M1 Infiltration Research Articles

Abstract 4126: CSF-1R inhibition blocks rhabdomyoscarcoma metastasis by polarizing macrophage differentiation

Abstract Colony Stimulating Factor 1 (CSF-1) is overexpressed by many tumors and predicts poor clinical outcome. CSF-1 expression results in the recruitment of CSF-1R (Fms)-expressing cells, which have been shown to suppress tumor immunity, thereby promoting cancer progression. PLX3397 is a novel, orally active small molecule inhibitor that selectively targets CSF-1R, KIT, and oncogenic FIT3. CSF-1R and KIT regulate the tumor, tumor microenvironment, and the pre-metastatic niche. However, it is unknown if targeting the myeloid CSF-1R expressing component of the tumor microenvironment regulates metastasis of pediatric tumors, such as rhabdomyosarcoma. Using the M3-9-M murine syngeneic orthotopic rhabdomyosarcoma model, we evaluated the effects of PLX3397 on primary tumor growth and metastatic progression. This highly metastatic rhabdomyosarcoma model has high infiltration of CSF-1R-expressing bone marrow-derived cells, which includes inflammatory monocytes, M2 macrophages, and myeloid-derived suppressor cells. Therefore, we hypothesized PLX3397 would have a direct effect on CSF-1R expressing M2 macrophages and inflammatory monocytes, resulting in decreased metastasis. Using both in vitro and in vivo models, we found that PLX3397 treatment decreased tumor-infiltrating M2 macrophages and skewed the differentiation of inflammatory monocytes toward M1 macrophages and development of immunocompetent mature dendritic cells. This resulted in impairment of spontaneous M3-9-M metastasis to secondary sites. In addition, we have found that the polarization of inflammatory monocytes reversed the T cell suppressive capability of tumor-derived myeloid cells. In conclusion, CSF-1R blockage with PLX3397 inhibited metastatic progression by decreasing the infiltration of M2 macrophages in the primary tumor and pre-metastatic niche. The polarization of inflammatory monocytes to dendritic cells restored the immune recognition and decreased metastasis. This data supports our rationale for testing PLX3397 in patients with high M2 infiltration and suggests value in this agent as an adjuvant setting for metastasis prevention in high-risk patients. Note: This abstract was not presented at the meeting. Citation Format: Justin Evans, Amber Giles, Caitlin Reid, Rosandra Kaplan. CSF-1R inhibition blocks rhabdomyoscarcoma metastasis by polarizing macrophage differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4126. doi:10.1158/1538-7445.AM2015-4126

Abstract 1273: Relation between tumor-associated macrophage (TAM) subsets and CD47 expression on squamous cell carcinoma of the head and neck (SCCHN) in tumor microenvironment

Abstract Introduction: In general, TAM is classified into M1 producing Th1 cytokines and M2 secreting IL-10. Numerous reports have demonstrated that M2 infiltration to tumor microenvironment associates with local immunosuppression and proliferation of cancer cells. In this study, we investigated phagocytic potentials of M1 and M2 in SCCHN in relation to the expression of CD47 molecule, which is known as “Don't eat me” signal against macrophage (Mϕ). Material and Methods: CD68 (pan-Mϕ marker) positive CD163 (M2 marker) negative M1 (derived with GM-CSF and IFN-γ) and CD68+CD163+M2 (derived with M-CSF and IL-10) were induced from positively selected CD14+ cells in PBMC from a healthy donor. Phagocytosis of CD47+/CD47-SCCHN cell lines labeled with CFSE by M1/M2 were assessed using multi-color flow cytometry. Suppressions of the phagocytosis by neutralizing anti-CD47 antibody were also evaluated. Furthermore, we examined CD68, CD163 and CD47 expressions immunohistochemically in 74 cases of oral squamous cell carcinoma, and relation between Mϕ counts or CD47 expression on cancer cells and clinicopathological parameters or prognoses. Results: M1 derived from PBMC showed higher phagocytic potential comparing to M2. Phagocytosis of M1 was dose-dependently inhibited by anti-CD47 neutralizing antibody, however M2 phagocytosis was not suppressed. In immunohistochemical analysis, while CD47 was expressed in almost half cases of oral cancer, CD47 expression did not correlate to the numbers of total Mϕ nor M2. Patients with high CD47 expression on cancer cells, increasing number of total Mϕ or M2 showed significantly shorter overall survival. Conclusion: Only M1 showed CD47-dependent phagocytosis of CD47-expressed cancer cells in vitro. While CD47 expression on patient samples did not correlate with number of total Mϕ or M2, each of them independently showed significant association with prognosis. Various mechanisms were presumed between CD47 expression on SCCHN cells and tumor-infiltrating Mϕ subsets. Citation Format: Koichi Sakakura, Hideyuki Takahashi, Kazuaki Chikamatsu. Relation between tumor-associated macrophage (TAM) subsets and CD47 expression on squamous cell carcinoma of the head and neck (SCCHN) in tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1273. doi:10.1158/1538-7445.AM2015-1273

Abstract 1248: Environmental factors (heavy metal, alcohol) enhance tumor malignant behaviors

Abstract Global cancer incidence and mortality increases dramatically word wild, epically in China, even though significant progress has been made against cancer. Cancer will account for the first most common cause of disease-related death in China. Abundant evidences demonstrated both genetic and environmental factors might contribute to human cancer initiation and progression. In our lab, we focus on role of gain-of-function mutant SHP-2 (genetic factor) and heavy mental contamination or alcohol drinking (environmental factors) on tumor progression. Here, we reported environmental factors like heavy mental arsenic and alcohol consumption promote digestive tumor and breast cancer malignant behaviors. The first, clinic data showed that an increasing arsenic concentration in III/IV TNM stage gastric cancer patient, then an enhanced cancer cell malignant behaviors (proliferation/ motility/ metastasis, etc) were found in gastric cancer or colon cancer cell lines chronic treated with 5-15 nm arsenic in vitro and in vivo. EMT might contribute to this promotion process induced by arsenic. The second: a positive correlation between alcohol consumption and advanced Tumor-Node-Metastasis (TNM) stages, higher vessel invasion and poorer prognosis in Hepatocellular carcinoma (HCC) were found. In vitro and in vivo experiments further indicated that alcohol promoted the progression and migration/invasion of HCC, breast cancer and lewis lung carcinoma (LLC). Specifically, we found that alcohol enhanced the migration/invasion of tumor cells and increased tumor angiogenesis. Consistently, higher expression of VEGF, MCP-1, activated NF-κB and more infiltrated M2 macrophage was observed in tumor tissues of alcohol-drinkers. Alcohol induced the accumulation of intracellular reactive oxygen species (ROS) and the activation of NF-κB signaling in HepG2 cells. Conversely, blockage of alcohol-mediated ROS accumulation and NF-κB signaling inhibited alcohol-induced expression of VEGF and MCP-1, the tumor growth, angiogenesis and metastasis. These results suggested that chronic moderate alcohol consumption may promote the progression and metastasis of tumor; the oncogenic effect may be at least partially mediated by the ROS accumulation and NF-ĸB-dependent VEGF and MCP-1 up-regulation. Even more, an increasing cancer stem cell population was found in tumor cell lines under low dose alcohol induction. Our results make us presume chronic stress induced by environmental factors like alcohol, heavy mental, etc might activate cell signal and enhance tumor progression in a universal way. Those studies will be benefited to tumor prevention. This study was supported by a grant from the National Natural Science Foundation of China (codes: 30873046, 30973424, and 81072663). Note: This abstract was not presented at the meeting. Citation Format: Siying Wang. Environmental factors (heavy metal, alcohol) enhance tumor malignant behaviors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1248. doi:10.1158/1538-7445.AM2015-1248

CXCL16 suppresses liver metastasis of colorectal cancer by promoting TNF-α-induced apoptosis by tumor-associated macrophages.

BackgroundInhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients.MethodsWe generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages.ResultsCXCL16 expression enhanced TNF-α-induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-κB-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF-α-induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF-α-induced apoptosis through the induction of M1 macrophages, which released TNF-α. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis.ConclusionsCollectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-949) contains supplementary material, which is available to authorized users.

Correlation of tumor-associated macrophages with clinicopathologic factors and angiogenesis in gastric cancer.

54 Background: Tumor associated macrophages (TAMs) are major components of tumor environment, and polarized into M1 and M2 type. M1 has been known as antitumorigenic, whereas M2 as protumorigenic. M2 have a significant role in tumor progression by promoting tumor cell invasion, migration and angiogenesis. We evaluated the M2 macrophages to investigate its importance in predicting clinical outcome or prognosis, and the relationship between M2 and angiogenesis in patients with gastric cancer. Methods: Formalin-fixed, paraffin-embedded blocks were obtained from the 88 patients with gastric cancer. CD163+ TAMs and CD105+ vessels were evaluated by immunohistochemical staining and the extent of CD163+ TAMs in tumor were divided into three groups: (A) infiltrated TAMs in cancer cell nest (nest TAMs); (B) infiltrated TAMs in tumor stroma (stroma TAMs); and (C) infiltrated TAMs along the invasive margin of a tumor (margin TAMs). Results: The increased stroma and margin TAMs (>72 and >102, respectively) were closely correlated with lymph node metastasis, TNM stage and lymphatic invasion (p<0.05 in 3 factors) and positive correlation existed between the stroma and margin TAMs (p<0.001). Disease-free survival rate analyzed using the Kaplan– Meier method was significantly lower in patient with high stroma and margin TMAs than patient with low stroma and margin TAMs (p=0.0039 and 0.0499, respectively). CD105+ vessels showed significant correlation with lymph node metastasis and lymphatic invasion.(p=0.006 and p<0.0001, respectively) Stromal, marginal and nest TAMs were positively correlated with CD105+ vessels (p=0.001). Conclusions: M2 infiltration in tumor stroma and tumor margin in gastric cancer showed relationship with aggressive phenotypes such as lymph node metastasis, TNM stage and lymphatic invasion and was correlated with angiogenesis. This study supports the view that the adverse prognostic impact of M2 involves tumor angiogenesis in gastric cancer.

Regulation of Apoptosis and Innate Immune Stimuli in Inflammation-Induced Preterm Labor

An innate immune response is required for successful implantation and placentation. This is regulated, in part, by the a2 isoform of V-ATPase (a2V) and the concurrent infiltration of M1 (inflammatory) and M2 (anti-inflammatory) macrophages to the uterus and placenta. The objective of the present study was to identify the role of a2V during inflammation-induced preterm labor in mice and its relationship to the regulation of apoptosis and innate immune responses. Using a mouse model of infection-induced preterm delivery, gestational tissues were collected 8 h after intrauterine inoculation on day 14.5 of pregnancy with either saline or peptidoglycan (PGN; a TLR 2 agonist) and polyinosinic-polycytidylic acid [poly(I:C); a TLR3 agonist], modeling Gram-positive bacterial and viral infections, respectively. Expression of a2V decreased significantly in the placenta, uterus, and fetal membranes during PGN+poly(I:C)-induced preterm labor. Expression of inducible NO synthase was significantly upregulated in PGN+poly(I:C)-treated placenta and uterus. PGN+poly(I:C) treatment disturbed adherens junction proteins and increased apoptotic cell death via an extrinsic pathway of apoptosis among uterine decidual cells and spongiotrophoblasts. F4/80(+) macrophages were increased and polarization was skewed in PGN+poly(I:C)-treated uterus toward double-positive CD11c(+) (M1) and CD206(+) (M2) cells, which are critical for the clearance of dying cells and rapid resolution of inflammation. Expression of Nlrp3 and activation of caspase-1 were increased in PGN+poly(I:C)-treated uterus, which could induce pyroptosis. These results suggest that the double hit of PGN+poly(I:C) induces preterm labor via reduction of a2V expression and simultaneous activation of apoptosis and inflammatory processes.

Tumor associated macrophages polarization dictates the efficacy of BCG instillation in non-muscle invasive urothelial bladder cancer

BackgroundTo evaluate the prognostic role of TAMs in patients affected by non-muscle invasive bladder cancer (NMIBC), undergone Trans Urethral Resection of Bladder (TURB) and Bacillus Calmette-Guerin (BCG) therapy.MethodsData from 40 patients (36 men, 4 women), mean age 69 years (40-83 years), treated for NMIBC with TURB and BCG instillation were collected. Two different groups were considered: group with and group without bladder cancer recurrence. Correlations between immunofluorescence measured Mtot, M1 and M2 infiltration and clinicopathological parameters were evaluated using Spearman and Mann–Whitney methods. The recurrence-free survival rate was calculated using the Kaplan-Meier method.ResultsCD68 positive cells (Mtot) were observed in all specimens tested. High Mtot, M1 and M2 infiltration was observed in patients with disease recurrence, even before endovescical BCG instillation. Significant value for M2 infiltration (p = 0,042) was found calculating significativity between two group medians before BCG therapy. p = 0,072 and p = 0,180 were observed correlating median of Mtot and M1 between two groups of patients respectively. Values of p = 0,44, p = 0,23 and p = 0,64 from correlation between DFS and Mtot, M1 and M2 median in patients before endovescical BCG instillation, were calculated respectively. Comparing DFS and Mtot, M1 and M2 median in patients group after endovescical BCG instillation significant values were obtained (p = 0,020; p = 0,02; and p = 0,029 respectively).ConclusionsM2 tumor infiltration could be a prognostic value of recurrence in patients with NMIBC.

Spleen-Derived Interleukin-10 Downregulates the Severity of High-Fat Diet-Induced Non-Alcoholic Fatty Pancreas Disease

Obesity is associated with systemic low-grade inflammation and is a risk factor for non-alcoholic fatty pancreas disease (NAFPD), but the molecular mechanisms of these associations are not clear. Interleukin (IL)-10, a potent anti-inflammatory cytokine, is released during acute pancreatitis and is known to limit inflammatory responses by downregulating the release of proinflammatory mediators. The origin of IL-10 that suppresses pancreatitis has not been investigated. Since obesity is known to reduce expression of proinflammatory cytokines in the spleen, we examined whether spleen-derived IL-10 regulates NAFPD caused by high-fat (HF) diet-induced obesity. The following investigations were performed: 1) IL-10 induction from spleen was examined in male mice fed a HF diet; 2) triglyceride content, expression of pro- and anti-inflammatory cytokines and infiltration of M1 and M2 macrophages were determined to evaluate ectopic fat accumulation and inflammatory responses in the pancreas of splenectomy (SPX)-treated mice fed HF diet; 3) exogenous IL-10 was systemically administered to SPX-treated obese mice and the resulting pathogenesis caused by SPX was assessed; and 4) IL-10 knockout (IL-10KO) mice were treated with SPX and ectopic fat deposition and inflammatory conditions in the pancreas were investigated. Obesity impaired the ability of the spleen to synthesize cytokines, including IL-10. SPX aggravated fat accumulation and inflammatory responses in the pancreas of HF diet-induced obese mice and these effects were inhibited by systemic administration of IL-10. Moreover, SPX had little effect on fat deposition and inflammatory responses in the pancreas of IL-10KO mice. Our findings indicate that obesity reduces IL-10 production by the spleen and that spleen-derived IL-10 may protect against the development of NAFPD.

Site-specific subtypes of macrophages recruited after peripheral nerve injury

After partial ligation of mouse sciatic nerve, the subtypes of macrophages were examined in the injured nerve and dorsal root ganglia (DRGs). Many M1 macrophages, which were inducible nitric oxide synthase (iNOS)-positive and arginase-1 (Arg-1)-negative, and neutrophils infiltrated the injured nerve. In contrast, almost all macrophages infiltrating the ipsilateral side of DRGs after the nerve injury were iNOS⁻/Arg-1⁺, M2 type. The infiltration of M1 and M2 macrophages was first observed in the injured nerve and ipsilateral DRGs on days 1 and 2, respectively. In addition, the macrophage infiltration preceded the activation of microglia in the ipsilateral dorsal horn of spinal cord. Thus, infiltrating macrophages after peripheral nerve injury may play unique roles dependent on the location in the development of neuropathic pain.

Placental ATPase Expression Is a Link Between Multiple Causes of Spontaneous Abortion in Mice

The a2 isoform of vacuolar ATPase (ATP6V0A2 referred to as a2V) plays a pivotal role in successful pregnancy and provides a microenvironment to maintain the delicate immunological balance at the feto-maternal interaction. We studied the expression of a2V mRNA in embryos and placenta of abortion-prone (female CBA × male DBA) murine matings or LPS (lipopolysaccharide)-treated mice. The expression of a2V was significantly higher in the placentas of nonabortion-prone (female BALB/c × male BALB/c and female CBA × male BALB/c) matings compared with the abortion-prone (female CBA × male DBA) mating. The expression of a2V was significantly decreased in the placentas treated with LPS in both female CBA × male DBA and female BALB/c × male BALB/c mating combinations with increased Lif, Il1b, and Tnf expression in the placenta. Decreased expression of a2V in the placenta is directly correlated with high percentages of pregnancy loss in abortion-prone mating (female CBA × male DBA) as well as in LPS-treated animals. The normal expression of placental a2V on Day 16 in the nonabortion-prone matings correlated with higher Mcp1 (monocyte chemotactic protein 1) gene expression, markedly higher infiltration of M1 and M2 macrophages, and no significant polarization patterns (M1/M2 = 1.2-1.6). However, in the abortion-prone mating, decreased placental a2V expression correlated with significantly lower Mcp1 gene expression with less infiltration of M1 and M2 macrophages and with polarization patterns skewed to M1 phenotypes (M1/M2 = 3.9-4.2). These data indicate that the higher expression of placental a2V is associated with dynamic infiltration of M1 and M2 macrophages through the induction of Mcp1 expression. This strengthens our hypothesis that a2V regulates the delicate cytokine and chemokine networks that coordinate the recruitment of macrophages for successful placental development and growth at the feto-maternal interface.

Abstract 3809: Tumor-released GPI-anchored proteins bind to mannose receptor and polarize tumor-infiltrating macrophages

Abstract Tumors polarize their microenvironment to escape immune-mediated destruction and to promote their growth. Macrophages respond to microenvironmental signals and can be activated toward a classical (M1) or an alternative (M2) phenotype. Tumor-associated macrophages (TAM) resemble to M2 macrophages with a phenotype characterized by the overexpression of IL10, scavenger receptor SR-A, CD68 and MR phenotype but hampered levels of IL-12. M2, in contrast to M1 macrophages, promote TH2 immune responses as well as tissue remodelling and M2 infiltration in solid tumors has been shown to significantly correlate with poor prognosis. Recent evidences suggest that macrophage differentiation towards TAM involves a “chemical conversation” via exchange of unknown soluble extracellular mediators between tumor cells and macrophages. In addition, previous work showed that the cross-linking mannose receptor (MR) with an anti-MR mAb could cause dendritic cell differentiation into APCs promoting T-cell anergy. We hypothesized that MR expressed by macrophages could be bound by tumor-released GPI-anchored glycoproteins through the GPI-anchor mannose residues freed by cleavage. We also hypothesized that MR binding by GPI-anchor could trigger macrophage polarization toward TAMs. To test our hypotheses, we established an in vitro model system of cell co-culture in transwell allowing chemical exchanges between human monocyte-derived macrophages and human ovarian cancer cell line Ovcar3 that sheds mesothelin, a cancer biomarker and a GPI-anchored glycoprotein. Macrophage phenotype changes were monitored by flow cytometry and qRT-PCR. We also isolated three novel recombinant antibodies (scFv) against MR by magnetic and flow sortings from a novel yeast-display human scFv library. We describe here for the first time that tumor-released mesothelin bound to macrophage cell surface in presence of calcium, and that mesothelin binding to macrophages could be blocked by the anti-MR scFv. Furthermore, macrophage phenotype switch toward M2 during coculture with tumor cells could be prevented by one of the anti-MR scFv. Finally, we demonstrated that M1 phenotype could be rescued during coculture with tumor cells with one of the anti-MR scFv. To further characterize the role of MR in vivo, we have implanted mannose receptor knock-out mice intraperitoneally (IP) with luminescent-labeled ovarian cancer cells that overexpress mesothelin. We are now monitoring the tumor growth by in vivo imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3809.

Abstract 5670: KLF5 Controls Macrophage Polarity in Chronic Inflammatory Diseases via S100 Proteins

Chronic kidney disease is a major risk factor for atherosclerotic cardiovascular disease. Chronic inflammation has been shown to be involved in both diseases. Therefore, the molecular mechanisms underlying them are likely to be closely linked with each other. We have previously demonstrated that the transcription factor KLF5 is involved in cardiovascular remodeling. In the present study we addressed if KLF5 also plays a role in chronic kidney disease. KLF5 expression levels were increased in various kidney disease models including unilateral ureteral obstruction (UUO) and KLF5 +/− mice ameliorated renal pathology induced by UUO. Genome-wide analyses of potential KLF5 target genes using ChIP-seq and microarrays identified S100A8 and S100A9. S100A8 and A9 are secretory proteins and form a heterodimer. S100A8/A9 induced macrophage accumulation in Matrigel plugs and direct injection of S100A8/A9 into kidneys resulted in infiltration of M1 classically activated macrophages. In KLF5 +/− mice S100A8/A9 expression was reduced and M1 macrophage infiltration was markedly suppressed in UUO. Interestingly, UUO induced infiltration of M2 alternatively activated macrophages during later stages of disease development. In KLF5 +/− mice macrophage activation was shifted to the M2 polarity. M2 macrophages produced anti-inflammatory and pro-fibrotic cytokines, including IL-10 and TGFb1. This shift in macrophage polarity is likely to be primarily accountable for the UUO phenotype of KLF5 +/− , in which tissue inflammation, apoptosis and destruction were ameliorated during early stages, while fibrosis was increased in later time points. Taken together, KLF5 appears to regulate tissue remodeling by controlling the macrophage polarity via inflammatory mediators including S100A8/A9. Because KLF5 is also essential for cardiovascular remodeling, the KLF5-S100 axis may play an important role in chronic inflammation in various chronic diseases, including cardiovascular, renal and metabolic diseases.

Tracking fluid flow during deep crustal anatexis: metasomatism of peridotites (Naxos, Greece)

Horizons of ultramafic lenses were metamorphosed with host felsic gneisses at upper amphibolite facies conditions during the M2 event on the island of Naxos, Greece. The synkinematic peak M2 Ol–Opx–Hbl–Chl–Spl assemblage of the Main, migmatite-associated, Ultramafic Horizon (MUH) retains mantle-like chemical and oxygen isotope compositions and thus shows no evidence of infiltration of fluids from the host rocks. A bimodal distribution of temperatures, grouped at 700 and 1,200 °C, is given by oxygen isotope Opx–Ol thermometry in the MUH meta-peridotites and indicates partial oxygen exchange during M2 superposed over previous mantle fractionation. The Agia Ultramafic Horizon (AUH), a coarser-grained and unfoliated peridotite, occurs within sillimanite gneisses in northwest Naxos and contains talc–enstatite and olivine domains. Recrystallization of the AUH peridotite during post-peak M2 infiltration of silica-rich, high δ18O fluids is indicated by lack of deformation, increased activity of silica required to stabilize the talc–enstatite assemblage, extremely high δ18O values of Ol and Opx and Δ18O(Opx–Ol) temperatures of 520–650 °C. The source for these fluids is inferred to be aplitic and pegmatitic dikes emanating from the migmatitic core of Naxos and intruding the AUH. At peak M2 temperatures and during anatexis of gneisses, volumes of fluid were small and fluid composition was locally buffered in the deeper part of the Naxos section. Crystallization of melts within the migmatitic core released siliceous fluids and initiated an episode of retrograde hydrous metamorphism in the overlying sequences, as observed in the AUH.