervision of a veterinarian to ensure the utmost care and compliance with animal welfare regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatement of ethics\u003c/h2\u003e \u003cp\u003e All animal experiments were performed in accordance with ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals (National Research Council, 8th edition, 2011) and approved by the Institutional Animal Care And Use Committee (IACUC) of Tianjin Second People's Hospital under Assurance Number LL-BG-032.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were determined by one-way ANOVA, followed by Tukey's multiple comparison test analysis. All statistical analyses were conducted using GraphPad Prism 7 (GraphPad).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe optimum concentration of PC-3R cells to OLP and RAPA\u003c/h2\u003e \u003cp\u003eIn this study, we employed the CCK-8 assay to investigate the cytotoxic effects of OLP on PC-3R cells, as well as to determine the tolerance threshold of these cells to RAPA. Treatment with low concentrations (0.1, 0.2, and 0.5 \u0026micro;M) of OLP resulted in only a slight decrease in the viability of PC-3R cells. However, higher concentrations (1, 2, and 5 \u0026micro;M) of OLP demonstrated significant inhibition of PC-3R cell growth at 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), as well as at 48 and 72 hours (Supplementary Fig.\u0026nbsp;1A). Importantly, all concentrations of RAPA showed no significant inhibition of growth in PC-3R cells at 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), as well as at 48 and 72 hours (Supplementary Fig.\u0026nbsp;1B). Based on the above-mentioned findings, RAPA or OLP at the concentration of 1 \u0026micro;M was used for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eOLP augments the inhibitory efficacy of RAPA on the proliferation and stemness of PC-3R cells\u003c/h2\u003e \u003cp\u003eBioluminescent imaging unveiled a notably reduced proliferation of PC-3R cells upon treatment with the combination of RAPA and OLP, in comparison to the RAPA monotherapy group. This suggests that OLP enhances the sensitivity of RAPA in PC-3R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, we conducted real-time PCR assay and observed that the combination of RAPA and OLP led to a reduction in the expression of pro-proliferative genes, including \u003cem\u003eIgf1\u003c/em\u003e, \u003cem\u003eHgf\u003c/em\u003e, \u003cem\u003eEgf\u003c/em\u003e, \u003cem\u003eIl6\u003c/em\u003e, \u003cem\u003eIl33\u003c/em\u003e, and \u003cem\u003eCsf3\u003c/em\u003e in PC-3R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Among these genes,the most significant down-regulation was observed in the expression of the \u003cem\u003eCsf3\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In parallel, the secretion of proliferation-related proteins, such as insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), epidermal growth factor (EGF), IL-6, IL-33 and granulocyte colony-stimulating factor (G-CSF), in PC-3R cells were down-regulated in combined treatment group compared to the monotherapy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Among these proteins, the most significant down-regulation was observed in the expression of G-CSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results indicate that the inhibition of PC-3R cell proliferation by the combination of RAPA and OLP is primarily mediated through the suppression of G-CSF expression.\u003c/p\u003e \u003cp\u003eMoreover, treatment with RAPA combined with OLP resulted in decreased expression levels of pluripotency-related genes, including \u003cem\u003eAldh1a1\u003c/em\u003e, \u003cem\u003eNanog\u003c/em\u003e and \u003cem\u003eLin28a\u003c/em\u003e in PC-3R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Among these genes, the most significant down-regulation was observed in the expression of \u003cem\u003eLin28a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Consistent findings were obtained at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). These results suggest that the combination of RAPA and OLP could attenuate the stemness of PC-3R cells primarily by suppressing the expression of LIN28A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eOLP augments the promoting efficacy of RAPA on oxidative stress and apoptosis of PC-3R cells\u003c/h2\u003e \u003cp\u003eSubsequently, we conducted \u003cem\u003ein vitro\u003c/em\u003e assays to investigate whether OLP can potentiate the apoptosis induced by RAPA in PC-3R cells. The combination of RAPA and OLP triggered the release of lactate dehydrogenase (LDH, a necrotic marker) in PC-3R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating strong cytotoxicity against RAPA-resistant PCa cells. This observation was supported by annexin V/PI assay, which demonstrated a higher percentage of apoptotic cells in the combination therapy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Activation of apoptosis frequently involves mitochondrial deregulation and oxidative stress [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our data revealed that the combined treatment with RAPA and OLP resulted in mitochondrial damage, as evidenced by an increase in collapsed mitochondrial membrane potential in PC-3R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In addition, we observed an elevation in oxygen free radical (OFR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and in lipid peroxidation markers, including 4-hydroxynonenal (4-HNE) and malonaldehyde (MDA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) in PC-3R cells treated with RAPA and OLP. This suggests the potential involvement of oxidative stress in initiating apoptosis. Importantly, these changes occurred concurrently with a decrease in intracellular levels of antioxidants, such as superoxide dismutase (SOD), glutathione (GSH), and the activity of glutathione peroxidase (GSH-Px) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Consistently, PC-3R cells treated with RAPA and OLP exhibited increased mRNA expression of pro-oxidant genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) and reduced expression of anti-oxidative genes, as revealed by real-time RT-PCR (Supplementary Fig.\u0026nbsp;2). We also observed that the combined treatment with RAPA and OLP significantly upregulated the expression of p53, a tumor suppressor protein known to positively regulate apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). In addition, PC-3R cells subjected to the combined treatment of RAPA and OLP exhibited increased expression and activity of caspase-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI and J), indicating the involvement of the intrinsic apoptotic pathway. Supporting this observation is the modulation of a broad spectrum of apoptotic genes, including cytochrome c (\u003cem\u003eCycs\u003c/em\u003e), \u003cem\u003eBax\u003c/em\u003e, \u003cem\u003eCasp3\u003c/em\u003e, \u003cem\u003eCasp9\u003c/em\u003e and poly (ADP-ribose) polymerase 1 (\u003cem\u003eParp1\u003c/em\u003e) in the combined treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Importantly, the expression of \u003cem\u003eBax\u003c/em\u003e gene was significantly down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). The same conclusion is confirmed at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). Taken together, these findings suggest that the combination of RAPA and OLP can induce PC-3R cell apoptosis via activation of oxidative stress and a mitochondrial-mediated pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eThe combination of OLP and RAPA indirectly inhibits angiogenesis by modulating PCa cells \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eNext, we aim to explore whether OLP can enhance the inhibitory effect of RAPA on angiogenesis. Our results showed that the combination of OLP and RAPA significantly down-regulated the expression of a series of angiogenesis-related genes compared with the RAPA group in HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This was accompanied by enhanced PHD activity in PC-3R cells exposed to OLP and RAPA (Supplementary Fig.\u0026nbsp;3). Among them, \u003cem\u003eVegfa\u003c/em\u003e and \u003cem\u003eAngpt1\u003c/em\u003e genes were down-regulated most significantly. The same conclusion is confirmed at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To further elucidate the mechanism underlying the anti-angiogenic property of the combined treatment, conditioned medium (CM) of PC-3R cells was harvested and used to treat HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). CM derived from RAPA-treated PC-3R cells had a weak impact on the viability and function of HUVECs. In contrast, CM from the combined treatment group markedly reduced cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), increased LDH release (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), impaired tubule formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and inhibited migratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) of HUVECs. In addition, exposure to CM derived from the combined treatment group significantly reduced the expression of VEGFA and Ang-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) and suppressed mRNA expression of \u003cem\u003eVegfa\u003c/em\u003e and \u003cem\u003eAngpt1\u003c/em\u003e in HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Thus, our data indicate that OLP can indirectly inhibit tumor angiogenesis mainly by inhibiting the expression of VEGFA and Ang-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOLP augments the inhibitory efficacy of RAPA on the mesenchymal transition (EMT) and migratory capacity of PC-3R cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEpithelial to mesenchymal transition (EMT) is a critical process involving phenotypic transition that confers invasive and migratory properties to PCa cells, ultimately leading to metastasis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To investigate whether the combination of RAPA and OLP could reverse the metastatic phenotype of PC-3R cells, we examined the expression profiles of EMT-related genes in PC-3R cells. Our results demonstrated that RAPA combined with OLP significantly decreased the mRNA expression of a wide range of mesenchymal markers (e.g. \u003cem\u003eVim\u003c/em\u003e and \u003cem\u003eCdh2\u003c/em\u003e) and transcription factors (e.g. \u003cem\u003eSnail1/2\u003c/em\u003e and \u003cem\u003eTwist1\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;4), while restoring the expression of epithelial markers such as \u003cem\u003eCdh1\u003c/em\u003e, \u003cem\u003eTjp1\u003c/em\u003e and \u003cem\u003eClaudin\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) in PC-3R cells. Subsequently, these findings were further validated by ELISA experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is well established that matrix metalloproteinases (MMPs) promote tumor invasion and metastasis by degrading the extracellular matrix, and this effect can be inhibited by tissue inhibitors of metalloproteinases (TIMPs) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Thus, we next investigated whether the combination of RAPA and OLP could suppress the invasion potential of PC-3R cells by regulating the expression of MMPs and TIMPs. We observed no change in mRNA expression of \u003cem\u003eMmp9\u003c/em\u003e, \u003cem\u003eTimp1\u003c/em\u003e, \u003cem\u003eTimp4\u003c/em\u003e, or \u003cem\u003eKiss1\u003c/em\u003e in PC-3R cells due to OLP and RAPA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and E). Most importantly, the expression of \u003cem\u003eMmp2\u003c/em\u003e was significantly down-regulated and \u003cem\u003eTimp3\u003c/em\u003e was markedly up-regulated after combined treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and E). The conclusions were further verified by ELISA experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and F). The above results indicate that RAPA combined with OLP could inhibit the migration and invasion of PC-3R cells mainly by regulating the expression of MMP2 and TIMP3.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eThe combination of RAPA and OLP inhibit growth of PCa via promoting apoptosis\u003c/h2\u003e \u003cp\u003eWe subsequently evaluated whether the combination of RAPA and OLP could effectively hinder tumor growth in a xenograft model. PC-3R cells expressing Fluc-GFP were implanted subcutaneously into the flanks of nude mice, which were then intratumorally injected with either saline, OLP, RAPA or a combination of OLP\u0026thinsp;+\u0026thinsp;RAPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Our findings indicated that RAPA alone exerted limited inhibitory effects on tumor growth, resulting in only a slight delay in tumor progression. However, the combination of RAPA and OLP induced a remarkable degree of tumor regression in all mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Furthermore, local administration of OLP and RAPA significantly enhanced the overall survival of xenograft mice compared to the OLP or RAPA groups alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to evaluate the inhibitory effect of the combination of RAPA and OLP on tumor growth, we conducted immunofluorescence experiments on tumor samples. Our results revealed a significant reduction in the number of ki-67\u0026thinsp;+\u0026thinsp;proliferating cells in the group treated with both OLP and RAPA compared to those treated with OLP or RAPA alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Additionally, real-time RT-PCR analysis demonstrated that the combined treatment downregulated the expression of several anti-apoptosis genes, including \u003cem\u003eBcl2\u003c/em\u003e, \u003cem\u003eBcl2l1\u003c/em\u003e, \u003cem\u003eBirc5\u003c/em\u003e, \u003cem\u003eAkt1\u003c/em\u003e and \u003cem\u003ePik3ca\u003c/em\u003e, among which \u003cem\u003eBcl2\u003c/em\u003e gene is the most significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Consistent with these findings, ELISA experiments confirmed the downregulation of anti-apoptosis protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These observations suggest that OLP primarily enhances the apoptotic effect of RAPA by potentiating its inhibition of \u003cem\u003eBcl2\u003c/em\u003e gene expression. Furthermore, we found that the combined treatment upregulated the expression of several pro-apoptotic genes, such as \u003cem\u003eBax\u003c/em\u003e, \u003cem\u003eBad\u003c/em\u003e, \u003cem\u003eBcl2l11\u003c/em\u003e, \u003cem\u003eCycs\u003c/em\u003e, \u003cem\u003eCasp3\u003c/em\u003e, \u003cem\u003eCasp9\u003c/em\u003e, and \u003cem\u003eParp1\u003c/em\u003e, with \u003cem\u003eBax\u003c/em\u003e being the most significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Notably, the expression trends of these proteins were similar to those of their respective mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Collectively, these findings indicate that OLP accelerates the apoptosis of PC-3R cells primarily by potentiating RAPA's promotion of Bax gene expression.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eThe combination of RAPA and OLP inhibit tumor growth by blocking angiogenesis\u003c/h2\u003e \u003cp\u003eTo assess whether OLP potentiates the anti-angiogenic effects of RAPA in PC-3R xenografts, we first performed real-time PCR analysis on tumor samples. Our findings revealed that the combined treatment of RAPA and OLP downregulated the expression of multiple pro-angiogenic genes, including \u003cem\u003eVegfa\u003c/em\u003e, \u003cem\u003eKdr\u003c/em\u003e, \u003cem\u003eFgf2\u003c/em\u003e, \u003cem\u003ePigf\u003c/em\u003e, \u003cem\u003ePdgfa\u003c/em\u003e, \u003cem\u003eHif1a\u003c/em\u003e, \u003cem\u003eCxcl12\u003c/em\u003e, \u003cem\u003eAngpt1\u003c/em\u003e, \u003cem\u003eAngpt2\u003c/em\u003e and \u003cem\u003eEdn1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Consistent with these observations, ELISA results demonstrated a significant downregulation of pro-angiogenic protein expression, particularly VEGFA and Ang-1, in PC-3R xenografts treated with the combination of RAPA and OLP. Collectively, these results suggest that the anti-angiogenic activity of RAPA and OLP is primarily achieved by suppressing the expression of VEGFA and Ang-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe combination of RAPA and OLP promote the differentiation of M2 macrophages into M1\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIt is well-established that macrophages can promote advanced tumor metastasis by releasing cytokines [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, to investigate the impact of RAPA combined with OLP on macrophage polarization, we evaluated the expression patterns of 17 genes related to M1 and M2 macrophages in PC-3R transplanted tumor following intratumoral injection of OLP and RAPA. Among these genes, six M1 macrophage gene expressions (\u003cem\u003eIl12a\u003c/em\u003e, \u003cem\u003eIl6\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eTnfa\u003c/em\u003e, \u003cem\u003eNos2\u003c/em\u003e and \u003cem\u003eCcl2\u003c/em\u003e) were prominently augmented (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, the gene expressions of several M2 macrophages (\u003cem\u003eIl10\u003c/em\u003e, \u003cem\u003eVegfa\u003c/em\u003e, \u003cem\u003eEgf\u003c/em\u003e, \u003cem\u003eArg1\u003c/em\u003e, \u003cem\u003eMrc1\u003c/em\u003e and \u003cem\u003eTgfb1\u003c/em\u003e) were significantly inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Notably, there were no significant differences in the expression of other genes (M1: \u003cem\u003eIl23a\u003c/em\u003e and \u003cem\u003eClec4e\u003c/em\u003e; M2: \u003cem\u003eRetnlb\u003c/em\u003e, \u003cem\u003eYm1\u003c/em\u003e and \u003cem\u003ePdgfa\u003c/em\u003e) before and after treatment with OLP and RAPA (Supplementary Fig.\u0026nbsp;5), indicating that the combination of RAPA and OLP does not regulate macrophage infiltration and polarization through these genes \u003cem\u003ein vivo\u003c/em\u003e. The ELISA assay demonstrated that the combination of RAPA and OLP enhances the secretion of cytokines by M1 macrophages and suppresses the secretion of cytokines by M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and D). Among the six cytokines influenced in the M2 phenotype, IL-10 and TGF-β play a crucial role in regulating the function of TAMs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, we evaluated the surface markers of M1 and M2 macrophages. The results revealed that the combined treatment of RAPA and OLP not only up-regulates the protein expression on the surface of anti-tumor M1 macrophages, but also downregulates the protein expression on the surface of tumor-promoting M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F). These findings suggest that the combination of RAPA and OLP effectively polarizes macrophages in PC-3R transplanted tumors towards the M1 phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eThe combination of RAPA and OLP inhibit tumor angiogenesis indirectly through macrophages \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eNumerous studies have demonstrated that M1 macrophages could inhibit angiogenesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To investigate the direct impact of the combination of RAPA and OLP on M1 phenotype gene expression profiles, we analyzed THP-1 cells. Our results demonstrated that the combined treatment significantly upregulated the gene expressions of \u003cem\u003eIl12a\u003c/em\u003e, \u003cem\u003eIl6\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eTnfa\u003c/em\u003e, \u003cem\u003eNos2\u003c/em\u003e and \u003cem\u003eCcl2\u003c/em\u003e compared to the RAPA group alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Notably, there were no significant differences in the expression of \u003cem\u003eIl23a\u003c/em\u003e and \u003cem\u003eClec4e\u003c/em\u003e genes (Supplementary Fig.\u0026nbsp;6), indicating that the combination of RAPA and OLP does not stimulate M1 macrophages to produce cytokines encoded by these two genes. To further validate the findings from RT-PCR, we conducted ELISA assays to assess the expression levels of M1 markers in the combined treatment group. These assays confirmed the upregulation of IL-12α, IL-6, IL-1β, TNF-α, iNOS, and CCL2, thereby reinforcing the conclusion that the combination of RAPA and OLP directly promotes M1 macrophage expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere have already been several reports indicating that TAMs could vastly promote tumor angiogenesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To investigate the impact of RAPA combined with OLP on tumor angiogenesis by modulating macrophage \u003cem\u003ein vitro\u003c/em\u003e, conditioned medium (CM) of THP-1 cells was harvested and used to treat HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). RT-qPCR was employed to measure the gene expression of angiogenesis markers (\u003cem\u003eEdn1\u003c/em\u003e, \u003cem\u003eAngpt1\u003c/em\u003e, \u003cem\u003eVegfa\u003c/em\u003e, \u003cem\u003ePdgfb\u003c/em\u003e, \u003cem\u003eFgf2\u003c/em\u003e, \u003cem\u003eEgln1\u003c/em\u003e and \u003cem\u003eHif1a\u003c/em\u003e) in HUVECs. The results demonstrated a significantly reduced expression of these markers in the MΦ\u0026thinsp;+\u0026thinsp;OLP\u0026thinsp;+\u0026thinsp;RAPA group compared to the OLP\u0026thinsp;+\u0026thinsp;RAPA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). The protein expression trend was consistent with the mRNA expression, indicating that the combination of RAPA and OLP indirectly suppresses angiogenesis by regulating macrophage secretion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eThe combination of RAPA and OLP affects macrophage polarization by regulating PC-3R cells \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eRecent publications have unequivocally demonstrated the promising therapeutic potential of targeting TAMs for cancer immunotherapy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, to ascertain whether the regulation of PC-3R cells by OLP and RAPA can induce the transformation of M2 macrophages into M1 macrophages \u003cem\u003ein vitro\u003c/em\u003e, we employed real-time PCR to assess the effect of the culture medium after treatment with RAPA combined with OLP on the polarization of TGF-β-treated M2 macrophages following PC-3R cell treatment. The findings revealed a significant upregulation of M1 macrophage markers in the TGF-β\u0026thinsp;+\u0026thinsp;PC-3R\u0026thinsp;+\u0026thinsp;OLP\u0026thinsp;+\u0026thinsp;RAPA group compared to the TGF-β\u0026thinsp;+\u0026thinsp;PC-3R\u0026thinsp;+\u0026thinsp;RAPA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA), while the expression levels of M2 phenotype markers were notably downregulated in the TGF-β\u0026thinsp;+\u0026thinsp;PC-3R\u0026thinsp;+\u0026thinsp;OLP\u0026thinsp;+\u0026thinsp;RAPA group compared to the TGF-β\u0026thinsp;+\u0026thinsp;PC-3R\u0026thinsp;+\u0026thinsp;RAPA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). These results suggest that the treatment of PC-3R cells with OLP and RAPA could more effectively promote the polarization of M2 macrophages towards the M1 phenotype compared to RAPA alone. Additionally, we assessed the expression of M1 and M2-related proteins in macrophages treated with OLP and RAPA using ELISA, yielding consistent conclusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eProstate cancer (PCa) remains a significant health concern worldwide due to its high incidence and mortality rates. Despite advancements in treatment modalities, including surgical intervention, radiation therapy, and androgen deprivation therapy, the management of PCa remains challenging, especially in cases where resistance to conventional therapies develops. In this context, the exploration of alternative therapeutic strategies is imperative to improve patient outcomes and reduce mortality rates.\u003c/p\u003e \u003cp\u003eThe present study aimed to investigate the efficacy of the PARP inhibitor Olaparib (OLP) in treating RAPA-resistant PCa, with a specific focus on understanding its underlying molecular mechanisms. Our findings demonstrate that OLP effectively suppresses the proliferation, stemness, invasion, angiogenesis, apoptosis resistance, and anti-oxidative stress capacity of PC-3R cells. These results suggest that OLP holds promise as a potential therapeutic agent for overcoming drug resistance and inhibiting tumor progression in PCa patients who have failed RAPA treatment.\u003c/p\u003e \u003cp\u003eOne of the key findings of this study is the ability of OLP to regulate macrophage polarization within the tumor microenvironment. Tumor-associated macrophages (TAMs) play a crucial role in tumor progression and therapeutic resistance by promoting angiogenesis, immunosuppression, and tissue remodeling. Our results indicate that OLP treatment alters the phenotype of TAMs, potentially shifting them from a pro-tumorigenic M2 phenotype to an anti-tumorigenic M1 phenotype. This modulation of TAM polarization by OLP could contribute to its anti-tumor effects and reversal of drug resistance in PCa.\u003c/p\u003e \u003cp\u003eThe mechanisms underlying the efficacy of OLP in RAPA-resistant PCa are likely multifaceted. Previous studies have implicated the role of PARP inhibitors in inducing synthetic lethality in tumors with homologous recombination deficiency (HRD), leading to enhanced sensitivity to DNA damage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Given the inherent genomic instability observed in PCa, particularly in advanced stages, OLP may exploit this vulnerability to induce tumor cell death. Moreover, OLP has been shown to inhibit the repair of DNA single-strand breaks (SSBs) and promote the accumulation of DNA double-strand breaks (DSBs), further sensitizing cancer cells to cytotoxic insults [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, the crosstalk between cancer cells and the tumor microenvironment (TME) is increasingly recognized as a determinant of therapeutic response and resistance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this context, our findings suggest that OLP-mediated modulation of TAMs may contribute to the restoration of sensitivity to RAPA in PC-3R cells. By reprogramming the immunosuppressive TME towards an anti-tumorigenic state, OLP may enhance the efficacy of RAPA and other anti-cancer therapies.\u003c/p\u003e \u003cp\u003eHowever, it is essential to acknowledge the limitations of our study. While our findings provide compelling evidence for the potential therapeutic utility of OLP in RAPA-resistant PCa, further preclinical and clinical studies are warranted to validate these findings in a broader patient population. Additionally, the precise mechanisms underlying the crosstalk between OLP-treated cancer cells and TAMs require further elucidation. Future research efforts should focus on deciphering the signaling pathways involved in this interaction to identify novel therapeutic targets and optimize treatment strategies for PCa patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study highlights the therapeutic potential of OLP in overcoming drug resistance and inhibiting tumor progression in RAPA-resistant PCa. By elucidating the molecular mechanisms underlying its efficacy, we provide a rationale for the clinical evaluation of OLP as a novel therapeutic approach for PCa patients who have failed conventional treatments. Moreover, our findings underscore the importance of considering the dynamic interplay between cancer cells and the TME in the development of effective anti-cancer therapies. Ultimately, the translation of these research findings into clinical practice holds the promise of improving outcomes and quality of life for PCa patients.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are particularly grateful to all the people who have given us help on our article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science and Technology Major Project (No.2023ZD0508702), Natural Science Foundation of Tianjin (23JCYBJC00950), Tianjin Health Science and Technology Project key discipline special (TJWJ2022XK034), Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-059B), Research project in key areas of TCM in 2024 (2024022).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKai Ye and Gang Shi have contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuqiang Mi and Rui Su supervised the project; Kai Ye and Gang Shi drafted the manuscript; Kai Ye, Gang Shi, Kunyan Qiao, Jian Xu, Qinghai Dai, Zhixiao Huo, Yu Cao, Wei Liu, Yue Hu and Lihua Yan performed the experiments; Kai Ye and Gang Shi performed the statistical analyses; Ping Li, Liang Xu, Yu Zhu, Rui Su, Yuqiang Mi revised the article. All authors read and approved the final article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Yuqiang Mi, Liang Xu, Rui Su or Ping Li.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eInformed consent:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHe J, Chen WQ, Li N, Cao W, Ye DW, Ma JH, Xing NZ, Peng J and Tian JH (2022) [China guideline for the screening and early detection of prostate cancer (2022, Beijing)]. 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Expert Opin Ther Targets 27:447-457. doi: 10.1080/14728222.2023.2230362\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":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Prostate cancer, Rapamycin, Olaparib, Tumor microenvironment, Macrophage polarization","lastPublishedDoi":"10.21203/rs.3.rs-5399970/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5399970/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProstate cancer (PCa) is the most common non-cutaneous malignancy and the second leading cause of cancer-related death in men. Despite its prevalence, treatment outcomes are often unsatisfactory, necessitating the search for more effective therapeutic approaches. mTOR inhibitor Rapamycin (RAPA) has shown promise in managing PCa, but the emergence of resistance often undermines its long-term effectiveness. Recent studies suggest that PARP inhibitor Olaparib (OLP) may overcome drug resistance in various tumor types. This study aims to assess the efficacy of OLP in treating RAPA-resistant PCa, with a specific focus on elucidating its underlying molecular mechanisms. This study utilized drug exposure and concentration escalation experiments to establish human RAPA-resistant PCa cell line (PC-3R) based on the human PCa cell line (PC-3). PC-3R cell lines were screened through a cloning assay. The efficacy of OLP in RAPA-resistant PCa, as well as its regulatory impact on tumor-associated macrophages (TAMs), was evaluated through a combination of real-time PCR, ELISA, immunohistochemistry, and fluorescence experiments. This study unveiled that the combination of OLP and RAPA effectively suppressed the proliferation, stemness, invasion, angiogenesis, apoptosis resistance, and anti-oxidative stress capacity of RAPA-resistant PCa. Additionally, it demonstrated the capacity of OLP to regulate macrophage polarization within the tumor microenvironment and reverse drug resistance to RAPA in PCa. The findings of this study lay a theoretical foundation for the potential utilization of OLP in the treatment of RAPA-resistant PCa, offering substantial academic significance and promising application prospects.\u003c/p\u003e","manuscriptTitle":"Olaparib reverses prostate cancer resistance to Rapamycin by promoting macrophage polarization towards the M1 phenotype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 19:44:11","doi":"10.21203/rs.3.rs-5399970/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-19T10:18:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-17T18:14:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141047250306161982205796353173062500185","date":"2024-12-24T15:42:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-23T18:05:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-23T18:02:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-07T15:10:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2024-11-06T06:17:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"efa9516d-cf36-40f5-92b0-f52d936dc3d5","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:05:11+00:00","versionOfRecord":{"articleIdentity":"rs-5399970","link":"https://doi.org/10.1007/s11010-025-05231-0","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2025-02-21 15:57:13","publishedOnDateReadable":"February 21st, 2025"},"versionCreatedAt":"2024-11-26 19:44:11","video":"","vorDoi":"10.1007/s11010-025-05231-0","vorDoiUrl":"https://doi.org/10.1007/s11010-025-05231-0","workflowStages":[]},"version":"v1","identity":"rs-5399970","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5399970","identity":"rs-5399970","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}