Myeloid PTEN loss affects therapeutic response by promoting stress granules assembly and impairing phagocytosis of macrophages in breast cancer

doi:10.21203/rs.3.rs-3616918/v2

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Myeloid PTEN loss affects therapeutic response by promoting stress granules assembly and impairing phagocytosis of macrophages in breast cancer

https://doi.org/10.21203/rs.3.rs-3616918/v2

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published 30 Jul, 2024

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Breast cancer (BRCA) has become the most common type of cancer in women. The improvement of therapeutic response remains a challenge. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a classic tumor suppressor with emerging new functions discovered in recent years, and myeloid PTEN loss has been reported to impair the anti-tumor immunity. In this study, we revealed a novel mechanism of myeloid PTEN potentially affecting anti-tumor immunity in BRCA. We identified accelerated stress granules (SGs) assembly under oxidative stress in PTEN deficient bone marrow-derived macrophages (BMDMs), due to the upregulation of EGR1 transcripting TIAL1. The activation of PI3K/AKT/mTOR (PAM) pathway also contributed to the promoted SGs formation. ATP consumption during SGs assembly in BMDMs impaired the phagocytosis of 4T1 cells, which might contribute to the damage of anti-tumor immunity. In BRCA neoadjuvant cohort, we observed poorer response in myeloid PTEN^lowpatients with G3BP1 aggregating as SGs in CD68+ cells, which was consistent with the mechanism that PTEN deficient macrophages tend to have easier SGs assembly with impaired phagocytosis in our study. Our results revealed an unconventional impact of SGs on BMDMs and might provided new perspectives for drug resistance and therapeutic strategies for BRCA patients.

Biological sciences/Cancer/Breast cancer

Biological sciences/Cell biology/Cytoskeleton/Actin

Stress granules

Breast cancer

Macrophages

PTEN

TIAL1

PAM pathway

Phagocytosis

Tumor microenvironment

As the most prevalent type of tumor worldwide^[1], BRCA is noted for the heterogeneity of the disease, especially the complexity of tumor microenvironment (TME)^[2]. Numerous patients suffer from poor responses and resistance to anti-tumor treatments even with the increasing refined oncology development. The integrated stress response (ISR)^{[3, 4]} is an evolutionarily conserved mechanism for cells to adapt to TME with comprehensive stresses including hypoxia, ROS, DNA damages and inflammation, among which oxidative stress is considered the leading stress generated by tumor cells, immune cells and even exogenous therapeutic drugs^[5-8]. As a crucial member of TME, macrophages is vital for the establishment of pre-ecological environment for the occurrence and metastasis of BRCA especially when they are in dysfunction, which is closely related to the responses to nutrient/oxygen deprivation or therapeutic drugs^{[8, 9]}. PTEN is a canonical gene with important regulatory functions both in tumors and immune cells. Studies found that myeloid PTEN loss inhibits anti-tumor immunity in breast cancer patients, revealing new functions of PTEN in macrophages and TME^[10].

Similar to tumor cells, macrophages assemble SGs to protect their survival when facing stresses^[11-15]. As a kind of non-membrane cell organelle and product of liquid-liquid phase separation (LLPS), SGs protect tumor cells from death which is considered a strategy for tumor resistance^[16-18]; however, the role and regulatory mechanism of SGs in immune cells remain unclear and require more researches.

In the tissue sections of BRCA cohort who received neoadjuvant treatment, we identified SGs in macrophages from the patients with low myeloid PTEN expression who were also relatively insensitive to treatment. By inducing SGs in BMDMs, we observed impaired phagocytosis against 4T1 cells accompanied with remarkable ATP consumption in macrophages. What’s more, PTEN-deficient macrophages were more likely to form SGs due to the upregulation of nucleating protein TIAL1 and the activation of the PAM pathway, and compensation of ATP or inhibition of PAM could reverse the damage to BMDM phagocytosis after SGs assembly. Our study explored the impact of SGs on the anti-tumor immunity of BMDMs and revealed an unexpected role of PTEN played in TME, thus providing new outlook for BRCA treatment, especially for patients with myeloid PTEN dysfunction.

Myeloid PTEN is related to SGs in macrophages and anti-tumor immune response in BRCA patients

PTEN inactivation is the most common type of tumor suppressor dysfunction in human cancers, with an approximate 40% rate among BRCA patients, especially in triple-negative breast cancer(TNBC)^[19]. PTEN status not only affects the behavior of cancer cells but also influences the microenvironment directly or indirectly by regulating immune responses^{[20, 21]}. PTEN dysfunction in different immune cells would shift the global balance of TME towards immunosuppression^{[22, 23]}.

Although been regarded as a tumor suppressor, high PTEN expression in general tissues didn’t represent a destined better survival among BRCA patients (Fig.1A, sFig. 1A &1B), which could be indicated by our analysis by collecting patients data from the TCGA-BRCA database. Then we measured PTEN expression in tumor cells and stromal cells separately and explored whether there’re distinctive links between PTEN level and therapeutic outcomes. We collected clinical data and pathological sections from BRCA patients cohort who received neoadjuvant chemotherapy (anthracycline and taxane based regime) in our hospital, and evaluated treatment efficacy by Miller-Payne (MP) scoring system^[24] (sTab. 1). Patients with 1~2 MP scores were defined as therapeutic resistance, whereas those scored 3~5 were defined as therapeutic sensitive. By immunohistochemical (IHC) staining and MOD measurement, we didn’t see explicit correlation between PTEN expression in tumor cells and MP score (Fig. 1B). However, we found that PTEN expression in CD68-positive cells were lower in patients with drug resistance, and patients who had higher myeloid PTEN expression tended to be more sensitive to the treatment, showing a significant correlation between myeloid PTEN expression and MP scores (Fig. 1C&D). Our previous work revealed a part of the mechanisms that myeloid PTEN could promote anti-tumor immunity by accelerating NLRP3 inflammasome activation^[10], while it was also noted that inflammasomes and stress granules were connected as diverging paths of cell fate under stress^[15]. So we asked whether stress granules were also involved in TME regulation by myeloid PTEN status.

Cells assemble SGs for self-protection under stress, among which oxidative stress leads in the TME. So we evaluated whether response to oxidative stress affects clinical outcomes firstly. Based on the patient data from the TCGA-BRCA database, we obtained the genesets related to positive or negative regulation of response to oxidative stress (GO:1902884 and GO:1902883) from the GO knowledgebase for enrichment analysis, and then classified the patients into high-ES and low-ES groups according to the ES cutoff value. The results indicated that samples in positive_regulation_high group had worse prognosis (sFig.1C), referring to a negative correlation between genes with positive regulation of response to oxidative stress and the prognosis of BRCA patients. The negative_regulation_of_response_to_oxidative_stress geneset showed no significant difference in OS data (sFig.1D). To explore our hypothesis, we attempted to identify the clinical evidence of SGs in macrophages from BRCA patients and explore the relevance between SGs and clinical outcomes of BRCA. By immunofluorescence staining of G3BP1 of the pathological sections from the BRCA cohort, we found G3BP1 distributed as rough specks and even droplets aggregates in CD68-positive cells in several myeloid PTEN^low samples, representing the formation of stress granules. In myeloid PTEN^high samples, G3BP1 staining presented fine sand-like distribution in cytoplasm with no droplet aggregates (Fig. 1E). It indicated that SGs formation might be easier in PTEN deficient macrophages and might participated in drug resistance mechanism of BRCA patients with corresponding myeloid PTEN status.

SGs assembly was accelerated in PTEN-deficient BMDMs.

According to the evidence of SGs in macrophages in myeloid PTEN^low BRCA samples, we presumed that PTEN-deficient macrophages might tend to form SGs more easily. For confirmation, we induced SGs by arsenite (Ars) in BMDMs from Lys2-Cre-Pten^f/f(Pten^mKO) and Pten^f/fmice and detected accumulated assembly of SGs in Pten^mKOBMDMs (Fig. 2A&B). Considering the conventional SGs assembly is triggered by phosphorylation of the eukaryotic translation initiation factor 2 subunit alpha (EIF2S1, eIF2α), we detected the phospholipid esterase activity of PTEN in the two groups of macrophages. However, no significant difference was found in the protein level of eIF2α or P-eIF2α between Pten^f/f and Pten^mKO BMDMs, indicating the eIF2α phosphorylation didn’t alter with PTEN status in BMDMs (Fig. 2C).

As a product of LLPS in cytoplasm, SGs formation is also driven by elevated concentrations of nucleating component proteins^{[25, 26]}. Next, we measured the protein level of some key RNA-binding proteins in SGs such as G3BP, TIA1/TIAL1 and DDX3X, and found a leading elevated expression of TIA1/TIAL1, especially TIAL1 (Fig.2E). Consistently, in the cohort of BRCA patients who received neo-adjuvant chemotherapy, the IHC staining of BRCA tissues also showed increased expression of TIAL1 in myeloid PTEN^low samples compared to those with normal or high PTEN expression in myeloid cells (Fig.2F&G). For therapeutic response, similar with myeloid PTEN, the expression of myeloid TIAL1 also presented a correlation with therapeutic efficacy of BRCA patients.

PTEN deficiency is accompanied by upregulated EGR1 in BMDMs

To investigate the mechanism of the up-regulation of TIAL1 expression by PTEN loss in BMDMs, we performed RNA-seq in BMDMs from Pten^f/f or Pten^mko mice and extracted the differential expressed genes(DEGs) (sTABLE. 2). The mRNA level of aforementioned SG component proteins were evaluated and the mRNA of TIAL1 was also the most significantly upregulated one(Fig.3A), suggesting the regulation in transcription of TIAL1 in Pten deficient BMDMs. We predicted the transcription factors (TFs) of TIAL1 using the ChEA software and ChIP-X database^[27] (Fig.3B). Meanwhile, we screened out all the TFs from DEGs between Pten^f/f and Pten^mkoBMDMs (sTABLE. 3), and get intersection genes with predicted TFs. Finally 6 potential TFs of TIAL1 in BMDMs were obtained (Fig.3C & sTABLE.4). We evaluated the expression differences of the 6 genes in the RNA sequencing data and found that EGR1 is the only gene that had a |Log2FC| over 1 (Fig.3D), which makes it the most likely gene that might upregulate TIAL1 transcription in BMDMs. For verification, we measured the protein level of EGR1 in BMDMs of Pten^f/f and Pten^mko mice, and found EGR1 expression was significantly increased after PTEN deletion(Fig.3E).

Upregulated EGR1 promoted the transcription of TIAL1 in PTEN deficient BMDMs

To investigate the transcription relationship between EGR1 and TIAL1, EGR1 CHIP-seq in BMDMs of Pten^f/f and Pten^mko mice were performed and showed that the peak of TIAL1 was mapped in the unique reads of Pten^mkogroup (Fig.4A), indicating TIAL1 might be the target gene of EGR1. This suggested that after PTEN deprivation, the increased EGR1 might bind to TIAL1 gene and regulate its transcription in BMDMs. We further clarified the regulation of EGR1 on TIAL1 by Dual-luciferase reporter gene assay. We found potential binding sites (5 '- gacgggcgg-3') of TIAL1 locating at TSS -416 to -428 bp using JASPAR software prediction(sTable. 5), and then constructed wild-type (TIAL1-wt) and Egr1 binding region mutated (TIAL1-mut) plasmids of TIAL1 promoter reporter gene using PGL3.0 vectors (FIG. 4B), followed by co-transfection with EGR1 overexpression plasmids to detect fluorescence intensity. The fluorescence was significantly enhanced in observed TIAL1-mut group (Fig.4C). In addition, we also conducted GO and KEGG enrichment analyses based on CHIP-seq results and found that the “response to stimuli” (Fig.3D) and “actin cell skeleton” (Fig.3E) were enriched.

SGs formation impaired phagocytosis of BMDMs by remarkable ATP consumption

Since we discovered evidence of SGs in CD68+ cells of myeloid PTEN^lowBRCA patients who are more therapeutic resistant, but not in myeloid PTEN^highpatients, we presumed that despite the protectiveness for survival, SGs might be detrimental to the anti-tumor function of BMDMs. Firstly, we detected several conventional inflammatory cytokines(TNF-α, IL-6 and MCP-1) in the cell culture media of BMDMs, and did not see significant differences after inducing SGs (sFig. 2A)，which is consistent with reported results^[15]. For the ability of antigen presentation, the MHC-II molecules expression on BMDMs didn’t show significant changes either (sFig. 2C&D). However, by co-culturing with 4T1 cells, we found apparent reduction of phagocytic efficiency after inducing SGs in BMDMs, which could be rescued by anisomycin, a compound that has been proved to inhibit SGs assembly^[15](Fig. 5A ~C).

Phagocytosis is regulated by multiple processes including receptor recognition on the membrane surface, remodeling of the cytoskeleton composed of myosin filaments, and ATP dependence during the whole process^[28-31]. The regulatory receptors include "eat-me" and "don't eat-me" receptors which promote or inhibit phagocytosis of macrophages^[32]. However, we didn’t see a significant difference in mRNA level of major regulatory receptors after SGs induction (sFig. 2E). Bio-physically, phagocytosis also requires the cytoskeleton remodeling or the formation of plasma membrane protrusions (such as filopodia or pleats) which are constructed by the polymerization of F-actin and used for target detecting. Inhibition of cytoskeleton remodeling or F-actin blockade hindered the formation of filopodia and impeded phagocytosis in BMDMs^[33]. So we labeled F-actin to display filopodia and found that the appearance of filopodia in untreated BMDMs were presented densely elongated and sharp, while cell membrance of arsenite treated BMDMs were more flattening with shortened and blunted filopodia, especially in cells with SGs (Fig. 5D). Filopodia in cells pre-treated with anisomycin were similar to the control group. This suggested SGs formation in macrophages might hinder the remodeling of the cytoskeleton and obstruct the extension of filopodia which resulted in the damage of phagocytosis.

Next, we sought to identify the reason of cytoskeleton remodeling failure. Since interactions between myosin polymerization and myosin motor proteins are indispensable in phagocytosis^[31] and some myosin subunits were reported inside RNA granules^[34], it was possible that some myosins were dysfunctional for being wrapped into SGs. Although structure prediction revealed the IDRs in some myosins (sFig. 2F), immunofluorescence of pan-myosin proteins didn’t detected any co-localization with G3BP1 (sFig.2H). Considering nearly all stages of cytoskeleton remodeling require ATP for energy, and macrophage phagocytois could be weakened by ATP deprivation^[33]; further more, SGs assembly and disassembly are both ATP-dependent processes^[35], we conducted intracellular ATP level measurement in BMDMs. It showed that ATP level were significantly declined in BMDMs with SGs induction than untreated cells, and inhibition of SGs recovered ATP consumption (Fig. 5E). Interestingly, it is completely different in tumors that SGs induction didn’t reduce ATP level in 4T1 cells (sFig. 2G), which was similar to some published studies^[36]. We supposed that when facing stresses, massive ATP supplied SGs assembly to ensure survival, while left insufficient ATP for cytoskeleton remodeling, targets capture and phagocytosis, a nonessential vital activity for survival. For confirmation, we replenished ATP upon BMDMs after SGs induction, and observed rescued phagocytosis to 4T1 cells, which is similar to the SGs inhibition group (Fig. 5F). It was indicated that ATP consumption by SGs assembly impaired 、phagocytosis of macrophages.

ATP complement or PAM pathway inhibition alleviated phagocytosis impairment by SGs in PTEN-deficient BMDMs

As an vital tumor suppressor, PTEN participates in various signaling pathways regulating cell growth and proliferation, in which the the most famous one is PI3K-AKT-mTOR(PAM) pathway^[37]. Studies showed that in tumor cells, PI3K and mTOR are involved in the regulation of SGs formation^{[38, 39]}. It was also noted that the relationship between mTOR-related pathway and SGs varied among different cell types or stress conditions, and the specific impact of mTOR pathway on SGs assembly remains controversial^[40]. To determine the role of PAM pathway in SGs formation in BMDMs, we treated cells with PI3K inhibitors, mTOR inhibitors, and pan-AKT inhibitors respectively before SGs induction. All the three inhibitors retarded SGs formation to a certain extent, especially PI3K inhibitor and mTOR inhibitor, which statistically shrank the gap between cells under different PTEN status (Fig. 6A). It revealed that PAM activation by PTEN deficiency also contributed to promoting SGs assembly. To further confirm the relationship among PAM pathway, SGs and phagocytosis, we co-cultured BMDMs and 4T1 cells again and detected intracellular ATP level along with phagocyte efficiency at the same time (Fig. 6B), it showed higher consumption of ATP and further weakening of the phagocytic function of BMDM with PTEN deficiency., which could be rescued by ATP replenishment or rapamycin pre-treatment (Fig. 6C-F).

TME has always been a spot in diagnosis and therapeutic research of BRCA. A high level of immune infiltration was confirmed to be associated with increased immune response and better survival in some subtypes of BRCA, but not always a consistency in pCR rate of neoadjuvant chemotherapy^[41], indicating further exploration needed. As a major cell population in TME, macrophages play a vital role in innate immunity against tumors. SGs formation is a conservative self-protective mechanism for eukaryotic cells in mammals, which assists cells’ survival under adverse environments, thus always be closely related to therapeutic resistance in cancer cells^{[14, 17, 18, 42-44]}, SGs of immune cells was reported to assist survival^[15] but the impact on immune functions remains vague. Our study revealed that in macrophages, this surviving strategy might sacrifice their anti-tumor capacity, which contributes to drug resistance in BRCA patients. Our research might provide new perspectives on the mechanisms of macrophages dysfunction, especially in patients with PTEN deficiency.

SGs are dynamic, reversible cellular organelles formed by concentrated stress proteins and nucleic acids in eukaryotic cells^{[13, 45, 46]}. Both assembly and disassembly of SGs are ATP-depended processes^{[35, 47]}, and ATP-dependent proteins/nucleic acids present in yeast and mammalian stress granules, including protein partners (such as Hsp70/Hsp40 and CCT complex) and various RNA and DNA unwindases^[35]. It might explain the noticeable ATP consumption by SGs in macrophages and subsequent obstruction of cellular skeleton remodeling in our study, which could be rescued by ATP replenishment. Interestingly, we observed an opposite ATP alteration trend in 4T1 cells that ATP level did not decline after arsenite, and even increased compared to untreated group, which is consistent with published findings in cancer cells^[36]. We presumed that it may be associated with the high metabolic characters of tumor cells which enable cells with more efficient ATP production than immortal cells, even in the case of stresses and SGs assembly; and the potential alternative pathways retained for ATP generating also maintain malignant biological features. This might explain the different impact of SGs assembly on intracellular ATP level between tumor cells and BMDMs, which deserves further exploration in future studies.

Classic SGs formation is initiated by the activation of stress sensors for adverse environmental stimuli, which are the four kinase families: HRI(the heme-regulated initiation factor 2α kinase), PKR(the interferon inducible double-stranded RNA dependent protein kinase), PERK(protein kinase R-like ER kinase), and GCN2(the general control nonderepressible 2)^[48-52]. The following phosphorylation of eIF2α causes protein translation arrest and biomolecules (such as mRNPs) condensate eventually lead to SGs assembly^{[11, 53]}. Therefore, multiple regulatory factors could affect SG formation such as phosphorylation of eIF2α, interactions between biomolecules, and plasm-concentration of nucleating proteins with RNA-specific binding domains. In our study, PTEN loss did not accelerate SGs formation by modulating eIF2α phosphorylation through its phospholipid esterase activity. Similar results were reported that PTEN or the PTENα-C297S mutant did not significantly influence the eIF2α phosphorylation status in Pten−/− MEFs upon H2O2 exposure^[54]. Instead, we identified two other major regulation mechanisms by PTEN loss in BMDMs promoting SGs formation. On one hand, the up-regulation of expression and transcription level of SG nucleating protein TIAL1 promote SGs assembly by lowering the threshold^[55] of protein-RNA-protein binding interactions. On the other hand, the activation of the PAM pathway caused by PTEN dysfunction also contributed to accelerated SGs formation. In our data, the inhibition of PI3K, AKT, or mTOR pathways led to SGs reduction, indicating PAM pathway also plays a regulatory role in SGs formation in BMDMs. In PAM pathway, MTOR exists in both mTORC1 and mTORC2 complexes with different functions regulating metabolic control throughout life^[56-58]. The mammalian mTORC1 responds to various stress factors including oxidative stress, DNA damage, and unfolded protein stress^{[59, 60]}. It’s reported that oxidative stress inhibited mTORC1 through the small protein REDD1 location^{[61, 62]}. What’s more, mTORC1 was proved to promote SGs assembly by phosphorylating and suppressing the activity of ribosome protein S6 kinase beta-1 (p70-S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1)^[38]. Overactivation of mTORC1 increases the number of SGs induced by arsenate or heat stress^[63]. Interestingly, SGs also inhibit mTORC1 expression in yeast and mammalian cells through various mechanisms. When mTORC1 is inhibited, the assembly of mTORC1-driven SGs may constitute a negative feedback and limit mTORC1 activation by stresses to help regulate cellular metabolic processes and homeostasis und er pressure^{[64, 65]}. Moreover, G3BP was recently found to recruit TSC complex to the lysosome membrane through a stress-independent mechanism, acting as a new key protein involved in modulating mTORC1 activity^[66]. PI3K and MAPK/p38 are also believed to promote SGs formation via mTORC1 activation^[67] in cancer cells, which is consistent with our findings that PTEN loss accelerated SGs assembly through the activation of PAM pathway in BMDMs.

There’re some limitations in this study. Our research focused on BMDMs, which is the most common type of macrophages in stroma. Polarization is a typical character of macrophages when facing various environmental features. Due to the complexity of TME component, macrophages polarization alters and is not strictly defined as M1 or M2 types. In our study, macrophages were activated by LPS and tended to act like M1 type. To confirm the polarization subtype, we detected polarization markers of BMDMs and did not see transition from M1 to M2. Considering functions and molecular characters differs among various polarization status, it is hard to explore every subtype of macrophages all at once. SGs in M2 and other subtypes of macrophages must call for more studies in the future. Additionally, both phagocytosis and SGs fomation are dynamic processes, which makes it difficult to detect and observe, especially in vivo and in real time. It remains a challenge to capture the evidence of relationship between SGs and phagocytosis in vivo, except for the observed G3BP1 aggregation in BRCA sections in our study, which was precious and rare.

In summary, our study found that when confronted with oxidative stress, the assembly of SGs in macrophages consumes tremendous ATP, which impaird cellular skeleton remodeling for phagocytosis. What’s more, PTEN loss in macrophages promoted SGs fomation by upregulating TIAL1 and PAM pathway activation, thus ultimately damaged the anti-tumor immunity in BRCA microenvironment (Fig. 7). Our findings may provide new strategies and theoretical basis for improving treatment and outcomes for BRCA patients, especially those with insufficient myeloid PTEN function.

Mice

The C57BL/6J mice were bought from Slaccas Animal Cooperation. The Pten^f/fmice and the LysM-cre mice (B6.129P2-Lyz2tm1(cre)Ifo/J) were gifts from Laboratory of Prof. Rongbin Zhou in University of Science and Technology of China (USTC). The two genotypes of mice were hybridized to obtain the myeloid PTEN deficient phenotype, which was the Lys2 Cre Pten^f/f (Pten^mKO) mouse. Mice were backcrossed for at least eight generations for purification. All the mice were specific pathogen free (SPF) and all of the mouse experiments were conducted on 10~20 weeks animals. The operations are compliant with all relevant ethical rules and regulations. This study was under approval of The Ethics Committee of University of Science and Technology of China(2021-N(A)-327).

Human tissue samples

27 BRCA patients treated with standard neoadjuvant chemotherapy (anthracycline and taxane based regime, anti-Her2 targeted therapy for HER2+ patients) in the First Affiliated Hospital of USTC were included in the BRCA cohort in this study. Tumor tissues were abtained at two time points. The biopsies before treatment were collected at the beginning of diagnosis and the sections were collected during radical mastectomy. The surgeries were underwent 3-4 weeks after at least 4 rounds of standard neoadjuvant treatment. The therapeutic effects were assessed by the pathological changes among the sections on the two time points, according to the Miller-Payne (MP) scoring system by multiple pathologists. The grading criteria was discribed in Supplementary Table. 7. Patients with MP Grade 1-2 response were classified as chemo-resistant, whereas patients scoring 3-5 were classified as chemo-sensitive. Detailed information including case number, MP grading, MOD of specific targets were summarized in Supplementary Table 1. All of the samples were obtained with informed consent under approval of the review board of the USTC (2021-N(H)-216). The study is compliant with all relevant ethical regulations regarding research involving human participants.

Reagents.

Arsenite and ATP was purchased from Sigma-Aldrich. Anisomycin was purchased from Absin(abs817944). CFSE was purchased from Invitrogen(65-0850-84). Actin-Tracker Green was obtained from Beyotime( C1033, 1:60), Cell Tracking Dye Kit -Red-Cytopainter was purchased from Abcam( ab138893). LPS from Invitrogen. Primary antibodies against G3BP1(Proteintech, 66486-1-Ig, 1:400(IF) or 1:5000(WB); BD Trausduction Laboratories, 611126, 1:400), G3BP2(Proteintech,16276-1-AP, 1:3000), TIA1 (Proteintech, 12133-2-AP, 1:3000),TIAL1 (Cell Signaling Technology, 8509S, 1:800 for IHC and 1:1000 for WB), FMR1 (Proteintech, 13755-1-AP, 1:3000), EGR1(Cell Signaling Technology, 4154S, 1:1000for WB and 1:50 for Chip-seq), CD68 (Cell Signaling Technology, 76437S, 1:400), DDX3 (Bethyl Laboratories, A300-474A, 1:1000),PTEN (Cell Signaling Technology, 9188S, 1:1000(for WB), and Proteintech, 66486-1-Ig, 1:200(for IHC)), MYH pan Monoclonal (Elabscience, E-AB-22021, 1:100), Beta Actin (Proteintech, 66009-1-Ig, WB 1:5000), eIF2α(Cell Signaling Technology, 5324T, 1:1000), Phospho-eIF2α(Cell Signaling Technology, 3597S, 1:1000), FITC anti-mouse I-A/I-E Rat IgG2b(Biolegend, 107605, 1:200) were used. Secondary antibodies including Alexa 488 Goat Anti-Mouse, Alexa 488 Goat Anti-Rabbit, Alexa 594 Goat Anti-Rabbit, Alexa 594 Goat Anti-Mouse, Peroxidase Goat Anti-Mouse HRP, Peroxidase Goat Anti-Rabbit HRP were all obtained from Jackson.

Cell culture.

The 293T cell line and fibroblast cell line L929 were cultured in DMEM medium (Gibco) added by 5% newborn calf serum (Gibco), 5% FBS and 1% penicillin-streptomycin (Beyotime Biotechnology) in the incubator set at 37 °C with 5% CO₂. The breast cancer cell line 4T1 (from BALB/c mice) was cultured in RPMI-1640 medium (Gibco), added by the same supplements as above. BMDMs were derived from bone marrow cells (BMCs) of C57BL/6 mice. After the removal of red blood cells, BMCs were cultured in DMEM medium with the same complements as above and added by 10% supernatant of L929 cells. The culture medium was refreshed every other day till the BMDMs were matured on day 7. All of the cell lines have been tested mycoplasma free.

BMDMs stimulation and stress granules induction

After 7 days of culturing and maturing,BMDMs were primed with LPS (100 ng/mL ) for 4h. BMDMs were then treated with arsenite (90 µM ) for 1h to induce stress granules. For SGs inhibition, cells were treated with anisomycin (25 ug/ml) for 20 minutes before arsenite. For PAM pathway inhibition, wortmannin(100 nM), rapamycin(100 nM) or AZD5356(3 μM) was given before arsenite for 2 hours. In ATP compensatory experiment, ATP was given before arsenite at 4M for 90 minutes. Cells were then collected to conduct immunofluorescence, coculturing or Flow cytometry. The cell extracts were processed for immunoblot or ATP detection and the cell supernatants were processed for cytokines detection using CBA kit.

Co-culturing of BMDMs and 4T1 cells

The 4T1 cells were digested with 0.1% trypsin-EDTA and terminated by the addition of antibiotic-free medium. After counting, the cells were washed by PBS to reach the concentration of 1×10⁶ cells/mL. Then they were stained by CFSE at a final concentration of 1×10⁶ cells/μL, incubated at 37°C for 20 min and stood on ice for 3 min to terminate staining, followed by centrifuging, washing with PBS and resuspended with complete medium. The mature BMDMs (d7) were digested, washed, counted, and resuspended in complete medium, added with Tracking dye Red (25×), and incubated at 37°C for 30 min. When staining finished, BMDMs were washed with PBS and resuspend with complete growth medium. BMDM and 4T1 were mixed in a 5:1 ratio and transferred to a round-bottomed 96-well plate with low adhesion, and incubated in the 37°C incubator for 4~6 hr. The phagocytosis could be observed and analyzed in the fluorescence channel under the microscope or flow cytometry.

Flow cytometry and cell sorting.

For BMDMs authentication, the mature BMDMs (d7) were blocked in 4 °C for 5 min and then stained with antibodies against F4/80 or CD11b in 4 °C for 20 min. For MHC-II molecules detection and measurement, BMDMs were were primed with LPS and pretreated with arsenite in need. Then cells were fixed and then stained with FITC/MHC-II (I-A/I-E) antibodies. For phagocytosis experiments, BMDMs were were primed with LPS and pretreated with arsenite or other agents in need. Single-cell suspensions of BMDMs were stained with Tracking dye Red (detected in PE channel), 4T1 cells were stained with CFSE (detected in FITC channel). After the washing step, samples were run by flow cytometry and the results were processed and analyzed by Flowjo(version 10.8.1). The gating strategy is described in Supplementary Fig. 2.

Confocal microscopy and immunofluores

The prepared BMDMs were pretreated with PHEM, then fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and washed with phosphate buffered saline (PBS). Then cells were permeated with with 0.2% Triton X-100 for 5 min and blocked with 1% BSA (TBST contained) for 1 h. Cells were incubated with primary antibodies at 4℃ overnight, secondary antibody at RT for 1h and DAPI for 3min. After all the staining, Dako Fluorescence Mounting Medium (Dako North America, S302380-2) was used for mounting. Confocal microscopy analyses were carried out using Zeiss LSM980. Images were acquired with ZEN (version 3.8) and analyzed by ImageJ (version 1.8.0).

Immunohistochemistry (IHC) of human biopsies

The human breast tumour tissue were paraffin sections which were consecutive serially sliced by the pathologists. After baked in a 60℃ oven for 25 min, paraffin removal by dixylene and hydration by ethanol, the sections were heated in sodium citrate (pH6.0) for antigen retrieval. Then the samples were treated with 3% methanol in H₂O₂ and blocked with goat serum and incubated with primary antibodies (anti-PTEN, 1:200, 66486-1-Ig, Proteintech; anti-CD68, 1:200, 76437S, Cell Signaling Technology; anti-TIAL1, 1:800, 8509S, Cell Signaling Technology.) at 4 °C overnight. After washing with PBS, the sections were incubated with secondary antibodies at RT for 30min. Then the DAB and haematoxylin were stained on the sections for observation. For Immunohistochemical quantification, four random fields in each sections were captured for analyzing.The integrated optical density (IOD) of PTEN in cells was calculated by the mean DAB intensity using ImageScope (version 12.4.6.5003) and the PTEN expression was calculated as MOD=IOD/(the total area of captured cells).

Quantitative PCR

The total RNA in cells was extracted using the TransZol Up Kit (Transgene, ET101-01). First-strand cDNA synthesis was synthetized using the HiScript II cDNA Synthesis kit (Vazyme, R212-02) and stocked at -80℃. Quantitative PCR was conducted using AceQ qPCR SYBR Green Master Mix (Vazyme, Q111-02). The primers used are listed in Supplementary Table 6. The program running and data collection were performed by PikoReal software (version 2.2).

ATP Detection

Byotime's S0026 ATP detection kit was used to detect the intracellular ATP levels. The lysate was added into the 6-well plate 200 μL per well on ice, without cell culture medium inside. The transferred cell lysate was centrifuged at 12000 G at 4℃ for 5 min, and get the supernatant. the samples or diluted standard substance were added into the test hole with working solution. The chemiluminescence instrument was used to measure the RLU value or CPM in the hole 2 seconds later. The concentration of ATP in the sample can be calculated by substituting the RLU value or CPM in the sample well into the standard curve. The data was analyzed by Excel software and GraphPad 9.

Cell lysis and immunoblotting

Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM Na2P2O4, 1 mM Na3VO4, 1 mM PMSF and proteinase inhibitor cocktail (Sigma). After boiled at 95 °C, the cell lysates were separated on 10-12% SDS-polyacrylamide gels for electrophoresis. Then the sample gel was transferred to 0.22-μm PVDF membranes (Millipore) in ice-bath. After blocked with 5% non-fat milk in Tris-buffered saline on a table concentrator for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at RT. Western Lightning Chemiluminescence Reagent Plus ChemiScope software (version 17.05.17A) was used to detect the signals and ImageJ was used to perform quantitative analysis.

CHIP-Sequencing

Cells were fixed with Formaldehyde/paraformaldehyde for 10 min at RT and added with glycine to quench crosslinking. After rinsing twice with PBS, cells were disrupted and spun at 1500 g for 5 min at 4 °C, then suspended with SDS lysis buffer and protease inhibitor complex. Cells were sonicate for 20 min (30 sec on, 30 sec off). 20 µl aliquot of cell lysate from each sample was saved as input DNA and Stored at -20 °C. Protease K was added into the control and input DNA respectively. The mixture was incubated at 55 °C water bath for 120 min(or 65℃ for1h). The samples were run with 100 bp DNA ladder on 1.2% agarose gel to check sonication efficiency. Samples were then spun at 20,000 g for 15 min at 4 °C and the supernatant wascollected for IP. The DNA concentration of each sample was measured by nanodrop. 25% of spike-in chromatin and the antibody were added into each sample sequentially and incubated at 4°C on rocker for O/N. The blocked magnetic Protein A/G beads were transfered into cell lysate and incubated for 2-4 hr 4°C on rocker, and washed using High-salt Wash buffer and Low-salt Wash buffer sequentially. ChIP Elution Buffer were added into each sample including input. DNA were purificated with Protease K and prepared for concentration detection using Qubit. The parameter of alignment: bowtie2 -x $database/genome -1 ${id}_1.fq -2 ${id}_2.fq -S ${id}.sam -p 18 -k 6 -X 700 --local; the parameter of call peak: macs2 callpeak -t ${id}.rmdup.bam -n $id -B --trackline -g mm -s 150 -q 0.05 -f BAMPE --keep-dup all.

Cytokines detection using CBA method

The lyophilized standards was prepared to dilutions according to the concentration gradient: Undiluted/1:2/1:4/1:8/1:16/1:32/1:64/1:128/1:256/Diluent and stored at 4°C. Antibody-capture microspheres was aspirated at the required amount and mixed well. Testing samples were diluted based on the cytokine concentration. 50 µL capture beads was added to the test tube, or 50 µL dilution fluid to the negative control tube, or 50 µL sample to each remaining tube. Then 50 µL PE detection reagent was added into all the tubes. After vortexing and mixing, the tubes were incubate at RT in the dark for 2 hr with one time of vortex per hour. After antibody labeling completed, 1 mL washing buffer was added to each test tube and centrifuge at 200 g for 5 minutes. 300 µL washing buffer was added into each tube with supernatant discarded. The microspheres were resuspended for testing. The results were analyzed by FCAP Array software.

Dual-luciferase reporter gene assay

The pasmid was pGL3-basic. 293T cells were seeded in 24-well plate and transfected when the density reaches about 70%. Plasmid or transfection reagent were added into 25ul opti-MEM and incubated at RT for 5 min, gently mixed and incubated at RT for another 20 min.. 450ul per well of serum-free and antibody-free DMEM were added to the obove solution to obtain the transfection mixture. The medium in the 24-well plate was replaced with the transfection mixture and discarded after 6 hr of incubation, then replaced with 500ul of complete medium per well and continued culturing for another 48 hr. After discarding the current medium, 200ul of mixed reporter gene cell lysis solution was added to each well and shaked on ice for 20 min, then centrifuged at 15,000×g for 5 minutes at 4°C. The supernatant was kept for testing. The sea pansy luciferase detection substrate (100X) and sea pansy luciferase detection buffer were prepared at the ratio of 1:100 and mixed to obtain the sea pansy luciferase working solution. 100ul of the samples were added and an equal volume of the reporter gene cell lysis solution was taken as the blank control. The detection wavelength of the multifunctional enzyme m arker was set to 560nm, and 100ul of firefly luciferase detection reagent was added and mixed well, then the RLU (relative light unit) was measured(RLU1). The detection wavelength was then set to 465nm, and 100ul of sea pansy luciferase working solution was added with the measurement of RLU(RLU2). With the sea pansy luciferase set as the internal reference, the activation degree of target reporter gene among different samples was calculated by taking the ratio of RLU1/RLU2.

Statistical and reproducibility

Experiments were independently conducted and repeated at least twice with similar results. Data were statistically analyzed with GraphPad Prism 8 and are presented as mean±s.e.m. P value calculation was described in corresponding figure legend. P<0.05 was considered to be significant.

Staining and analysis of immunohistochemistry and immunofluorescent were blindly performed. Immunofluorescence images were processed by Image J and immunohistochemistry information were collected by ImageScope(version 12.4.6.5003). The sample size of the patients cohort was not predetermined with statistical method.

Funding

This study was supported by National Natural Science Funds of China (Grant No. 82172775), the Fundamental Research Funds for the Central Universities(WK9110000058 and YD9110002032).

Funders only provide financial support.

Acknowledgements

We thank Rongbin Zhou at the University of Science and Technology of China(USTC) for providing the Pten^f/fmice and the LysM-cre mice; and Wen Pan at USTC for providing materials and reagents of the MHC-II detection experiment.

Declaration of interest

None.

Author contributions

Yan Li, Zhenye Yang and Yueyin Pan, conceptualization; Yan Li, Gang Wang, Chaoqiang Han and Hui Hua, investigation; Chao Xu, Rutong Zhang and Xiaoxi Feng, resources; Jian Chen and Haiyang Yu, bio-informational analysis; Menghao Dong, clinical data collection; Yan Li, visualization, writing and editing; Zhenye Yang and Yueyin Pan, supervision; Yan Li and Yueyin Pan, funding acquisition.

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The authors declare no competing interests.

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Myeloid PTEN loss affects therapeutic response by promoting stress granules assembly and impairing phagocytosis of macrophages in breast cancer

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