The role of macrophage in endometriosis and endometriosis-associated ovarian cancer

21 Endometriosis affects approximately 176 million women worldwide and carries a high risk of 22 malignant transformation. Understanding the mechanism of endometriosis occurrence and development 23 is crucial for its treatment, particularly in preventing malignant transformation. Macrophages are 24 among the most important immune cells in promoting endometriosis occurrence and development. 25 Defects in their function can result in incomplete clearance of retrograde endometrial debris. M2 26 macrophages can promote the formation of endometriotic lesions and induce an anti-inflammatory 27 environment that promotes the development and malignant transformation of endometriosis. This 28 review summarizes the role of macrophages in endometriosis and endometriosis-associated ovarian 29 cancer. The review also covers existing treatment methods and strategies of targeted macrophage 30 immunotherapy for endometriosis. 31

32 Endometriosis; M1; M2; macrophage; endometriosis associated ovarian cancer; immunotherapy. 33 34 35 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript 1.Overview of endometriosis and endometriosis-associated cancer 36 1.1 Endometriosis 37 Endometriosis (EMS), in which endometrial tissue is abnormally located outside the uterus, is a 38 common female inflammatory reproductive disorder, associated with chronic pelvic pain and 39 subfertility. It impacts nearly 5%-10% of fertile women, equivalent to 176 million women 40 worldwide[1]. According to Sampson’s theory of retrograde menstruation, EMS results from the reflux 41 of endometrial fragments through the fallopian tubes during menstruation, which then implants on the 42 peritoneum and ovary[2]. The revised American Society for Reproductive Medicine classification is 43 one of the most frequently used systems to classify EMS[3]. In general, the lesion location and 44 infiltration depth of surrounding tissue are bases for staging of EMS, corresponding to superficial 45 peritoneal lesion, deep infiltrating EMS, and endometrioma[4]. 46 EMS is an estrogen-dependent inflammatory disease[5]. In recent years, endocrine/paracrine 47 alterations and immune aspects, such as complements, cytokines, growth factors, hormones, and immune 48 cells, have been proposed to be involved in the pathophysiology of EMS (Figure 1). For example, 49 neutrophils produce biochemical factors that aid in peritoneal immune inflammation and angiogenesis. 50 The cytotoxicity of natural killer (NK) cells in endometriosis is inhibited to eliminate endometrial cells 51 in the abdominal cavity, thereby resulting in immune escape. The imbalance between T helper type 1 52 (Th1)/ T helper type 2 (Th2) cells causes cytokine secret aberrantly, which induces lesion progression[6-53 8]. Overall, in the abdominal cavity microenvironment, the dysfunction of the immune system promotes 54 the adhesion and invasion of ectopic endometrial cells, thus contributing to poor clearance, implantation, 55 angiogenesis, and proliferation of endometrial debris [5] [8, 9]. Although there is little information 56 available on the clinical application of immune therapy in EMS, these findings provide novel insights 57 into targeting related immune factors for EMS treatment[10]. The role and transformation of macrophage 58 engaging in EMS will be elaborated in detail in this review. 59 1.2 Endometriosis-associated cancer 60 EMS is a benign disease but is potential for malignant transformation. It shares features with cancer, 61 including metastasis-like behavior, tissue invasion, proliferation, angiogenesis, and reduced 62 apoptosis[11]. Growing evidence has suggested that the occurrence and development of various cancers 63 are associated with the malignant progression of EMS, such as endometrioid carcinoma, clear cell 64 carcinoma, non-Hodgkin’s lymphoma, brain tumors, and endocrine cancers[12]. Notably, a strong 65 genetic correlation and a positive association between EMS and ovarian cancer have been confirmed by 66 Mortlock and Kvaskoff et al respectively[4, 11]. A study by He ZX et al. showed that 2.9% of women 67 with ovarian endometriosis were found to have endometriosis-associated ovarian cancer[13]. Meanwhile, 68 Hermens’s study revealed a significant higher incidence of endometriosis-associated endometrioid 69 ovarian cancer in women with histologically proven endometriosis[14]. Therefore, EMS-related ovarian 70 cancer is the research focus. Atypical EMS, including hyperplasia and/ or atypia type, is associated with 71 ovarian cancer. Pathologic diagnosis of endometriosis is characterized by the presence of 2 of the 3 72 following histologic features: endometrial stromal cells, endometrial-type glands, and findings consistent 73 with chronic bleeding, such as hemosiderin-laden macrophages[15]. In contrast to the histological 74 features of typical endometriosis, atypical endometriosis is defined as EMS with a localized proliferation 75 of crowded glands lined by atypical epithelium resembling endometrial intraepithelial neoplasia, or 76 alteration in endometriotic cyst lining with stratification, disorganization and cytologic atypia by the 77 World Health Organization[16]. Epidemiological studies have shown that endometriosis is associated 78 with an increased risk of epithelial ovarian cancer (EOC)[4]. The subtypes of ovarian cancer that have a 79 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript high association with endometriosis are endometrioid ovarian cancer (ENOC) and clear cell ovarian 80 cancer (CCOC), whereas high-grade serous ovarian cancer (HGSOC) is considered to be less associated 81 with endometriosis[4]. Concurrent endometriosis is observed in 21%-51% of CCOC patients and 23%-82 43% of ENOC patients [4]. Studies have shown this association. When ovarian cancer coexists with EMS, 83 there are three possible mechanisms: ① they are caused by the same risk factors; ② cancer cells develop 84 from endometriotic cells; or ③ they are caused by different risk factors. This study focuses on the 85 mechanisms of endometriosis-associated ovarian cancer (EAOC) by which cancer cells develop from 86 EMS[4]. 87 The development of endometriosis-associated ovarian cancer is a multifactorial process that 88 involves genetic and environmental factors, including gene mutation resulting from long-term 89 inflammation in the abdominal cavity, immune disorder, and DNA damage (Figure 1)[5]. EMS lesions 90 in the abdominal cavity periodically and repeatedly bleed, leading to overload of iron in the 91 microenvironment. High iron concentration promotes ferroptosis of normal cells in the 92 microenvironment such as immune cells and stromal cells, thus promotes the development of EMS[17, 93 18]. This process is associated with intracellular reactive oxygen species(ROS) production, lipid 94 peroxidation, and mitochondrial damage[19]. Additionally, activation of inflammatory pathways and 95 angiogenesis can lead to EMS progression and iron-mediated DNA damage[20]. 96 Shared somatic mutations between benign endometriotic lesions and adjacent tumors suggest that 97 these lesions may serve as the cellular precursors of EAOC. Several genes, including A T-rich 98 interaction domain 1A (ARID1A), Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatase 99 and tensin homolog (PTEN), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha 100 (PIK3CA), and P53, which also play important roles in uterine endometrioid carcinomas and EMS-101 related cancer development, are involved in the development of endometriosis[5, 11, 21]. Mutations in 102 these specific genes have a high potential to trigger the progression of endometriosis to EAOC. Studies 103 have shown that patients with endometriosis have increased peritoneal fluid concentrations of ovarian 104 cancer markers carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4) [22]. 105 Estrogen excess is common in both EMS and ovarian cancer, and the decrease in estrogen receptor 106 levels can increase estrogen levels, which may lead to an increase in carcinogenesis[23]. Estrogen-107 positive is common in endometrioid adenocarcinoma. The use of contraceptives has been demonstrated 108 to decrease the risk of ovarian cancer, regardless of the presence of endometriosis. Hepatocyte nuclear 109 factor 1-beta (HNF1-β) is generally expressed in clear cell and serous carcinomas, as well as in 110 precursor lesions of endometriosis like borderline and atypical endometriosis[24],[25]. This suggests 111 that endometriosis is associated with a higher risk of ovarian cancer due to genetic and epigenetic 112 changes in gene expression, pathways, and other factors. 113 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript 114 Figure 1 Pathogenesis of endometriosis and endometriosis-associated ovarian cancer. 115 EMS results from the reflux of endometrial fragments through the fallopian tubes during menstruation 116 and subsequent implantation on the peritoneum and ovary. Estrogen, complements, cytokines, growth 117 factors, hormones, and immune cells interact to influence the peritoneal microenvironment, leading to 118 poor clearance, implantation, angiogenesis, and proliferation of endometrial debris. Endometriosis-119 associated ovarian cancer is caused by complicating factors including gene mutations, immune 120 dysregulation, and DNA damage. Mutations in some specific genes have a high potential to trigger the 121 progression of endometriosis to EAOC. In the peritoneal cavity, periodically and repeatedly bleeding 122 EMS lesions lead to ferroptosis, which is associated with intracellular ROS production, lipid peroxidation, 123 and mitochondrial damage. In addition, activation of inflammatory pathways and angiogenesis may lead 124 to EMS progression and iron-mediated DNA damage. NK cells, natural killer cells; Th1 cells, T helper 125 type 1 cells; Th2 cells, T helper type 2 cells; Th17 cells, T helper type 17 cells; E2, estradiol; COX2, 126 cyclooxygenase 2; IL-1β, interleukin-1 beta; IL-8, interleukin-8; TNF-α, tumor necrosis factor alpha; 127 PGE2, prostaglandin E2; Tf, Transferrin; ROS, reactive oxygen species; ARID1A, A T-rich interaction 128 domain 1A; KRAS, Kirsten rat sarcoma viral oncogene homolog; PTEN, phosphatase and tensin 129 homolog; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha. (Created by 130 BioRender). 131 2. Overview of macrophage in endometriosis 132 Macrophages are typically classified as either classically activated 'M1' macrophages or 133 alternatively activated 'M2' macrophages based on their function (Figure 2). Surface markers including 134 CD68, toll-like receptor 4 (TLR4), CD14, human leukocyte antigen DR (HLA-DR), CD80, and CD86 135 express on human M1 macrophages (F4/80, major histocompatibility complex Ⅱ (MHC II), CD86, and 136 inducible nitric oxide synthase (iNOS) on mouse macrophages) [26, 27]. These macrophages express 137 pro-inflammatory markers, secrete pro-inflammatory cytokines such as interleukin-1 (IL-1), interleukin-138 6 (IL-6), interleukin-8 (IL-8), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), interleukin-12 139 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript (IL-12), interleukin-23 (IL-23), and NOS, and promote inflammation[26]. The surface markers for 140 human M2 macrophages are CD68, TLR4, CD163, CD40, CD206, CD14low, HLA-DRlow, CD80low, and 141 CD86low (for mice are F4/80+, MHC II+, CD206+ and arginase1 (Arg1)+ )[27, 28]. Conversely, M2 142 macrophages secrete anti-inflammatory cytokines, such as interleukin-10 (IL-10), transforming growth 143 factor beta (TGF-β), and vascular endothelial growth factor (VEGF), and are involved in homeostasis, 144 wound healing, and immune regulation[29]. According to physiological or pathological conditions, 145 macrophages alter their transcription factors and phenotype in response to signals from the local 146 microenvironment. 147 Although the M1/M2 classification system is a useful tool for studying macrophage activation, it is 148 now recognized that macrophages in vivo exhibit a broad spectrum of tissue- and disease-specific 149 phenotypes. Therefore, the M1/M2 system cannot fully represent the diversity and complexity of 150 macrophage phenotypes. With the in-depth study of macrophages, they are classified as resident 151 macrophages or recruited macrophages based on their origin (Figure 2). Resident macrophages originate 152 from the yolk sac, fetal liver, and erythrocyte-marrow progenitor (EMP) cells produced during primitive 153 hematopoiesis in embryos at embryonic stages 7.5 and 8.25[30]. After the formation of the blood 154 circulation, macrophages derived from EMPs are implanted in fetal tissues. These macrophages are 155 partially or completely replaced by monocytes from the fetal liver (excluding microglia) and differentiate 156 into tissue macrophages[31]. Resident macrophages derived from fetal liver monocytes can persist 157 through out adulthood and undergo self-renewal with specific tissue macrophage characteristics, such as 158 microglia, and langerhans cells[32]. In certain organs, such as the intestine and dermis, tissue 159 macrophages that originate from fetal liver monocytes are gradually replaced by monocytes that originate 160 from bone marrow[33]. 161 The macrophages involved in the development of EMS are primarily endometrial macrophages, 162 peritoneal macrophages, and monocyte-supplemented macrophages[28]. Abdominal cavity 163 macrophages are one of the most studied macrophage populations in mice and one of the most 164 important cell populations involved in the development of EMS due to their ease of isolation. 165 Abdominal cavity macrophages can be classified according to their origin into resident macrophages 166 (peritoneal macrophages) and monocyte-derived macrophages (Figure 2). Peritoneal macrophages in 167 mice are classified into two subpopulations, "large" (larger size) peritoneal macrophages (LpM) and 168 "small" (smaller size) peritoneal macrophages (SpM), based on differential expression of F4/80 and 169 MHC II. LpM is characterized by F4/80high and MHC IIlow[34]. The LpM is the most abundant 170 macrophage subpopulation in peritoneal luminal homeostasis and plays an important role in 171 immunosurveillance, B1b cell recruitment and maintenance, and intestinal immunoregulation. It has a 172 specific transcription factor, GA TA-6, which controls self-renewal, proliferation, and phenotype[34]. 173 The SpM is a subpopulation of F4/80low MHC IIhigh macrophages and conventional dendritic cells. 174 Their differentiation is dependent on interferon regulatory factor 4 (IRF4), and they are replaced over 175 time by lymphocyte antigen 6 complex (Ly6C)high classical monocytes in a cysteine-cysteine motif 176 chemokine receptor 2 (CCR2)-dependent manner[34]. SpM have been implicated in the inflammatory 177 response, but their role in the homeostatic abdominal cavity remains unclear. Chronic inflammation, 178 fibrosis and angiogenesis are inherent features of EMS in which macrophages play an important role. 179 EMS lesions are formed through tissue repair, regeneration, angiogenesis, and nerve fiber 180 reconstruction involving macrophages[35]. Learning the complex characterization of macrophage is 181 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript vital for us to learn the development of EMS and better intervene in it. 182 Figure 2 The origin of macrophages in endometriosis. 183 Macrophages are typically classified as 'M1' macrophages and 'M2' macrophages based on their function. 184 M1 macrophages express pro-inflammatory markers, secrete pro-inflammatory cytokines and promote 185 inflammation. Conversely, M2 macrophages secrete anti-inflammatory cytokines, and are involved in 186 homeostasis, wound healing, and immune regulation. Depending on the origin, macrophages in the 187 abdominal cavity were classified as tissue resident macrophages and recruited macrophages. Tissue 188 resident macrophages originate from the yolk sac, fetal liver, and erythrocyte-marrow progenitor cells 189 produced during primitive hematopoiesis in embryos. Resident macrophages differentiate into "large" 190 (larger size) peritoneal macrophages (LpMs) in the abdominal cavity, which mainly exhibit M1 191 phenotype. SpMs are smaller size peritoneal macrophages derived from monocytes, which tend to be M2 192 phenotype. LpM, "large" (larger size) peritoneal macrophages; SpM, "small" (smaller size) peritoneal 193 macrophages; MHC Ⅱ, major histocompatibility complex Ⅱ; TIM4, T-cell immunoglobulin mucin-4; 194 EMS, endometriosis; Ly6C, lymphocyte antigen 6 complex; TLR4, toll-like receptor 4; HLA-DR, human 195 leukocyte antigen-DR. (Created by BioRender). 196 2.1 Endometrial macrophages 197 The human endometrium is highly regenerative and can undergo approximately 400 menstrual 198 cycles[36]. The menstrual cycle consists of four phases: menstrual, regenerative, proliferative, and 199 secretory phase. In the menstrual phase, the functional layer of the endometrium is shed, and an 200 inflammatory microenvironment is formed in the uterus, recruiting various immune cells[37]. 201 Macrophages play a crucial and irreplaceable role in this process[37]. 202 Macrophages in the endometrium are mainly divided into resident and recruited macrophages. 203 Tissue-resident macrophages are present in the three layers of the uterus, including the endometrium, 204 myometrium, and periuterine[26]. They are particularly abundant in the endometrium. Monocyte influx 205 occurs during the secretory and menstrual phases in the normal circulating endometrium (Figure 3). They 206 are regulated by cysteine-cysteine motif chemokine ligand 2 (CCL2) and C-X3-C motif chemokine 207 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript receptor 1 (CX3CR1) and influenced by sex hormones, particularly estrogen and progesterone[38]. 208 Ultimately, these monocytes differentiate into macrophages in the uterus[28]. The number of 209 macrophages in the endometrium, regulated by estrogen and progesterone, fluctuates during the natural 210 menstrual cycle[26]. They account for approximately 1-2% of all cells in the endometrium during the 211 proliferative phase, increasing slightly during the secretory phase to approximately 3-5%, and reaching 212 a peak of 6-15% during the menstrual phase[26]. The duration of resident and recruited macrophages and 213 their immune response during the menstrual cycle determine the outcome of the endometrial healing 214 process, including the degree of fibrosis and tissue regeneration. During menstruation, macrophages play 215 an important role in removing uterine tissue debris and secreting inflammatory cytokines and growth 216 factors[39]. This process is predominantly carried out by the M1 macrophages in the first phase of the 217 immune response. During the proliferative phase of the adaptive immune response, macrophages secrete 218 anti-inflammatory cytokines and growth factors to promote tissue regeneration[26]. This process mainly 219 involves the M2 macrophages. Additionally, macrophages in the endometrium perform regulatory repair 220 of the endometrium without scar formation to maintain tissue integrity during the natural menstrual cycle. 221 Endometrial macrophages may play a role in various processes of normal pregnancy, as well as in the 222 maintenance of immune tolerance. However, their function in the uterus has not been clearly 223 demonstrated. 224 The inflammatory features of endometriosis are not limited to the pelvic and abdominal cavities or 225 the vicinity of the ectopic lesion but also affect the native endometrium. A previous study found that the 226 number of M1 macrophages was higher in stage I-II endometriosis than in healthy controls, while the 227 number of M2 macrophages was elevated in the eutopic endometrium of women with stage III-IV 228 endometriosis. Additionally, a study reported that M2 macrophages were the predominant phenotype in 229 healthy endometrium, whereas the population of endometrial M2 macrophages in women with 230 endometriosis was lower than in controls at all cycle stages[6]. 231 The predominance of M2 macrophages in the endometrium of healthy women is due to the need 232 for the uterus to maintain an anti-inflammatory environment that facilitates embryo implantation[40]. 233 In contrast, endometrial macrophages from EMS patients exhibited a more pro-inflammatory 234 phenotype[41]. They secreted higher levels of IL-1, IL-6, IL-8, and HGF compared to normal controls. 235 Monocyte chemoattractant protein-1 (MCP-1) and macrophage inhibitory factors are involved in the 236 formation of the pro-inflammatory environment. Endometrial CD91+ macrophages overexpressed 237 signal regulatory protein α (SIRPα), a phagocytosis inhibitor, and CD64, which is associated with 238 inflammation environment[42]. The incomplete clearance of endometrial debris during menstruation 239 and decreased phagocytosis by macrophages allow tissue debris to "reflux" outside the uterine cavity, 240 leading to the formation of endometriosis-associated lesions (Figure 4)[39]. 241 Studying the effect of endometrial resident macrophages on EMS can be challenging due to their 242 low numbers and the fact that immune cells often infiltrate the uterus during menstrual cycle and are 243 non-uniformly distributed in both the epithelium and stromal layer[43, 44]. So, it is important to 244 consider these factors when conducting research on this population. Further accumulation of evidence 245 is essential to gain a clear and comprehensive understanding of the precise role of uterine macrophages 246 involved in these physiological and pathological events. 247 2.2 Abdominal cavity macrophages 248 As previously mentioned, one of the pathogenic mechanisms of EMS is retrograde menstruation. 249 Senescent erythrocytes accumulate and die in the pelvic cavity, leading to iron overload[45]. The iron 250 overload in peritoneal macrophages exacerbated phagocytosis and clearance of erythrocytes, which 251 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript affects macrophage activation[2]. Macrophage dysfunction, which includes reduced antigen 252 presentation, phagocytosis, and cytotoxic activity, as well as activation of the nuclear factor-κB (NF-253 κB) pathway, ultimately leads to immune escape of refluxing endometrial debris and an increased 254 inflammatory microenvironment and chemotaxis in the abdominal cavity . 255 The two subpopulations of abdominal cavity macrophages, LpM and SpM, play different role in 256 EMS development. It has been demonstrated that macrophages from EMS lesions in mice are derived 257 from LpMs and monocytes in eutopic endometrial tissue. In addition, abdominal cavity inflammation 258 due to EMS triggers sustained recruitment of monocytes and increased CCR2+ LpM[28]. However, it 259 has also been shown that the proportion of SpMs rises immediately after abdominal cavity injections of 260 endometrial tissues, whereas LpMs show the opposite trend[46]. Thus, the effect of macrophages in the 261 abdominal cavity, which are typed according to their origin, on the development of EMS is 262 controversial and needs to be verified by more experiments. 263 In EMS peritoneal macrophages, M1 and M2 macrophages have been studied in more detail. The 264 M2 phenotype, required for angiogenesis and ectopic lesion growth, is a peritoneal macrophage type 265 involved in the development of EMS (Figure 4)[47]. Compared with healthy women, patients with EMS 266 have decreased M1 macrophages and increased M2 macrophages in the abdominal cavity, while the 267 number of M1/M2 macrophages change contrarily in the endometrium. In EMS patients, anti-268 inflammatory M2 macrophages are the predominant phenotype in the abdominal cavity, whereas pro-269 inflammatory M1 macrophages play a leading role in the ectopic lesion. Research has shown that ectopic 270 endometrial homogenates or sera can increase the percentage of CD163+ macrophages and IL-10, while 271 decreasing the percentage of CD86+ macrophage cells and IL-12. This is achieved by up-regulating 272 suppressor of mother against decapentaplegic family member 2 (SMAD2)/SMAD3 in macrophages[48]. 273 Activation of estrogen receptor beta (ERβ) in endometrial stromal cells (ESCs) is involved in 274 macrophage recruitment through NF-κB/CCL2 signaling and contributes to the pathogenesis of EMS by 275 promoting macrophage differentiation into the M2 phenotype[47]. The presence of M2 macrophages in 276 the abdominal cavity and ectopic lesions indicates that this cell population plays a role in creating an 277 anti-inflammatory environment that allows for lesion establishment and growth. 278 The polarization and function of M2 macrophages are affected by many factors. Research has shown 279 that patients with EMS exhibit an increased proportion of T helper type 17 (Th17) cells[46, 49, 50]. Th17 280 cells promote M2 macrophage polarization and lead to the recruitment of bone marrow-derived SpM 281 precursors and CD206+ SpM[51]. Additionally, in vitro, it enhances the expression of IL-8, which 282 promotes inflammation and fibrosis in EMS by inducing neutrophil migration and MCP-1 secretion, 283 making macrophage migrate towards EMS lesions[52, 53]. Via the transporter legumain pseudogene 1 284 (LGMNP1), ectopic endometrial stromal cell-derived extracellular vesicular can act as intercellular 285 messengers to mediate upregulation of legumain (LGMN) mRNA expression in macrophages, thus 286 reprogramming macrophages into M2 phenotype in vitro[54]. Lactate was found to be a key factor 287 driving macrophage M2 polarization to promote ESC invasion in vitro and in vivo, and activation of 288 methyltransferase 3 (Mettl3) and its target gene, tribbles pseudokinase 1 (Trib1), promotes M2 289 macrophage polarization through the extracellular regulated protein kinases (ERK)/ signal transducer 290 and activator of transcription 3 (STA T3) signaling pathway when inhibition of glycolysis significantly 291 reduces EMS progression[55]. Nanovesicles derived from M1 macrophages directly or indirectly 292 inhibited the migration and invasion of ESCs obtained from patients with endometriosis and reduced the 293 formation of tubular structures and inhibited the development of EMS by reprogramming M2 294 macrophages[56]. 295 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript In addition, M2 macrophages in the abdominal cavity and lesions may exacerbate pain in women 296 with endometriosis by promoting the growth of nerve fibers. Ectopic lesions in the abdominal cavity 297 trigger the immune system and induce subsequent inflammation, which supports the survival and 298 growth of ectopic endometrial tissue fragments. Netrin-1, an extracellular guidance cue for neuronal 299 navigation, promotes neurovascularization in endometriosis. Studies have shown that netrin-1 300 expression peaks in peritoneal macrophages in endometriosis-associated infertility. Netrin-1 induces 301 the formation of ovarian endometriotic angiogenesis by interacting with CD146 in vascular endothelial 302 cells[57]. Additionally, netrin-1 promotes endometriotic protrusions by up-regulating microtubule-303 associated protein 4 (MAP4), tau protein, and calcitonin gene-related peptide (CGRP) through another 304 receptor, neogenin, which contributes to the growth and sensitization of endometriotic lesions[57]. 305 Cytokines and chemokines secreted by macrophages also play a role in EMS development (Figure 306 4). It has been found that IL-33 secreted by macrophages can accelerate the progression of EMS[58]. Co-307 culturing with macrophages or using IL-33/ suppression of tumorigenicity 2 (ST2) can stimulate the 308 viability and migration of ectopic endometrial stromal cells (eESCs). Macrophage-derived IL-33 309 upregulates solute carrier family 7 member 11 (SLC7A11) in eESCs through the p38/ c-Jun N-terminal 310 kinase (JNK)/ activating transcription factor 3 (ATF3) pathway, reduce intracellular iron levels and lipid 311 peroxidation, which ultimately result in protection against ferroptosis in eESCs[58]. Exosome from EMS 312 patients’ peritoneal macrophages transports long non-coding RNA (lncRNA) CHL1-AS1 to promote 313 eESC proliferation, migration, and invasion, inhibits apoptosis by downregulating mir-610 and 314 upregulating Murine double minute 2 (MDM2)[59]. Additionally, ESC can influence macrophages to 315 promote EMS progress. Co-culturing with ESC, macrophage secreted cysteine-cysteine motif chemokine 316 ligand 20 (CCL20) and activated cysteine-cysteine motif chemokine receptor 6 (CCR6), which induced 317 the proliferation and migration of ESC as well as impaired the function of lysosome, thereby blocking 318 the autolysosomal degradation of ESC[60]. 319 Figure 3 Macrophages in endometrium. 320 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript During the menstrual cycle, monocytes in the uterus differentiate into macrophages. This process is 321 regulated by CCL2 and CX3CR1 and influenced by sex hormones, especially estrogen and progesterone. 322 In the physiological process, M1 macrophages remove uterine tissue debris and secrete inflammatory 323 cytokines and growth factors in the first phase of the immune response. M2 macrophages secrete anti-324 inflammatory cytokines and growth factors to promote tissue regeneration during the proliferative phase 325 of the adaptive immune response. CCL2, cysteine-cysteine motif chemokine ligand 2; CX3CR1, C-X3-326 C motif chemokine receptor 1. (Created by BioRender). 327 Figure 4 Macrophages in endometriosis. 328 In the abdominal cavity, lactate and IL-17A drive M1 macrophages to polarize to the M2 phenotype 329 via the SMAD2/SMAD3 pathway. Activation of ERβ in ESCs leads to macrophage recruitment 330 through the NF-κB/CCL2 pathway, which promotes macrophage differentiation into the M2 331 phenotype. M2 macrophages secrete cytokines, exosomes, and chemokines targeting ESCs, which 332 promote ESC invasion. In the endometrium, M1 macrophages secrete a number of pro-inflammatory 333 cytokines, including IL-1, IL-6, IL-8, HGF, and MCP-1. Additionally, they overexpress SIRPα, 334 which contributes to the formation of a pro-inflammatory environment. IL-17A, interleukin-17A; 335 SMAD2/SMAD3, SMAD family member 2/3; ERβ, estrogen receptor beta; NF-κB, nuclear factor-336 kappa B; CCL2, cysteine-cysteine motif chemokine ligand 2; IL-10, interleukin-10; IL-33, 337 interleukin-33; CCL20, cysteine-cysteine motif chemokine ligand 20; SIRPα, signal regulatory 338 protein α; IL-1, interleukin-1; IL-6, interleukin-6; IL-8, interleukin-8; HGF, hepatocyte growth 339 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript factor; MCP-1, monocyte chemoattractant protein-1. (Created by BioRender). 340 3 Macrophages in EAOC 341 EOC typically spreads through the abdominal cavity, attaching to the abdominal wall and spreading 342 easily within the cavity[61]. It is noteworthy that the abdominal cavity contains a significant number of 343 macrophages. Macrophages play an essential role in the development of EMS into EAOC[62]. However, 344 it is unclear how EOC evades macrophage-mediated anti-tumor immune surveillance. 345 3.1 The evolution of macrophages in EOC 346 Tumor macrophage’s functional phenotype and distribution are regulated by the dynamic nature of 347 the tumor microenvironment (TME), responding to tissue-specific and tumor-specific stimuli. During the 348 early stages of carcinogenesis, macrophages exhibit a higher degree of similarity to pro-inflammatory 349 M1-like phenotype when they infiltrate into the tumor from the periphery, and participate in tumor 350 antigen capturing, phagocytosis, and production of inflammatory factors[63]. As the tumor grows, stimuli 351 in the TME change, leading to alterations in the infiltration and polarization of macrophages. In ovarian 352 cancer, the TME predominantly drives the polarization of macrophages to an M2-like phenotype, which 353 were known as tumor associated macrophages (TAMs)[64]. A higher proportion of M2 macrophages and 354 a lower proportion of M1 macrophages are linked to a poor prognosis in ovarian cancer patients[64]. In 355 human EOC and ascites, TAMs constitute the most abundant infiltrating immune cell population[65]. 356 TAMs exhibit limited tumoricidal activity and promote immunosuppression, tumor cell invasion and 357 metastasis, and angiogenesis. TAMs of ovarian cancer can also directly mediate cell-cell interactions via 358 immune checkpoint receptors and their ligands, leading to functional impairment of T cell responses. 359 Furthermore, TAMs in ovarian cancer promote extracellular stromal remodeling, which contributes to 360 the invasion of various tumor cells. 361 3.2 Function of TAMs in TME 362 TAMs promote the development of ovarian cancer through a variety of ways. Inhibitory monocytes 363 and macrophages were recruited to the TME by producing cytokines, chemokines, growth factors, and 364 other molecules, including IL-6, leukemia inhibitory factor, CCL2, colony stimulating factor 1 (CSF1), 365 tumor necrosis factor (TNF), cysteine-cysteine motif chemokine ligand 2 (CCL22), C-X-C motif 366 chemokine ligand 12 (CXCL12), VEGF, periostin (POSTN), and Semaphorin 4D[63]. To promote tumor 367 growth and metastasis, macrophages are promoted to differentiate into M2 macrophages. Ovarian cancer 368 cells secrete macrophage colony-stimulating factor (M-CSF) to polarize TAMs towards the M2 369 phenotype. In EOC, the AT-rich interaction domain 1A (ARID1A)644delG/ E4F transcription factor 1 (E4F1) 370 complex induces the expression of histone deacetylase 6 (Hdac6) through GATA3 activation. The 371 overexpression of Hdac6 regulates the trafficking of IL-10 to the extracellular environment, promoting 372 M2 polarization of macrophages[66]. TAMs promote tumor growth by releasing cytokines, enzymes, and 373 chemokines that enhance the formation of ovarian cancer spheroids and the adhesion of cancer cells to 374 metastatic sites, allowing them to evade immune cell attack. TAMs secrete CCL18 to induce epithelial 375 mesenchymal transition (EMT) of ovarian cancer by upregulating the expression of Zinc finger E-box 376 binding homeobox 1 (ZEB1) and other EMT-related transcription factors. ZEB1, in turn, promotes the 377 transcription of M-CSF[67]. TAMs secrete epidermal growth factor (EGF) and upregulate αMβ2 integrin 378 on TAMs and intercellular cell adhesion molecule-1 (ICAM-1) on ovarian tumor cells, promoting binding 379 between tumor cells and TAMs. Additionally, TAM-secreted EGF activates EGFR on tumor cells, which 380 upregulates VEGF/ vascular endothelial growth factor receptor (VEGFR) signaling in surrounding tumor 381 cells, supporting tumor cell proliferation and metastasis[68]. M2 macrophages regulate vascular 382 permeability through the very late antigen 4 (VLA4)/ vascular cell adhesion molecule 1 (VCAM1) 383 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript pathway, which determines ovarian tumor ascites development. The study found that M2 macrophages 384 reduce VCAM1 levels in endothelial cells and regulate VLA4 expression when in direct contact with 385 them. However, overexpression of VLA4 or VCAM1 leads to increased vascular permeability[69]. 386 Macrophages can promote POSTN expression in ovarian cancer cells through TGF-β. POSTN released 387 by ovarian cancer cells can recruit macrophages. POSTN enhances integrin/ERK/NF-κB signaling 388 through autocrine effects on cancer cells to produce macrophage-attracting and mobilizing cytokines, 389 including macrophage inflammatory protein 1 beta (MIP-1β), MCP-1, tumor necrosis factor alpha (TNF-390 α), and regulated on activation normal T cell expressed and secreted (RANTES), leading to the 391 polarization of Tohoku Hospital Pediatrics-1 (THP-1) to M2 macrophages in vitro. This process may 392 potentially promote tumor growth[70, 71]. 393 3.3 Important molecules shared by EMS and EAOC 394 C-X-C motif chemokine receptor 3 (CXCR3) is expressed in various immune cell subsets, stromal 395 cells (including vascular endothelial cells), and some types of tumor cells[72]. CXCR3B is a variant of 396 CXCR3, and C-X-C motif chemokine 4 (CXCL4) is its major functional ligand. CXCL4 directly interacts 397 with VEGF and fibroblast growth factor-2 (FGF-2) to exert inhibitory effects on these angiogenic factors. 398 CXCL4 and its receptor CXCR3B have been found to be down-regulated in clear-cell ovarian cancer. 399 The evidence indicates that the CXCL4-CXCR3B axis may be disrupted in this type of cancer. In a 400 separate study, CXCL4 and its variant CXCL4L1 were significantly down-regulated in EAOC compared 401 to endometriosis[72]. CXCL4 was strongly expressed in CD68+ -infiltrating macrophages of 402 endometriosis. CXCL4 deficiency may be involved in the specific inflammatory microenvironment of 403 ovarian cancer, which arises from endometriosis. However, the inhibition of CXCL4 in cancer lesions 404 may be partially attributed to TAMs[72]. 405 Heme oxygenase 1 (HO-1) is a type II detoxifying enzyme that catalyzes the rate-limiting step in 406 heme degradation, producing carbon monoxide (CO), free iron, and biliverdin[73]. As a substrate for 407 HO-1, heme has been shown to be a potent pro-oxidant. It has been demonstrated to accelerate 408 inflammatory damage and promote cell death. However consequently, HO-1 is regarded as a pivotal 409 negative mediator of inflammatory cell and tissue damage[74]. HO-1 has been demonstrated to confer 410 an anti-inflammatory protective effect by modulating necroptosis and pyroptosis. On the contrary, in the 411 context of ferroptosis, HO-1 may play a role in promoting cell death by enhancing iron release. 412 Furthermore, HO-1 has been implicated in the co-regulation of autophagy[74-76]. HO-1 is mainly 413 expressed in macrophages. Macrophage recruitment and interaction with endometriotic cells were 414 thought to play a key role in the initiation and progression of endometriosis. Endometriotic cells secret 415 TGF-β1 to promote HO-1 expression in macrophage, thus protecting endometriotic cells from oxidative 416 damage and promoting cell survival[77]. Meanwhile, dysregulation of heme metabolism mediated by 417 HO-1 may be a crucial factor in the initiation and growth of EAOC. Compared to endometriosis tissues, 418 HO-1 is increasing expressed in ovarian clear cell carcinoma tissues. The expression of HO-1 in 419 macrophage favors M2 differentiation, promotes fibrosis of mature endometriosis lesions, decreases 420 heme levels in the tumor microenvironment, thereby supporting ovarian tumor growth[78]. In addition, 421 HO-1 expression has been observed in tumor-infiltrating macrophages and has been associated with 422 cancer progression, invasiveness, and aggressiveness[79]. Although HO-1 plays a promoting role in EMS 423 and EAOC, in principle, the downregulation of HO-1 results in elevated intracellular reactive oxygen 424 species (ROS) levels and DNA damage, which may contribute to tumorigenesis. In a subset of cancers, 425 such as lung cancer, HO-1 expression in macrophages has been observed to be reduced[80]. Yamada et 426 al observed a lower number of M2 macrophages expressing HO-1 in EAOC patients compared to those 427 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript with ovarian endometriosis. The sustained decrease of HO-1 expressing M2 macrophages may have an 428 important role in promoting the malignant transformation of endometriosis[81]. Consequently, the role 429 of HO-1 in the pathogenesis of tumor progression remains a subject of debate. 430 CD47 is a glycoprotein expressed on nearly all cell types. This molecule is referred as the "do not 431 eat me signal" as it functions by binding to SIRPα as a ligand, thereby inhibiting phagocytosis by 432 phagocytes, particularly macrophages[82]. In the current clinical landscape, the exploration of strategies 433 that target the SIRPα-CD47 innate immune checkpoint has emerged as a promising avenue for cancer 434 treatment[83, 84]. According to a study, the high expression of CD47 in ovarian tissue is associated with 435 the tumor's stage and grade[85]. The study found that CD47 was overexpressed in ovarian clear cell 436 carcinoma cells, which led to a reduction in macrophage phagocytosis and promoted the growth and 437 migration of human endometrioid (TOV-112D) and clear cell (TOV-21G) cancer cell lines by binding to 438 SIRPα[85]. 439 Oncogenic miR-1246 plays a significant role in chemotherapy resistance by inhibiting caveolin 1 440 (Cav1) in M2 macrophages. Additionally, exosomal miR-1246 promotes tumor progression in the tumor 441 microenvironment through M2 oncogenic macrophages[86]. Research has shown that miR-21, an 442 oncogene, partially regulates M2 macrophage polarization, promoting chemoresistance and inhibiting 443 apoptosis in ovarian cancer cells[64]. Additionally, ovarian cancer patients with high expression of 444 hypoxia-inducible factor 1a (HIF-1a) had higher CD163+ cell infiltration and inter-tumor miR-223 levels. 445 Exosomes-enriched miR-223 released from hypoxic macrophages promoted drug resistance of EOC cells 446 both in vivo and in vitro through the PTEN- phosphatidylinositol 3-kinase (PI3K)/ protein kinase B 447 (PKB/AKT) pathway[87]. 448 Under certain conditions, EMS can transform into EAOC. In the occurrence and development of 449 EMS and EOC, macrophages tend to polarize to M2 macrophages, weakening their phagocytic ability 450 and promoting disease progression[81]. Therefore, macrophages may play a key role in the 451 transformation of EMS into EAOC. Studying the functions of macrophages between EMS and EAOC 452 can aid in comprehending the mechanism of malignant transformation of EMS and developing new 453 immunotherapy methods. 454 4. Therapeutic strategy targeting macrophages in EMS and EAOC 455 In EMS lesions, endometriotic cells inhibit its apoptotic progression, while promoting invasion, 456 adhesion and proliferation pathways. In the meantime, angiogenesis and neurogenesis also participate in 457 pathogenesis of EMS[3]. Though a number of studies had unraveled the critical mechanisms of how 458 macrophages involve in the sophisticated progression from occurrence of EMS lesions to malignancy 459 transformation, whether targeting macrophages or macrophage-related markers could be applicable 460 options under clinical conditions remains in doubt[88, 89]. However, for the multiple drawbacks of 461 existing treatments of EMS, such as limited indications, adverse effects, unsatisfactory potencies and 462 long-term medication, the exploration of advanced macrophage-related therapies is extremely 463 demanding[3]. Here, we summarize the mainstream therapies of EMS and make a brief illustration of 464 their indications and drawbacks. Then we focus on reported macrophage-related therapeutic strategies 465 and propose its future perspective (Figure 5). 466 4.1 Existing therapies for EMS 467 The prevalently applied therapies of EMS include hormonal treatments, non-hormonal treatments 468 and surgery. Hormonal treatments are the mainstay of EMS therapies. By perturbing hormonal 469 fluctuations, hormonal treatments create amenorrhea and prevent inflammation from deterioration[90, 470 91]. Patients who were diagnosed with EMS and not desiring for pregnancy immediately would be 471 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript prescribed hormonal treatments, which include combined contraceptives, progestogens, Gonadotropin 472 releasing Hormone (GnRH) agonists, GnRH antagonists and aromatase inhibitors[3]. However, all these 473 drugs have distinct side effects to some extents, like nausea for combined contraceptives, weight gain for 474 progestogens as well as bone marrow density decline for GnRH agonists, GnRH antagonists and 475 aromatase inhibitors. More importantly, hormonal treatments only relieve the pain rather than radically 476 cure the EMS lesions, making it a long-term remedy[3, 90]. 477 Non-hormonal treatments mainly consist of conventional analgesics, like non-steroidal anti-478 inflammatory drugs (NSAIDS), and neuro-modulatory drugs, like analgesic tricyclic antidepressant, 479 selective serotonin uptake inhibitors and anticonvulsants[3].Clinicians only use these drugs to soothe 480 short-term sense of pain. On the other hand, the efficacy of NSAIDS on pain caused by EMS lacks high-481 quality evidence[92]. Also, long-term administration of NSAIDS and neuro-modulatory drugs may lead 482 to some severe adverse effects, like gastrointestinal bleeding and perforation for NSAIDS as well as 483 central nervous system and circulatory system toxicity for neuro-modulatory drugs. 484 Surgery treatment is offered when patients cannot tolerate medication treatments or have a plan for 485 pregnancy in the near future. For those suffering from ureteric or bowel obstruction due to EMS, surgery 486 is an inevitable option[91]. However, patients who accepted surgery have high rates of symptom and 487 lesion recurrence. This probably attributes to the continuous reflux of menstruation and sensitization of 488 central nervous system. What’s more, patients could also be afflicted with some surgery-related 489 complications like postoperative infection, rectovaginal fistula, neurogenic bladder and so on[90]. 490 4.2 Existing therapies for EAOC 491 According to several comprehensive reviews, the correlation between the morbidity of EMS and 492 ovarian cancer has been well corroborated[1, 93]. The major subtypes of EAOC include clear-cell ovarian 493 cancer and endometrioid ovarian cancer, which have relatively worse prognosis with 494 chemoresistance[94]. The standard treatment of EAOC is debulking operation followed by adjuvant 495 chemotherapy with paclitaxel and carboplatin or neoadjuvant chemotherapy with interval debulking 496 surgery, resembling that of common EOC, and the efficacy is apparently unsatisfying, especially for 497 patients with recurrence or advanced stage lesions[94, 95]. Efforts have been made in recent years to 498 ameliorate therapies by application of hyperthermic intraperitoneal chemotherapy (HIPEC)[96], 499 adjustment in the cycle and frequency of chemotherapies, combined use of antiangiogenic drugs like 500 bevacizumab[97], poly-adenosine diphosphate ribose polymerase (PARP) inhibitors like Olaparib and 501 rucaparib[98] and immunotherapies represented by checkpoint inhibiting[94]. However, a latest 502 published clinical trial found that addition of HIPEC to cytoreductive surgery did not improve 503 progression-free and overall survival in patients with advanced epithelial ovarian cancer[99]. Also, 504 whether dose-dense or conventional three-weekly paclitaxel administration would be more beneficial to 505 patients still entails further verification[100]. As two of the most promising drugs, bevacizumab and 506 PARP inhibitors display several drug-related toxic effects on the hematological system, gastrointestinal 507 system and urinary system[101]. With limited potency in BRCAwt and homologous recombination 508 proficient ovarian cancer, PARP inhibitors are restricted in use to some extents[95]. Furthermore, two 509 individual clinical studies on avelumab, a PD-L1 monoclonal antibody, do not support its use in the 510 frontline treatment setting[102, 103]. To conclude, though improvements have been witnessed in clinical 511 trials regarding these emerging solutions, more advanced and convincing approaches are still demanding. 512 4.3 Macrophage-related therapeutic strategies in EMS 513 There is an altered immune and inflammatory milieu in EMS lesions, where macrophages, the 514 critical modulator in EMS pathology, are recruited and produce an array of adhesion molecules 515 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript (fibronectin, intercellular adhesion molecule1), growth factors (insulin-like growth factor I, platelet-516 derived growth factor, vascular endothelial growth factor) and pro-inflammatory cytokines (IL-1, IL-6, 517 IL-8, IL-2, tumor necrosis factor)[1]. Ultimately, the aberrant inflammatory environment causes the 518 incomplete apoptosis and phagocytosis of shed endometrial cells during menses as well as implantation, 519 survival and proliferation of ectopic endometrium, resulting in chronic pain and infertility[26]. 520 Throughout the years, many therapeutic strategies targeting specific molecules or functions of 521 macrophages have been reported. 522 4.3.1 Strengthening phagocytosis of macrophages 523 The inefficient phagocytosis of macrophages in ectopic EMS lesions and peritoneal fluid is 524 necessary for the survival, adhesion and invasion of endometriotic cells in retrograde menstruation[26, 525 104]. Thus, the recovery of phagocytosis by macrophages may be a promising therapeutic strategy for 526 EMS patients. 527 CD47 blockade 528 SIRPα is an inhibitory receptor commonly expressed by macrophages and upregulated by ectopic 529 endometrial stromal cells during EMS[105]. By conjugating with CD47, which is also enriched in EMS 530 milieu, the immunoreceptor tyrosine-based inhibition motifs (ITIM) of SIRPα transduce inhibitory 531 signals, curb macrophage activation and suppress phagocytosis[106]. Li et al. found that CD47 blockade 532 not only significantly strengthened phagocytosis of macrophages towards human ESCs both in vitro and 533 in vivo, but abrogated the apoptosis tolerance of Human endometrial stromal cells (HESCs)[105, 107]. 534 Furthermore, CD47 blockade could alter the macrophage polarization by reducing M2 phenotype[105]. 535 Therefore, immunotherapy based on the CD47-SIRPα signaling pathway holds potential for treating 536 endometriosis. 537 Elevating cytosolic Ca2+concentration 538 Elevation of Ca2+concentration in cytoplasm is indispensable for macrophage efferocytosis, which 539 is critical for instant clearance of apoptosis cells[108, 109]. Another study designed a multifunctional 540 nanoparticle concomitantly loaded with calcium carbonate (CaCO3) and a specialized anti-inflammatory 541 mediator[110]. With concrete enhancement of efferocytosis by increasing the expression of T cell 542 immunoglobulin and mucin domain containing 4 (TIM4) and CD36, this nanoparticle also significantly 543 inhibited the formation of ectopic lesions in vivo[110]. Another study incubated mesoporous silica 544 nanoparticle immobilized with glucosaminyl muramyl dipeptide (GMDP) on the surface with 545 macrophage and observed significant higher expression of membrane scavenger proteins including CD36 546 and CD204[111]. Nevertheless, whether these treatments could be qualified therapeutic strategies for 547 EMS patients requires further investigation in clinical conditions. 548 Herbal extracts 549 Ferulic acid, ligustrazine and tetrahydropalmatine are separately isolated from components of a 550 traditional Chinese formula, which is commonly used for irregular menses. The combination use of 551 ferulic acid, ligustrazine and tetrahydropalmatine evidently enhance the phagocytosis ability of 552 macrophages in vitro and repress the growth of ectopic endometrial lesions[112]. 553 4.3.2 Reducing infiltration of macrophages 554 Several studies have corroborated the correlation between the increase of macrophage infiltration 555 and severity of EMS[47, 113, 114]. These excessively recruited macrophages induce angiogenesis, 556 fibrosis and neuroinflammation rather than phagocyting ectopic endometriotic debris. Over the years, 557 researchers have been seeking for adequate methods to reduce infiltrated macrophages in EMS lesion. 558 S1PR1 inhibitors 559 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript Sphingosine 1-phosphate receptor 1 (S1PR1), a kind of G protein-coupled receptor involved in 560 numerous immune-related diseases including multiple sclerosis, autoimmune encephalitis and diffuse 561 large B-cell lymphoma, was found significantly upregulated in endometriotic lesions[115, 116]. To 562 explore if S1PR1 blocking could inhibit EMS, Zhang et al. administered a broad-spectrum S1P modulator 563 and the specific S1PR1 modulator in EMS mice and observed strongly suppressed infiltration of 564 macrophages as well as attenuated local inflammation in lesions[117]. 565 Vitamin D receptor agonists 566 Vitamin D receptor (VDR) is a nuclear ligand-inducible transcription factor that interacts with the 567 active version of vitamin D, 1,25(OH)2D and regulates a vast number of physiological processes[118]. 568 With the prevalent expression on endometrial cells and all arrays of immune cells, VDR mediates distinct 569 anti-inflammatory effects by inhibiting NF-κB pathway. Endometrial cells with deficient NF-κB 570 signaling downregulate IL-8 production and reduce recruitment of macrophages, while peritoneal 571 macrophages with repressed NF-κB signaling decrease excretion of inflammatory cytokines[118, 119]. 572 Mariani et al. created an EMS model based on Balb/c mice and administered a VDR agonist called 573 elocalcitol, which possesses low calcemic liability and anti-inflammatory properties well defined in 574 benign prostatic hyperplasia[120]. Consequently, mice treated with elocalcitol have lower lesion weight, 575 lower macrophage recruitment in abdominal cavity, lower levels of IL-1α and IL-1β in collected 576 peritoneal fluid as well as lower inflammatory cytokines production by peritoneal macrophages[119]. 577 However, no relevant clinical trials on EMS are currently available. 578 4.3.3 Suppressing inflammatory capacities of macrophages 579 Estrogen receptor-inflammatory axis modulation 580 Estrogen receptor (ER) is expressed both by endometrial cells and mature macrophages in 581 endometrium as well as peritoneal fluid. In EMS patients, ER expression in endometriotic lesions is 582 significantly higher[121-123]. Endometrial tissue with enhanced ER activity recruit CD163+ 583 monocyte/macrophage cells to promote ectopic lesion growth[121]. Endometrial stromal cells with 584 elevated ER signaling upregulate CD200 production in ectopic endometrial tissue and curb macrophage 585 phagocytosis[122]. On the other hand, the expression of ER on macrophages in peritoneal fluid has a 586 positive correlation with production of inflammatory cytokines both in healthy individuals and EMS 587 patients[124]. The activation of ER signaling of macrophages also stimulates the production of brain-588 derived neurotrophic factor and neurotrophin 3, increasing the infiltration of nerve fibers in ectopic 589 lesions[125]. Protopanaxadiol, an aglycone of ginsenosides, was reported to induce downregulation of 590 ER in eutopic endometrium of EMS patients and alleviates interferon-gamma (IFN-γ) and IL-12 591 production from macrophage in abdominal cavity at the same time, which collaboratively restricted the 592 growth of ectopic lesions[126]. In 2015, two ER ligands called chloroindazole (CLI) and 593 oxabicycloheptene sulfonate (OBHS) that displayed potent antiestrogenic and anti-inflammatory 594 capacities were introduced. By conjugating with ER on endometrial cells and macrophages, CLI and 595 OBHS restrained the endometrial production of cytokines including IL-6, CCL2, CCL5 and TNF-α, 596 reduced the recruitment of macrophages in endometriotic lesions and finally suppressed the 597 endometriotic-like lesion formation in C57BL/6 mouse concurrently treated with uterine fragments and 598 estradiol[127]. However, no relevant clinical trials have been reported. 599 Neuron-inflammatory axis modulation 600 In the early 2000’s, people found that stimulation of the vagus nerve could suppress inflammatory 601 response in Lewis rats[128]. Following studies on neuroimmune nexus established an intrinsic model 602 of cholinergic anti-inflammatory pathway that precisely modulates systemic inflammatory state[129]. 603 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript In brief, vagus nerve controls a subpopulation of sympathetic neurons in the celiac and superior 604 mesenteric ganglia, which in turn sends messages to splenic nerve. The noradrenergic nerves in spleen 605 stimulate CD4+T cells by excreting norepinephrine (NE) and promote the production of acetylcholine 606 (Ach) by CD4+T cells. Finally, Ach diffuses towards macrophages, attaches to α7 -nicotinic 607 acetylcholine receptor (α7nAChR) on the surface of macrophages and inhibits the synthesis and release 608 of pro-inflammatory cytokines. 609 The strategies modulating catabolite gene activator protein (CAP) activation mainly include vagus 610 nerve stimulation (VNS) and pharmacological stimulation of α7nAChR. Interestingly, women with EMS 611 tend to have lower vagal activity as compared with controls[130]. In an EMS Balb/c mouse model, VNS 612 reduces total EMS lesion weight while vagotomy ablates this beneficial effect[130]. A study examining 613 IL-1β expression in human peritoneal mononuclear cells (PFMC) stimulated by lipopolysaccharide (LPS) 614 with or without α7nAChR agonists found a significant decrease of IL-1β expression in α7nAChR agonist 615 group[131]. The following study on EMS Balb/c mice model also observed a significant shrinkage of 616 EMS lesions when α7nAChR agonist was administered[131]. Though there is currently no reported 617 clinical trial concerning VNS and α7nAChR agonist, it could possibly be one of the promising therapeutic 618 strategies for patients with EMS in the future. 619 PGE2 receptor inhibition 620 The concentration of PGE2 is largely increased in peritoneal fluid of EMS patients, leading to the 621 growth and survival of ectopic endometriotic cells[132, 133]. NSAIDS was designed to inhibit the 622 activity of cyclooxygenase 2 (COX2), the rate limiting enzyme of PGE2 production. However, its 623 concurrent inhibition of COX1 may cause some adverse effects, restricting the dosage of NSAIDS. Thus, 624 researchers have resorted to PGE2 receptor antagonization in recent years and revealed myriad benefits 625 of concurrent inhibition of PGE2 receptor 2 (EP2) and PGE2 receptor 4 (EP4) including decreasing 626 angiogenesis and innervation of EMS lesions, alleviating pelvic pain by suppressing inflammation state 627 of dorsal root ganglia as well as restoring endometrial functional receptivity[134]. In the latest study, 628 Tomako et al. demonstrated that the combination of EP2 and EP4 antagonist also significantly decreased 629 PGE2-induced IL-6, VEGF, CXCL2 and CXCL3 production in peritoneal macrophages in vitro, making 630 it a potential therapeutic scheme[135]. 631 Herbal extracts 632 Throughout the years, people from different regions tried using processed extract from special local 633 plants to cure EMS and found these methods efficacious. For example, Euterpe oleracea is a common 634 plant found in the Amazon region of Brazil known for its high level of myriad beneficial phytochemicals, 635 which endow Euterpe oleracea with antioxidant, antinociceptive, anti-inflammatory and anti-cancer 636 activities[136]. In an EMS Sprague-Dawley rat model, Euterpe oleracea extract was administered 637 through gastric tube daily for 30 consecutive days and a significant decline of macrophage infiltration in 638 endometriotic lesion was observed. Simultaneously, the inflammatory profile measured by COX2, PGE2 639 and NO as well as angiogenesis process gauged by matrix metalloproteinase-9 (MMP-9), VEGF and 640 VEGFR-2 were ablated, leading to regression of lesion sites[136]. Another study on a Japan-derived 641 herbal implied that its ingredients, represented by ferulic acid, could suppress IL-8 production of patient-642 derived peritoneal macrophages[137]. Moreover, dehydrocostus lactone, an active compound extracted 643 from the roots of Aucklandia lappa, was found to promote lesion regression by inhibiting the expression 644 of M2 markers and NF-κB pathway activity in EMS-associated macrophages (EAMs)[138]. However, a 645 further exploration of latent mechanisms and verification of its effectiveness and viability in clinical 646 condition is lacking. 647 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript 4.3.4 Adjusting phenotypes of macrophages 648 LPSlow-trained macrophages 649 The paradigm of modifying innate immune cells’ capacity to respond to a second stimulation based 650 on metabolic changes and epigenetic reprogramming is called “innate immune memory” or “trained 651 immunity”[139-141]. Mohamed et al. treated peritoneal macrophages with low doses of 652 lipopolysaccharide (LPSlow) for in vivo immune training. In a mice model of endometriosis, 653 intraperitoneal injection of LPSlow-trained macrophages leads to a decrease in the expression of CXCR4, 654 CCR2, and CD206, an increase in IL-10 expression, and the inhibition of endometriotic lesion 655 growth[142]. This study presents a novel therapeutic approach for endometriosis, involving the 656 manipulation of innate immune memory to target immune dysfunction. 657 Niclosamide 658 Niclosamide is originally an effective anthelmintic drug against human tapeworm (cestode) 659 infection. However, the following studies unraveled its efficacy in multiple diseases including metabolic 660 syndrome, bacterial infection and cancer[143]. In the past decade, the outstanding efficacy of 661 Niclosamide in EMS treatment without disrupting reproductive abilities promoted further exploration on 662 its mechanisms, in which the correlation between Niclosamide and macrophages was discovered[144-663 146]. Niclosamide alleviates macrophage-induced cell viability and inflammatory factors secretion in 664 human ESCs and endometriotic epithelial cells[145, 146]. During the progression of EMS, EMS-like 665 lesions could ablate embryo-derived Tim4+ macrophages in peritoneal fluid and were replenished by 666 CCR2+ monocyte-derived macrophages, which show strikingly different functions and disrupt the 667 homeostasis of local immune environment[147]. Zhao et al. further revealed that Niclosamide could 668 stabilize the composition of peritoneal macrophages from different origins, partly reverse the gene 669 expression of peritoneal macrophages dysregulated by EMS-like lesions and reconstitute the interaction 670 between macrophages and B cells[147]. Another study found that Niclosamide was capable of reducing 671 pro-inflammatory GA TA6+ LpMs, thereby suppressed aberrant inflammation in the peritoneal fluid, 672 EMS-like lesions, pelvic organs and dorsal root ganglion[148]. Nevertheless, current research has only 673 been conducted in rodents. 674 4.3.5 Other 675 Extracellular ATP 676 The presence of elevated levels of extracellular adenosine triphosphate (eATP) has been shown to 677 induce the processes of apoptosis and pyroptosis in endometriotic epithelial cells, with this effect being 678 mediated through the MAPK/JNK/Akt pathway, thereby impeding their progression. The administration 679 of eA TP has been observed to result in a substantial reduction in the size of endometriosis lesions, 680 accompanied by a notable increase in macrophage infiltration and the resolution of functional defects in 681 macrophages. Furthermore, the presence of eATP in conjunction with macrophages fosters an 682 inflammatory milieu, thereby inciting pyrodeath and the subsequent apoptosis of EM epithelial cells. 683 Consequently, eATP treatment emerges as a promising candidate for non-hormonal treatment of 684 endometriosis[149]. 685 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript Figure 5 Therapies for endometriosis and endometriosis related ovarian cancer. 686 Prevalently applied therapies of EMS include hormonal treatments, non-hormonal treatments and surgery. 687 Hormonal treatment is a long-term remedy using contraceptives, progestogens, GnRH agonists, GnRH 688 antagonists and aromatase inhibitors. Non-hormonal treatments consist of conventional analgesics, 689 neuro-modulatory drugs and surgery. Macrophage-related therapeutic strategies in EMS includes 690 extracellular A TP , LPSlow-trained macrophages and targeting CD47-SIRPα. Standard treatment of EAOC is 691 debulk operation followed by adjuvant chemotherapy with paclitaxel and carboplatin or neoadjuvant 692 chemotherapy with interval debulking, and surgery. The ameliorative therapies of EAOC includes HIPEC, 693 adjustment in the cycle and frequency of chemotherapies, combine use of antiangiogenic drugs or PARP 694 inhibitors and immunotherapies represented by checkpoint inhibiting. EMS, endometriosis; EAOC, 695 endometriosis-associated ovarian cancer; GnRH, Gonadotropin releasing Hormone; ATP, adenosine 696 triphosphate; LPS, lipopolysaccharide; SIRPα, signal regulatory protein α; HIPEC, hyperthermic 697 intraperitoneal chemotherapy; PARP, poly-adenosine diphosphate ribose polymerase. (Created by 698 BioRender). 699 5. Concluding remarks 700 Since menarche, women experience monthly menstruation until menopause. During this time, 701 incomplete removal of menstrual reflux and endometrial debris may lead to the formation of EMS. 702 Dysfunctions in phagocytosis by peritoneal macrophages and phenotypic switching to M2 macrophages 703 promote the development of endometriotic lesions, angiogenesis, and neurogenesis. Additionally, M2 704 macrophages contribute to the malignant transformation of EMS. Analyzing the role of macrophages in 705 EMS is crucial for understanding the pathogenesis of EMS, developing targeted treatments involving 706 macrophages, and reducing the incidence of EMS-associated tumors. 707 708 Financial Support 709 This study was supported by the grants from the National Natural Science Foundation of China 710 (82073165), Natural Science Foundation of Guangdong province (2214050008966, 711 20257625412,26842516959), the Medical Science and Technology Research Foundation of Guangdong 712 Province (A2023124). 713 714 https://doi.org/10.1017/erm.2025.10012 Published online by Cambridge University Press Accepted Manuscript Author’s Contributions 715 MQL, CRS, LHK and L YS drafted the manuscript. MQL and CRS drew the figures. HZZ, HXR and 716 ZQB revised draft. HW, ZL Y and YZY made forms. MQL, LHK, LYS, CRS, HW, ZLY , YZY , LYR and 717 DZH searched the literature. WS AND ZW discussed the concepts of the manuscript and approved the 718 version to be submitted. 719 720 Conflict of Interest 721 The authors declare no competing interests. 722 723 Ethical Standards 724 The authors assert that this work does not involve human or animal experimental procedures. 725 726

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