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Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth

Abstract

The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells’ adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites1. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs2. Clearly, the dynamic interaction between metastatic tumour cells and extrinsic signals at individual metastatic organ sites critically effects the subsequent metastatic outgrowth3,4. Yet, it is unclear when and how disseminated tumour cells acquire the essential traits from the microenvironment of metastatic organs that prime their subsequent outgrowth. Here we show that both human and mouse tumour cells with normal expression of PTEN, an important tumour suppressor, lose PTEN expression after dissemination to the brain, but not to other organs. The PTEN level in PTEN-loss brain metastatic tumour cells is restored after leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN messenger RNA and protein downregulation is epigenetically regulated by microRNAs from brain astrocytes. Mechanistically, astrocyte-derived exosomes mediate an intercellular transfer of PTEN-targeting microRNAs to metastatic tumour cells, while astrocyte-specific depletion of PTEN-targeting microRNAs or blockade of astrocyte exosome secretion rescues the PTEN loss and suppresses brain metastasis in vivo. Furthermore, this adaptive PTEN loss in brain metastatic tumour cells leads to an increased secretion of the chemokine CCL2, which recruits IBA1-expressing myeloid cells that reciprocally enhance the outgrowth of brain metastatic tumour cells via enhanced proliferation and reduced apoptosis. Our findings demonstrate a remarkable plasticity of PTEN expression in metastatic tumour cells in response to different organ microenvironments, underpinning an essential role of co-evolution between the metastatic cells and their microenvironment during the adaptive metastatic outgrowth. Our findings signify the dynamic and reciprocal cross-talk between tumour cells and the metastatic niche; importantly, they provide new opportunities for effective anti-metastasis therapies, especially of consequence for brain metastasis patients.

Main

The remarkable phenotypic plasticity observed in metastasis is indicative of co-evolution occurring at specific metastatic organ microenvironments5,6. To obtain insights into how disseminated tumour cells acquire essential traits from metastatic microenvironments for successful outgrowth, we analysed public gene expression profiles of clinical metastases from distinct organs as well as organ-specific metastases from mice injected with various cancer cells (Extended Data Fig. 1a–c). Notably, PTEN mRNA was markedly downregulated in brain metastases compared to primary tumours or other organ metastases. Our immunohistochemistry (IHC) analyses of PTEN expression confirmed a significantly higher rate of PTEN loss (defined by an immunoreactive score (IRS) of 0–3)7 in brain metastases (71%) than in unmatched primary breast cancers (30%) (Fig. 1a). PTEN loss was also detected at a significantly higher frequency in brain metastases (71%) than in matched primary breast cancers (37%) of an independent patient cohort (Fig. 1b).

Figure 1: Brain microenvironment-dependent reversible PTEN downregulation in brain metastases.
figure1

a, Representative IHC staining and histograms of PTEN protein levels in primary breast tumours (n = 139) and unmatched brain metastases (mets) (n = 131) (Chi-square test, P < 0.001). b, Histograms of PTEN protein levels in primary breast tumours and matched brain metastases from 35 patients (Chi-square test, P = 0.0211). c, PTEN western blots (left) and brain metastasis counts 30 days after intracarotid injection (right) of MDA-MB-231Br cells transfected with control or PTEN shRNAs. Macromets: >50 μm in diameter; micromets: ≤50 μm (mean ± s.e.m., Chi-square test, P = 0.1253). d, PTEN IHC staining of tumours derived from clonally expanded PTEN-normal sublines. ICA, intracarotid artery; MFP, mammary fat pad. e, Western blot and quantitative reverse transcriptase PCR (qRT–PCR) of PTEN expression in the indicated parental (P) and brain-seeking (Br) cells under culture (3 biological replicates, with 3 technical replicates each). f, Schematic of in vivo re-establishment of secondary (2°) brain metastasis, MFP tumour, and their derived cell lines. g, PTEN qRT–PCR (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each) and PTEN IHC in HCC1954Br secondary tumours and cultured cells.

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To test a possible role for PTEN loss in brain metastasis8,9, we intracarotidly injected PTEN-knockdown tumour cells and assessed experimental brain metastasis; unexpectedly, neither incidence nor size of brain metastases was increased (Fig. 1c). Furthermore, patients with PTEN-normal or PTEN-loss primary tumours had comparable levels of brain-metastasis-free survival, and patients with or without brain metastases had similar PTEN levels in their primary tumours (Extended Data Fig. 1d, e). Thus, the observed PTEN loss in brain metastases was unlikely to be derived from PTEN-low primary tumours. To investigate whether PTEN loss in brain metastasis is a secondary non-genetic event imposed by the brain microenvironment, we injected five PTEN-normal breast cancer cell lines either into mammary fat pad (MFP) or intracarotidly to induce brain metastasis. Notably, the PTEN level was significantly decreased in brain metastases compared to the respective MFP tumours or lung metastases (Extended Data Fig. 2a, b). We repeated the injections with cells clonally expanded from single PTEN-normal tumour cells, and observed similar phenotypes (Fig. 1d), suggesting that PTEN-loss brain metastases were not selected from pre-existing PTEN-low cells in the primary tumours. Surprisingly, established sublines from PTEN-low brain metastases (primary Br cells) regained PTEN expression in culture comparable to parental cells (Fig. 1e). Analogously, two in-vivo-selected brain-seeking sublines exhibited similar PTEN levels to their matched parental cells in vitro (Extended Data Fig. 2c). Re-injecting the cultured PTEN-normal primary brain sublines conferred a distinct PTEN loss in secondary brain metastases, but not in secondary MFP tumours, and PTEN levels in secondary brain subline cells were fully restored again in culture (Fig. 1f, g and Extended Data Fig. 2d), indicating a reversible non-genetic PTEN loss in the brain tumour microenvironment (TME).

To explore how the brain TME regulates PTEN in metastatic cells10,11,12, we co-cultured tumour cells with primary glia (>90% astrocytes)13, cancer-associated fibroblasts (CAFs), or NIH3T3 fibroblasts. Co-culture with glia led to a significant decrease of PTEN mRNA and PTEN protein (Fig. 2a, b and Extended Data Fig. 2e, f) in all tumour cells, but did not affect PTEN promoter methylation or activity (Extended Data Fig. 2g, h). This prompted us to examine whether glia reduce PTEN mRNA stability through microRNAs (miRNAs). Five miRNAs (miR-17, miR-19a, miR-19b, miR-20a and miR-92) in the miR-17~92 cluster were functionally demonstrated to target PTEN (refs 14, 15, 16, 17), and Mirc1tm1.1Tyj/J mice have a floxed miR-17~92 allele18. We knocked out the miR-17~92 allele in situ in Mirc1tm1.1Tyj/J mice by intracranial injection of astrocyte-specific Cre adenovirus (Ad-GFAP-Cre), then intracarotidly injected syngeneic mouse melanoma B16BL6 cells to form brain metastases (Fig. 2c). Astrocyte-specific depletion of PTEN-targeting miRNAs blocked PTEN downregulation (Fig. 2d) in the brain metastasis tumour cells in vivo without significantly altering other potential miRNA targets (Extended Data Fig. 3a), and significantly suppressed brain metastasis growth compared to the control group (Fig. 2d, e), indicating a tumour cell non-autonomous PTEN downregulation by astrocyte-derived PTEN-targeting miRNAs. Astrocyte-specific depletion of PTEN-targeting miRNAs also suppressed intracranially injected tumour cell outgrowth (Extended Data Fig. 3b–f). To examine which PTEN-targeting miRNA primarily mediates the PTEN loss in tumour cells when co-cultured with astrocytes, the luciferase activities of the wild-type and mutated PTEN 3′-untranslated region (UTR) (containing various miRNA binding site mutations) in tumour cells were assessed (Fig. 2f). Compared with CAF co-culture, astrocyte co-culture inhibited luciferase activity of wild-type PTEN 3′-UTR, which was rescued by the miR-19a binding site mutation (position 1), but not by other mutations, indicating the major role of miR-19a in astrocyte-mediated PTEN mRNA downregulation in tumour cells. Furthermore, PTEN mRNA (Fig. 2g and Extended Data Fig. 3g) and PTEN protein (Fig. 2h and Extended Data Fig. 3h) were not downregulated in tumour cells co-cultured with primary astrocytes from Mirc1tm1.1Tyj/J mice in which PTEN-targeting miRNAs were depleted (Extended Data Fig. 3i).

Figure 2: Astrocyte-derived miRNAs silence PTEN in tumour cells.
figure2

a, PTEN mRNA in the indicated tumour cells after 2–5 days co-culture with GFAP-positive primary glia or vimentin (vim)-positive primary CAFs or NIH3T3 fibroblasts (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). b, Western blot of PTEN protein under co-culture as in a (3 biological replicates). c, Schematic of astrocyte-specific miR-17~92 deletion by GFAP-driven Cre adenovirus (Ad-GFAP-Cre) in Mirc1tm1.1Tyj/J mice. d, Representative image of tumour sizes and PTEN IHC of brain metastases. e, Quantification of brain metastases volume (mean ± s.d., t-test, P = 0.0024). f, PTEN 3′-UTR luciferase activity after co-culture (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). bp, base pairs. g, qRT–PCR analyses of miR-19a and PTEN mRNA in MDA-MB-231 cells after 48 h co-culture with primary astrocytes from Mirc1tm1.1Tyj/J mice pre-infected (48 h) by adenovirus (Ad-βGLuc or Ad-GFP-Cre) (mean ± s.e.m., t-test, P < 0.001, 3 biological replicates, with 3 technical replicates each). h, Western blot of PTEN protein in MDA-MB-231 cells, co-cultured as in g.

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After co-culture with Cy3-labelled miR-19a-transfected primary astrocytes, we detected significantly more Cy3+ epithelial cell adhesion molecule (EpCAM)-positive tumour cells over time than under CAF co-culture (Fig. 3a and Extended Data Fig. 4a), suggesting that miR-19a is intercellularly transferred from astrocytes to tumour cells. miRNAs are transferable between neighbouring cells through gap junctions or small vesicles19,20. Treating tumour cells with a gap junction channel inhibitor, carbenoxolone disodium salt, had no significant effect on miR-19a intercellular transfer (data not shown), while adding astrocyte-conditioned media to tumour cells led to an increase in miR-19a levels and a subsequent PTEN downregulation (Extended Data Fig. 4b–d). Recognizing the involvement of exosomes in neuronal function and glioma development21, we postulated that exosomes may mediate miR-19a transfer from astrocytes to tumour cells. Indeed, transmission electron microscopy detected spherical, membrane-encapsulated particles between 30 and 100 nm, typical of exosome vesicles, in astrocyte-conditioned media22 (Fig. 3b). Additionally, the astrocyte-conditioned media contained significantly more CD63+, CD81+ and TSG101+ exosomes22 than the CAF-conditioned media (Fig. 3c and Extended Data Fig. 4e, f). Moreover, the exosomes from astrocytes contained 3.5-fold higher levels of miR-19a than those from CAFs (Extended Data Fig. 4g). Adding exosomes purified from conditioned media of Cy3-miR-19a-transfected astrocytes led to miR-19a transfer into cultured tumour cells (Fig. 3d). Furthermore, treating tumour cells directly with astrocyte-derived exosomes led to a dose-dependent increase of miR-19a and a subsequent decrease of PTEN mRNA in tumour cells (Fig. 3e). To determine whether astrocyte-released exosomes are required for miR-19a transfer, we blocked astrocyte exosome secretion by treating astrocytes with either an inhibitor of exosome release, dimethyl amiloride (DMA), or a short interfering RNA (siRNA) targeting Rab27a, a mediator of exosome secretion23 (Extended Data Fig. 5a–c). Both exosome blockades decreased the transfer of miR-19a from astrocytes to tumour cells and restored the PTEN mRNA levels (Fig. 3f, g). Furthermore, we intracranially injected Rab27a/b short hairpin RNA (shRNA) lentiviruses to block exosome secretion in mouse brain parenchyma (brain metastasis stroma), and then inoculated B16BL6 melanoma cells to the same sites (Fig. 3h). Inhibiting Rab27a/b reduced TSG101+ and CD63+ exosomes, blocked PTEN downregulation in tumour lesions (Fig. 3i and Extended Data Fig. 5d–g), and significantly decreased tumour outgrowth (Fig. 3j). Conversely, intracranial co-injection of tumour cells with astrocyte-derived exosomes (Fig. 3k) rescued PTEN downregulation in tumour cells (Fig. 3l) and metastatic outgrowth (Fig. 3m) in mouse brains injected with Rab27a/b shRNA (Extended Data Fig. 5h, i). Collectively, exosome-mediated miR-19a transfer from astrocytes to tumour cells is critical for tumour PTEN downregulation and aggressive outgrowth in the brain.

Figure 3: Intercellular transfer of PTEN-targeting miR-19a to tumour cells via astrocyte-derived exosomes.
figure3

a, Intercellular transfer of miR-19a. Top, light microscopy and fluorescent images of HCC1954 cells 12 and 60 h after co-culture with astrocytes loaded with Cy3-labelled miR-19a. Bottom, flow cytometry analysis of Cy3-miR-19a in tumour cells 60 h after co-culture (mean ± s.e.m., t-test, P < 0.05, 3 biological replicates). b, c, Transmission electron microscopy of exosome vesicles in astrocyte-conditioned media (b), confirmed by western blot for CD63, CD81 and TSG101 exosome markers released by 1 × 106 CAFs or astrocytes (c). d, Representative data showing presence of Cy3-miR-19a in HCC1954 breast cancer cells after adding exosomes purified from Cy3-miR-19a-transfected astrocytes for 24 h. Bottom, flow cytometry analysis of Cy3-miR-19a-positive HCC1954 cells after treatment with supernatant (without exosomes), or exosomes purified from Cy3-miR-19a-transfected astrocytes. Negative control is HCC1954 cells without treatment. Positive control is Cy3-miR-19a-transfected astrocytes (3 biological replicates). e, Histogram of miR-19a and PTEN mRNA in HCC1954 cells 48 h after addition of media, astrocyte supernatant, or exosomes purified from astrocyte-conditioned media (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). f, g, Histograms of miR-19a and PTEN mRNA in HCC1954 cells after 48 h co-culture in conditioned media from vehicle- or DMA-treated (4 h) astrocytes (f) and control- or Rab27a-siRNA-transfected (48 h) astrocytes (g) (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). hj, Schematics of in vivo experiments (h), IHC analyses of PTEN and exosome marker expression (i) and changes of tumour volume (j) (mean ± s.d., t-test, n = 7, P = 0.0157). B, brain; M, metastases; shRab27a/b, shRNA against Rab27a/b. km, Schematics showing in vivo rescue of exosome effect by pre-incubation of tumour cells with astrocyte-derived exosomes (k), IHC analyses of PTEN and exosome marker expression (l) and changes of tumour volume (m) (mean ± s.d., t-test, n = 8, P = 0.0091).

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We next explored how PTEN loss promotes brain metastasis. Doxycycline-inducible PTEN knockdown (Extended Data Fig. 6a) before intracarotid injection did not alter tumour cell extravasation into the brain parenchyma (Extended Data Fig. 6b, c). To test whether restoring PTEN expression after tumour cell extravasation inhibits metastatic outgrowth, we selected subclones of human breast carcinoma cells that selectively metastasize to the brain (MDA-MB-231Br) stably expressing either a doxycycline-inducible PTEN-coding sequence without the 3′-UTR miRNA binding sites, or red fluorescent protein (RFP) controls (Fig. 4a and Extended Data Fig. 6d). PTEN induction 7 days post-intracarotid injection after extravasation of tumour cells markedly extended the overall survival of brain metastases-bearing mice (Fig. 4a and Extended Data Fig. 6e). Collectively, PTEN loss primes brain metastasis outgrowth after tumour cell extravasation and PTEN restoration suppresses the outgrowth.

Figure 4: Brain-dependent PTEN loss instigates metastatic microenvironment to promote metastatic cell outgrowth.
figure4

a, Prolonged mouse survival by restoration of PTEN expression. Top, doxycycline (Dox)-inducible RFP (left) and PTEN expression (right) in 231Br cells. Middle, schematic of brain metastasis assay with doxycycline-induced RFP or PTEN expression. Bottom, overall survival of mice bearing brain metastases of 231Br cells with induced PTEN re-expression or RFP expression (log-rank test, n = 12, P < 0.0001). b, Cytokine array of 231Br cells with doxycycline-induced RFP or PTEN expression. c, Overall survival of mice bearing brain metastases of 231Br cells transfected with control or CCL2 shRNAs (shControl or shCCL2, respectively) (log-rank test, n = 8, P = 0.027). d, Western blot analysis of NF-κB p65 nuclear translocation after knocking down PTEN. Cyto, cytosol; nuc, nuclear. e, Histogram showing CCL2 mRNA levels detected by quantitative PCR after PTEN knockdown with shRNA (shPTEN) (mean ± s.e.m., t-test, P < 0.001, 3 biological replicates, with 3 technical replicates each). f, Light and fluorescent microscopy images and quantification of mCherry-labelled tumour cells with or without BV2 microglia co-culture under 2-day serum starvation (mean ± s.e.m., t-test, P = 0.031, 3 biological replicates, with 3 technical replicates each). g, FACS analyses of Annexin V+ apoptotic zsGreen-labelled 231Br cells under doxorubicin treatment with or without BV2 microglia co-culture (mean ± s.e.m., t-test, P = 0.004, 3 biological replicates). h, Immunofluorescence staining of IBA1+ myeloid cells in brain metastases of 231Br cells containing control (shControl) or CCL2 shRNA (shCCL2) (mean ± s.e.m., t-test, P < 0.01, 3 biological replicates, with 3 technical replicates each). i, j, IHC analyses showing decreased proliferation (Ki-67, i) and increased apoptosis (TUNEL staining, j) in brain metastases after shRNA-mediated CCL2 knockdown in vivo (mean ± s.e.m., t-test). k, PTEN and CCL2 expression in matched primary breast tumours and brain metastases. Left, representative IHC staining of PTEN and CCL2. Right, quantification of PTEN and CCL2 expression in 35 cases of matched primary breast tumours and brain metastases (mean ± s.d.).

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Autocrine and paracrine signalling have decisive roles in metastasis seeding and outgrowth. Although PTEN restoration only led to a trend of reduced Akt and P70S6K phosphorylation (pAkt and pP70S6K, respectively; Extended Data Fig. 6f), cytokine array analyses revealed markedly reduced CCL2 secretion in PTEN-expressing tumour cells compared to controls (Fig. 4b); whereas PTEN knockdown increased CCL2 expression (Extended Data Fig. 6g). Moreover, the overall survival of brain metastasis-bearing mice with CCL2-knockdown MDA-MB-231Br cells was significantly extended compared to controls (Fig. 4c and Extended Data Fig. 6h, i). Mechanistically, PTEN induction decreased NF-κB p65 phosphorylation (Extended Data Fig. 7a, b) along with reduced CCL2 secretion (Fig. 4b), whereas PTEN knockdown increased p65 nuclear translocation, an indicator of NF-κB activation, and CCL2 expression (Fig. 4d, e), partly through Akt activation (Extended Data Fig. 7c). Furthermore, CCL2 mRNA and CCL2 protein expression in brain-seeking tumour cells was inhibited by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) (Extended Data Fig. 7d–f), indicating that NF-κB activation is crucial for PTEN-loss-induced CCL2 upregulation.

CCL2 is a chemo-attractant during inflammation24. CCL2 receptor (CCR2)-expressing brain-derived IBA1-positive (IBA1+) primary myeloid cells and BV2 microglial cells (Extended Data Fig. 8a, b) migrate towards CCL2, which was blocked by CCR2 antagonists25 (Extended Data Fig. 8c, d). Functionally, co-culturing with BV2 cells enhanced proliferation and inhibited apoptosis of breast cancer cells (Fig. 4f, g). In vivo, CCL2-knockdown brain metastases had decreased IBA1+/CCR2+ myeloid cell infiltration (Fig. 4h), corresponding to their reduced proliferation and increased apoptosis (Fig. 4i, j). Furthermore, IHC staining of human primary breast tumours and matched brain metastases for PTEN and CCL2 (Figs 1b and 4k, respectively) revealed a significantly (P = 0.027) higher CCL2 expression in brain metastases than in primary tumours (Extended Data Fig. 9a). Importantly, severe PTEN loss in brain metastases corresponded to higher CCL2 expression (Extended Data Fig. 9b), which significantly correlated with IBA1+ myeloid cell recruitment (Extended Data Fig. 9c), validating that PTEN downregulation in brain metastatic tumour cells contributes to CCL2 upregulation and IBA1+ myeloid cell recruitment in clinical brain metastases.

Taken together, our data unveiled a complex reciprocal communication between metastatic tumour cells and their TME, which primes the successful outgrowth of cancer cells to form life-threatening metastases (Extended Data Fig. 10). Beyond a tumour cell autonomous view of metastasis, our findings highlighted an important plastic and tissue-dependent nature of metastatic tumour cells, and a bi-directional co-evolutionary view of the ‘seed and soil’ hypothesis. Notably, although clinical application of CCL2 inhibitor for metastasis treatment requires careful design26, our data of brain metastasis inhibition by stable ablation of PTEN-loss-induced CCL2 demonstrated the potential of CCL2-targeting for therapeutic intervention of life-threatening brain metastases.

Methods

Reagents and cell culture

All common chemicals were from Sigma. Pyrrolidinedithiocarbamic acid was from Santa Cruz Biotechnology. Exo-FBS exosome-depleted FBS was purchased from System Biosciences (SBI). PTEN (9188), pAkt(T308) (9275), pAkt(S473) (4060), Pan Akt (4691), and Bim (2933) antibodies were from Cell Signaling. CD9 (ab92726), Rab27a (ab55667), AMPK (ab3759), CCL2 (ab9899), MAP2 (ab11267), and pP70S6K (ab60948) antibodies were from Abcam. Tsg101 (14497-1-AP) and Rab27b (13412-1-AP) antibodies were from Proteintech. CD81 (104901) antibody was from BioLegend. E2F1 (NB600-210) and CCR2 (NBP1-48338) antibodies were from Novus. GFAP (Z0334) antibody was from DAKO. IBA1 antibody was from WAKO. Cre (969050) antibody was from Novagen. NF-κB p65 (SC-109) and CD63 (SC-15363) antibodies were from Santa Cruz. DMA (sc-202459) and CCR2 antagonist (sc-202525) were from Santa Cruz. MK2206 (S1078) was from Selleckchem. PDTC (P8765) was from Sigma-Aldrich. Human breast cancer cell lines (MDA-MB-231, HCC1954, BT474 and MDA-MB-435) and mouse cell lines (B16BL6 mouse melanoma and 4T1 mouse breast cancer) were purchased from ATCC and verified by the MD Anderson Cancer Center (MDACC) Cell Line Characterization Core Facility. All cell lines have been tested for mycoplasma contamination. Primary glia was isolated as described13. In brief, after homogenization of dissected brain from postnatal day (P)0–P2 neonatal mouse pups, all cells were seeded on poly-d-lysine coated flasks. After 7 days, flasks with primary culture were placed on an orbital shaker and shaken at 230 r.p.m. for 3 h. Warm DMEM 10:10:1 (10% of fetal bovine serum, 10% of horse serum, 1% penicillin/streptomycin) was added and flasks were shaken again at 260 r.p.m. overnight. After shaking, fresh trypsin was added into the flask and leftover cells were plated with warm DMEM 5:5:1 (5% of fetal bovine serum, 5% of horse serum, 1% penicillin/streptomycin) to establish primary astrocyte culture. More than 90% of isolated primary glial cells were GFAP+ astrocytes. Primary CAFs were isolated by digesting the mammary tumours from MMTV-neu transgenic mouse. 231-xenograft CAFs were isolated by digesting the mammary tumours from MDA-MB-231 xenograft. For the mixed co-culture experiments, tumour cells were mixed with an equal number of freshly isolated primary glia, CAFs or NIH3T3 fibroblast cells in six-well plate (1:3 ratio). Co-cultures were maintained for 2–5 days before magnetic-bead-based separation. For the trans-well co-culture experiments, tumour cells were seeded in the bottom well and freshly isolated primary glia, CAFs or NIH3T3 cells were seeded on the upper insert (1:3 ratio). Co-cultures were maintained for 2–5 days for the further experiments. Lentiviral-based packaging vectors (Addgene), pLKO.1 PTEN-targeting shRNAs and all siRNAs (Sigma), Human Cytokine Antibody Array 3 (Ray biotech), and lentiviral-based vector pTRIPZ-PTEN and pTRIPZ-CCL2 shRNAs (MDACC shRNA and ORFome Core, from Open Biosystems) were purchased. The human PTEN-targeting shRNA sequences in the lentiviral constructs were: 5′-CCGGAGGCGCTATGTGTATTATTATCTCGAGATAATAATACACATAGCGCCTTTTTT-3′ (targeting coding sequence); 5′-CCGGCCACAAATGAAGGGATATAAACTCGAGTTTATATCCCTTCATTTGTGGTTTTT-3′ (targeting 3′-UTR). The human PTEN-targeting siRNA sequences used were: 5′-GGUGUAAUGAUAUGUGCAU-3′ and 5′-GUUAAAGAAUCAUCUGGAU-3′. The human CCL2-targeting siRNA sequences used were: 5′-CAGCAAGUGUCCCAAAGAA-3′ and 5′-CCGAAGACUUGAACACUCA-3′. The mouse Rab27a-targeting siRNA sequences used were: 5′-CGAUUGAGAUGCUCCUGGA-3′ and 5′-GUCAUUUAGGGAUCCAAGA-3′. Mouse pLKO shRNA (shRab27a: TRCN0000381753; shRab27b: TRCN0000100429) were purchased from Sigma. For lentiviral production, lentiviral expression vector was co-transfected with the third-generation lentivirus packing vectors into 293T cells using Lipo293 DNA in vitro Transfection Reagent (SignaGen). Then, 48–72 h after transfection, cancer cell lines were stably infected with viral particles. Transient transfection with siRNA was performed using pepMute siRNA transfection reagent (SignaGen). For in vivo intracranial virus injection, lentivirus was collected from 15 cm plates 48 h after transfection of packaging vectors. After passing a 0.45 μm filter, all viruses were centrifuged at 25,000 r.p.m (111,000g) for 90 min at 4 °C. Viral pellet was suspended in PBS (200-fold concentrated). The final virus titre (1 × 109 UT ml−1) was confirmed by limiting dilution.

Isolation of tumour cells from co-culture

Cell isolation was performed based on the magnetic bead-based cell sorting protocol according to manufacturer’s recommendation (Miltenyi Biotec Inc.). After preparation of a single-cell suspension, tumour cells (HCC1954 or BT474) were stained with primary EpCAM-FITC antibody (130-098-113) (50 μl per 107 total cells) and incubated for 30 min in the dark at 4 °C. After washing, the cell pellet was re-suspended and anti-FITC microbeads (50 μl per 107 total cells) were added before loading onto the magnetic column of a MACS separator. The column was washed twice and removed from the separator. The magnetically captured cells were flushed out immediately by firmly applying the plunger. The isolated and labelled cells were analysed on a Gallios flow cytometer (Beckman Coulter). For EpCAM-negative MDA-MB-231 tumour cells, FACS sorting (ARIAII, Becton Dickinson) was used to isolate green fluorescent protein (GFP)+ tumour cells from glia or CAFs.

Isolation of CD11b+ cells from mouse primary glia

Isolation of primary glia was achieved by homogenization of dissected brain from P0–P2 mouse pups. After 7 days, trypsin was added and cells were collected. After centrifugation and re-suspension of cell pellet to a single-cell suspension, cells were incubated with CD11b+ microbeads (Miltenyl Biotec) (50 μl per 107 total cells) for 30 min at 4 °C. The cells were washed with buffer and CD11b+ cells were isolated by MACS Column. CD11b+ cells were analysed by flow cytometry and immunofluorescence staining.

Western blotting

Western blotting was done as previously described. In brief, cells were lysed in lysis buffer (20 mM Tris, pH 7.0, 1% Triton X-100, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA and protease inhibitor cocktail). Proteins were separated by SDS–PAGE and transferred onto a nitrocellulose membrane. After membranes were blocked with 5% milk for 30 min, they were probed with various primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature, and visualized with enhanced chemiluminescence reagent (Thermo Scientific).

qRT–PCR

In brief, total RNA was isolated using miRNeasy Mini Kit (Qiagen) and then reverse transcribed using reverse transcriptase kits (iScript cDNA synthesis Kit, Bio-rad). SYBR-based qRT–PCR was performed using pre-designed primers (Life Technologies). miRNA assay was conducted using Taqman miRNA assay kit (Life Technologies). For quantification of gene expression, real-time PCR was conducted using Kapa Probe Fast Universal qPCR, and SYBR Fast Universal qPCR Master Mix (Kapa Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). The relative expression of mRNAs was quantified by 2−ΔΔCt with logarithm transformation. Primers used in qRT–PCR analyses are: mouse Ccl2: forward, 5′-GTTGGCTCAGCCAGATGCA-3′; reverse: 5′-AGCCTACTCATTGGGATCATCTTG-3′. Mouse Actb: forward: 5′-AGTGTGACGTTGACATCCGT3′; reverse: 5′-TGCTAGGAGCCAGAGCAGTA-3′. Mouse Pten: forward: 5′-AACTTGCAATCCTCAGTTTG-3′; reverse: 5′-CTACTTTGATATCACCACACAC-3′. Mouse Ccr2 primer: Cat: 4351372 ID: Mm04207877_m1 (Life technologies)

miRNA labelling and transfection

Synthetic miRNAs were purchased from Sigma and labelled with Cy3 by Silencer siRNA labelling kit (Life Technologies). In brief, miRNAs were incubated with labelling reagent for 1 h at 37 °C in the dark, and then labelled miRNAs were precipitated by ethanol. Labelled miRNAs (100 pmoles) were transfected into astrocytes or CAFs in a 10-cm plate. After 48 h, astrocytes and CAFs containing Cy3-miRNAs were co-cultured with tumour cells (at 5:1 ratio).

PTEN promoter methylation analysis and luciferase reporter assay of PTEN promoter activity

Genomic DNA was isolated by PreLink genomic DNA mini Kit (Invitrogen), bisulfite conversion was performed by EpiTect Bisulphite Kit and followed by EpiTect methylation-specific PCR (Qiagen). Primers for PTEN CpG island are 5′-TGTAAAACGACGGCCAGTTTGTTATTATTTTTAGGGTTGGGAA-3′ and 5′-CAGGAAACAGCTATGACCCTAAACCTACTTCTCCTCAACAACC-3′. Luciferase reporter assays were done as previously described27. The wild-type PTEN promoter driven pGL3-luciferase reporter was a gift from A. Yung. The pGL3-PTEN reporter and a control Renilla luciferase vector were co-transfected into tumour cells by Lipofectamine 2000 (Life Technologies). After 48 h, tumour cells were co-cultured with astrocytes or CAFs. Another 48 h later, luciferase activities were measured by Dual-Luciferase Report Assay Kit (Promega) on Luminometer 20/20 (Turner Biosystems). The PTEN 3′-UTRs with various miRNA binding-site mutations were generated by standard PCR-mediated mutagenesis method and inserted downstream of luciferase reporter gene in pGL3 vector. The activities of the luciferase reporter with the wild-type and mutated PTEN 3′-UTRs were assayed as described above.

Exosome isolation and purification

Astrocytes or CAFs were cultured for 48–72 h and exosomes were collected from their culture media after sequential ultracentrifugation as described previously. In brief, cells were collected, centrifuged at 300g for 10 min, and the supernatants were collected for centrifugation at 2,000g for 10 min, 10,000g for 30 min. The pellet was washed once with PBS and purified by centrifugation at 100,000g for 70 min. The final pellet containing exosomes was re-suspended in PBS and used for (1) transmission electron microscopy by fixing exosomes with 2% glutaraldehyde in 0.1 M phosophate buffer, pH 7.4; (2) measure of total exosome protein content using BCA Protein Assay normalized by equal number of primary astrocytes and CAF cells; (3) western blotting of exosome marker protein CD63, CD81 and Tsg101; and (4) qRT–PCR by extracting miRNAs with miRNeasy Mini Kit (Qiagen).

Transmission electron microscopy

Fixed samples were placed on 100-mesh carbon-coated, formvar-coated nickel grids treated with poly-l-lysine for about 30 min. After washing the samples on several drops of PBS, samples were incubated on drops of buffered 1% gluteraldehyde for 5 min, and then washed several times on drops of distilled water. Afterwards, samples were negatively stained on drops of millipore-filtered aqueous 4% uranyl acetate for 5 min. Stain was blotted dry from the grids with filter paper and samples were allowed to dry. Samples were then examined in a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 Kv. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp.).

Flow cytometry analysis of exosome marker proteins, Annexin V and CCR2

For exosome detection, 100 μl exosomes isolated from 10-ml conditioned media of astrocytes or CAFs were incubated with 10 μl of aldehyde/sulfate latex beads (4 μm diameter, Life Technologies) for 15 min at 4 °C. After 15 min, PBS was added to make sample volume up to 400 μl, which was incubated overnight at 4 °C under gentle agitation. Exosome-coated beads were washed twice in FACS washing buffer (1% BSA and 0.1% NaN3 in PBS), and re-suspended in 400 μl FACS washing buffer, stained with 4 μg of phycoerythrin (PE)-conjugated anti-mouse CD63 antibody (BioLegend) or mouse IgG (Santa Cruz Biotechnology) for 3 h at 4 °C under gentle agitation and analysed on a FACS Canto II flow cytometer. Samples were gated on bead singlets based on FCS and SSC characteristics (4 μm diameter). For Annexin V apoptosis assay, after 24 h doxorubicin (2 μM) treatment, the cells were collected, labelled by APC-Annexin V antibody (Biolegend) and analysed on a FACS Canto II flow cytometer. CD11b+ and BV2 cells were stained with CCR2 antibody (Novus) at 4 °C overnight; they were then washed and stained with Alexa Fluor 488 anti-rabbit IgG (Life Technologies) at room temperature for 1 h. The cells were then analysed on a FACS Canto II flow cytometer.

In vivo experiments

All animal experiments and terminal endpoints were carried out in accordance with approved protocols from the Institutional Animal Care and Use Committee of the MDACC. Animal numbers of each group were calculated by power analysis and animals are grouped randomly for each experiment. No blinding of experiment groups was conducted. MFP tumours were established by injection of 5 × 106 tumour cells in 100 μl of PBS:Matrigel mixture (1:1 ratio) orthotopically into the MFP of 8-week-old Swiss nude mice as done previously28. Brain metastasis tumours were established by ICA injection of tumour cells (250,000 cells in 0.1 ml HBSS for MDA-MB-231, HCC1954, MDA-MB-435, 4T1 and B16BL6, and 500,000 cells in 0.1 ml HBSS for BT474.m1 into the right common carotid artery as done previously29). Mice (6–8 weeks) were randomly grouped into designated groups. Female mice are used for breast cancer experiments, both female and male are used for melanoma experiments. Since the brain metastasis model does not result in visible tumour burdens in living animal, the endpoints of in vivo metastasis experiments are based on the presence of clinical signs of brain metastasis, including but not limited to, primary central nervous system disturbances, weight loss, and behavioural abnormalities. Animals are culled after showing the above signs or 1–2 weeks after surgery based on specific experimental designs. Brain metastasis lesions are enumerated as experimental readout. Brain metastases were counted as micromets and macromets. The definition of micromets and macromets are based on a comprehensive mouse and human comparison study previously published30. In brief, ten haematoxylin and eosin (H&E)-stained serial sagittal sections (300 μm per section) through the left hemisphere of the brain were analysed for the presence of metastatic lesions. We counted micrometastases (that is, those ≤ 50 μm in diameter) to a maximum of 300 micrometastases per section, and every large metastasis (that is, those > 50 μm in diameter) in each section. Brain-seeking cells from overt metastases and whole brains were dissected and disaggregated in DMEM/F-12 medium using Tenbroeck homogenizer briefly. Dissociated cell mixtures were plated on tissue culture dish. Two weeks later, tumours cells recovered from brain tissue were collected and expanded as brain-seeking sublines (Br.1). For the astrocyte miR-19 knockout mouse model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected with Ad5-GFAP-Cre virus (Iowa University, Gene Transfer Vector Core) 2 μl (MOI 108 U μl−1) per point, total four points at the right hemisphere (n = 9). Control group (n = 7) was injected with the same dose Ad5-RSV-βGLuc (Ad-βGLuc) at the right hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week after virus injection, mice were intracarotidly injected with 2 × 105 B16BL6 tumour cells. After two weeks, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, and IHC staining were evaluated. Only parenchymal lesions, which are in close proximity of adenovirus injection, were included in our evaluation. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For the intracranial tumour model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected as described above. Seven mice were used in the experiment. One week later, these mice were intracranially injected with 2.5 × 105 B16BL6 tumour cells at both sides where adenoviruses were injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above.

For the Rab27a/b knockdown mouse model, seven C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total three points at the right hemisphere; concentrated control lentivirus containing pLKO.1 scramble were injected at the left hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week later, mice were intracranially injected with 5 × 104 B16BL6 tumour cells at both sides where they had been infected. After one week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, IHC staining were evaluated. When performing metastases size quantification, only parenchymal lesions that were in close proximity to the adenovirus injection sites were included in the analyses. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For exosome rescue experiments, eight C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total 3 points at both hemispheres. One week later, these mice were intracranially injected with 5 × 104 B16BL6 tumour cells with 10 μg exosome isolated from astrocyte media at the right sides where they had been injected with lentivirus; 5 × 104 B16BL6 tumour cells with vehicle were injected at the left sides where lentivirus had been injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above.

For in vivo extravasation assay, equal numbers of cells labelled with GFP-control shRNA and RFP-PTEN shRNA (Open Biosystems) were mixed and ICA injected. After cardiac perfusion, brains were collected and sectioned through coronal plan on a vibrotome (Leica) into 50-μm slices. Fluorescent cells were then counted. For inducible PTEN expression in vivo, mice were given doxycycline (10 μg kg−1) every other day. To quantify brain metastasis incidence and tumour size, brains were excised for imaging and histological examination at the end of experiments. Ten serial sagittal sections every 300 μm throughout the brain were analysed by at least two pathologists who were blinded to animal groups in all above analyses.

Reverse-phase protein array

Reverse-phase protein array of PTEN-overexpressing cells was performed in the MDACC Functional Proteomics core facility. In brief, cellular proteins were denatured by 1% SDS, serial diluted and spotted on nitrocellulose-coated slides. Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. The signal obtained was amplified using a Dako Cytomation-catalysed system and visualized by DAB colorimetric reaction. The slides were analysed using customized Microvigene software (VigeneTech Inc.). Each dilution curve was fitted with a logistic model (‘Super curve fitting’ developed at the MDACC) and normalized by median polish. Differential intensity of normalized log values of each antibody between RFP (control) and PTEN-overexpressed cells were compared in GenePattern (http://genepattern.broadinstitute.org). Antibodies with differential expression (P < 0.2) were selected for clustering and heat-map analysis. The data clustering was performed using GenePattern.

Patient samples

Two studies in separate cohorts were conducted. The first one was a retrospective evaluation of PTEN in two cohorts. (1) Archived formalin-fixed and paraffin-embedded brain metastasis specimens (n = 131) from patients with a history of breast cancer who presented with metastasis to the brain parenchyma and had surgery at the MDACC (Supplementary Information). Tissues were collected under a protocol (LAB 02-486) approved by the Institutional Review Board (IRB) at the MDACC. (2) Archived unpaired primary breast cancer formalin-fixed and paraffin-embedded specimens (n = 139) collected under an IRB protocol (LAB 02-312) at the MDACC (Supplementary information). Formal consent was obtained from all patients. The second study was a retrospective evaluation of PTEN, CCL2 and IBA1 in the matched primary breast tumours and brain metastatic samples from 35 patients, of which there are 12 HER2-positive, 14 triple-negative and nine oestrogen-receptor-positive tumours according to clinical diagnostic criteria (Supplementary Information). Formalin-fixed, paraffin-embedded primary breast and metastatic brain tumour samples were obtained from the Pathology Department, University of Queensland Centre for Clinical Research. Tissues were collected with approval by human research ethics committees at the Royal Brisbane and Women’s Hospital (2005/022) and the University of Queensland (2005000785). For tissue microarray construction, tumour-rich regions (guided by histological review) from each case were sampled using 1-mm cores. All the archival paraffin-embedded tumour samples were coded with no patient identifiers.

IHC and immunofluorescence

Standard IHC staining was performed as described previously28. In brief, after de-paraffinization and rehydration, 4 μm sections were subjected to heat-induced epitope retrieval (0.01 M citrate for PTEN). Slides were then incubated with various primary antibodies at 4 °C overnight, after blocking with 1% goat serum. Slides underwent colour development with DAB and haematoxylin counterstaining. Ten visual fields from different areas of each tumour were evaluated by two pathologists independently (blinded to experiment groups). Positive IBA1 and Ki-67 staining in mouse tumours were calculated as the percentage of positive cells per field (%) and normalized by the total cancer cell number in each field. TUNEL staining was counted as the average number of positive cells per field (10 random fields). We excluded necrotic areas in the tumours from evaluation. Immunofluorescence was performed following the standard protocol recommended by Cell Signaling. In brief, after washing with PBS twice, cells were fixed with 4% formaldehyde. Samples were blocked with 5% normal goat serum in PBS for 1 h before incubation with a primary antibody cocktail overnight at 4 °C, washed, then incubated with secondary antibodies before examination using confocal microscope. Pathologists were blinded to the group allocation during the experiment and when assessing the outcome.

Bioinformatics and statistical analysis

Publicly available GEO data sets GSE14020, GSE19184, GSE2603, GSE2034 and GSE12276 were used for bioinformatics analysis. The top 2 × 104 verified probes were subjected to analysis. Differentially expressed genes between metastases from brain and other sites (primary or other metastatic organ sites) were analysed by SAM analysis in R statistical software. The 54 commonly downregulated genes in brain metastases from GSE14020 and GSE19184 were depicted as a heat-map by Java Treeview. For staining of patient samples, we calculated the correlation by Fisher’s exact test. For survival analysis of GSE2603, the patient samples were mathematically separated into PTEN-low and -normal groups based on K-means (K = 2). Kaplan–Meier survival curves were generated by survival package in R. Multiple group IHC scores were compared by Chi-square test and Mantelhaen test in R. All quantitative experiments have been repeated using at least three independent biological repeats and are presented as mean ± s.e.m. or mean ± s.d.. Quantitative data were analysed either by one-way analysis of variance (ANOVA) (multiple groups) or t-test (two groups). P < 0.05 (two-sided) was considered statistically significant.

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Acknowledgements

We thank M.-C. Hung, H.-K. Lin and Z. Lu for reading the manuscript, A. Yung for PTEN promoter constructs, MD Anderson Cancer Center (MDACC) shRNA and ORFome, FACS, histology and high-resolution electron microscopy support, the animal core facilities (NIH CA16672) for technical support, and members of the Yu laboratory for helpful discussions. Thanks to A. Matsika for histological review and tissue microarray construction. This work was supported partially by DOD Center of Excellence grant (P.S.S.) subproject W81XWH-06-2-0033 (D.Y.), NIH Pathway to Independence Award 5R00CA158066-05 (S.Z.), DOD Postdoctoral Fellowship W81XWH-11-1-0003 (C.Z.), Isaiah Fidler Fellowship in Cancer Metastasis (F.J.L.), PO1-CA099031 project 4 (D.Y.), RO1-CA112567-06 (D.Y.), R01CA184836 (D.Y.), Susan G. Komen Breast Cancer Foundation Promise Grant KG091020 (D.Y.), METAvivor Research Grant (D.Y.), Breast and Ovarian Cancers Moon Shot program, China Medical University Research Fund, and Sowell-Huggins Pre-doctoral Fellowship (L.Z.) and Professorship (D.Y.) in Cancer Research. D.Y. is the Hubert L. & Olive Stringer Distinguished Chair in Basic Science at the MDACC.

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Affiliations

Authors

Contributions

L.Z., S.Z. and D.Y. developed original hypothesis and designed experiments. L.Z., S.Z., J.Y., F.J.L., Q.Z., W.-C.H., P.L., M.L., X.W., C.Z., K.E., H.W., D.P., M.C., J.H.M., P.S.S. and D.Y. performed experiments and/or analysed data. J.S., S.L., S.H., A.A.S., K.D.A. and P.S.S. provided critical reagents and/or clinical samples. S.Z., L.Z., F.J. and D.Y. wrote and edited the manuscript. D.Y. supervised the study.

Corresponding author

Correspondence to Dihua Yu.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Organ-specific loss of PTEN in brain metastases.

a, Schematics of microarray analyses. Patients’ brain metastases exhibited a discrete gene expression profile with 650 genes significantly downregulated compared to bone or lung metastases (GSE14020). Cancer cells were injected into immunodeficient mice to produce orthotopic primary tumours (MDA-MB-231 cells for mammary tumour, PC14 for prostate tumour, A375SM for melanoma) and experimental brain metastases (all three lines). Brain metastases derived from these three cancer cell lines exhibited 2,161 commonly downregulated genes compared to their respective primary tumours (GSE19184). PTEN is one of only 54 commonly downregulated genes in brain metastases of both data sets. b, Heat-maps showing expression of 54 commonly downregulated genes (see a) in clinical brain metastases versus lung metastases and bone metastases. c, Heat-maps showing expression of the 54 genes (see a) in cell-line-induced primary tumours versus experimental brain metastases. d, Kaplan–Meier survival analyses showing no significant differences in brain metastasis-free survival between breast cancer patients with primary tumours expressing normal PTEN or low PTEN mRNA in GEO cDNA microarray set GSE2603 (P = 0.74). e, PTEN mRNA levels detected in primary breast tumours from patients with or without brain metastasis relapse. Three GEO cDNA microarray data sets (GSE2034, GSE2603 and GSE12276) with clinical annotation were analysed. Relative PTEN expression levels were compared by t-test.

Extended Data Figure 2 PTEN expression in different metastatic organ microenvironments and in vitro culture condition.

a, Breast cancer cell lines (MDA-MB-231, HCC1954, BT474, and MDA-MB-435) were cultured and injected either to the MFP to form primary tumour or intracarotidly to form brain metastases. Cell pellets and tumour tissues were stained for PTEN expression using anti-PTEN antibodies as described previously28. b, IHC staining of PTEN in brain metastases, paired lung metastases and primary tumour derived from either MDA-MB-231 or 4T1 cells. PTEN expression level was analysed based on an IRS scoring system. c, PTEN mRNA levels between parental MDA-MB-231 and CN-34 breast cancer cell lines (blue) and their brain-seeking sublines (red). Normalized PTEN-specific probe intensity values were extracted from cDNA microarray data set GSE12237. Dot plot shows the mean probe intensity derived from independent RNA samples. d, PTEN qRT–PCR (mean ± s.e.m., t-test) and PTEN IHC in MDA-MB-231Br secondary tumours and cultured cells (3 biological replicates, with 3 technical replicates each). e, f, qRT–PCR (e) and western blot (f) analysis of PTEN mRNA expression (mean ± s.e.m., t-test) or protein expression in MDA-MB-231 cells after co-culture with either primary mouse CAFs isolated from MDA-MB-231 xenograft tumours or primary mouse glia isolated from mouse brain (3 biological replicates, with 3 technical replicates each). g, Representative methylation-specific PCR of PTEN promoter and quantification under co-culture with glia or CAF (mean ± s.e.m., t-test, 2 biological replicates, with 2 technical replicates each). h, PTEN promoter activity measured by luciferase reporter in HCC1954 cells after co-culture with either CAF or glia cells for 48 h (mean ± s.e.m., t-test, P = 0.5271, 3 biological replicates, with 3 technical replicates each).

Extended Data Figure 3 Cre-mediated depletion of PTEN-targeting microRNAs in astrocytes.

a, IHC analyses of the expression of AMP-activated protein kinase (AMPK), pro-apoptotic protein BIM and transcription factor E2F1 (mean ± s.d., t-test) in brain metastasis tumours with/without pre-knocking out the miR-17˜92 cluster in the brain microenvironment. B, brain tissue; M, brain metastases. b, Schematic of experimental design. The Ad-GFAP-Cre adenovirus was injected intracranially to the right hemisphere of the Mirc1tm1.1Tyj/J mouse, and the control adenovirus (Ad-βGLuc) was injected intracranially to contralateral side of the brain. B16BL6 cells were then injected intracranially to both sides. c, IHC analysis of Cre expression in the brain astrocytes. d, IHC analysis of PTEN expression in the tumour cells. e, Quantification of PTEN expression in tumour cells (mean ± s.d., t-test). f, Quantification of intracranial tumour outgrowth by volume (mean ± s.e.m., t-test). g, qRT–PCR analyses of miR-19a and PTEN mRNA in tumour cell HCC1954 after 48 h co-culture with primary astrocytes from Mirc1tm1.1Tyj/J mice pre-infected (48 h) with adenovirus (Ad-βGLuc or Ad-GFP-Cre) (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). h, Western blot of PTEN protein in the indicated tumour cells co-cultured as in g. i, Knockdown of miR-17˜92 allele in cultured primary astrocytes. miR-17˜92 cluster is flanked by loxP site in Mirc1tm1.1Tyj/J mouse. Primary astrocytes were isolated from Mirc1tm1.1Tyj/J mouse brain then infected by adenovirus encoding for βGLuc or GFP-Cre protein. Concentrated adenovirus particles of indicated volume (same MOI ˜108 U ml−1) encoding βGLuc or GFP-Cre proteins were added to 106 astrocytes. Left, representative image showing the infection efficiency. Right, bar diagram showing the relative miR-19a expression (one of the five miRNA genes in the miR-17~92 cluster) three days after adenovirus infection (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each).

Extended Data Figure 4 Contact-independent downregulation of PTEN in tumour cells by miR-19a from astrocyte-derived exosomes.

a, Flow cytometric detection of Cy3-miR-19a and FITC-EpCAM in tumour cells 60 h after co-culture with Cy3-miR-19a-transfected astrocytes and CAFs. b, c, Tumour cells were co-cultured with conditioned media from astrocytes or CAFs for 60 h. RT–PCR analyses of the PTEN-targeting miR-19a level (b) and PTEN mRNA level (c) in tumour cells (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). d, Western blot detecting PTEN protein levels in HCC1954 cells after culture with conditioned media from either astrocytes or CAFs for 60 h. e, Flow cytometry detecting CD63+ exosomes extracted from CAF- or astrocyte-conditioned media. f, Histogram showing the exosome protein level detected from CAF- and astrocyte-conditioned media normalized by cell number (mean ± s.e.m., t-test, P < 0.0001, 3 biological replicates, with 3 technical replicates each). g, RT–PCR analyses of miR-19a level in exosomes extracted from CAF- or astrocyte- conditioned media normalized by equal cell numbers (mean ± s.e.m., t-test, P < 0.0001, 3 biological replicates, with 3 technical replicates each).

Extended Data Figure 5 Inhibition of exosome release by DMA, Rab27a siRNA or Rab27 shRNAs.

a, Exosome-releasing inhibitor (DMA) treatment reduced exosome secretion from astrocytes compared to vehicle treated astrocytes. Astrocytes were treated with DMA (25 μg ml−1) or vehicle for 4 h; exosomes were concentrated from astrocyte-conditioned media and total proteins from exosomes were examined by BCA assay (normalized to total cell numbers) (mean ± s.e.m., t-test, P = 0.038, 3 biological replicates, with 3 technical replicates each). b, Knockdown of Rab27a in astrocytes by siRNA. Two siRNAs targeting mouse Rab27a were transiently transfected into astrocytes, and the Rab27a mRNA level was examined by RT–PCR 48 h after transfection (mean ± s.e.m., t-test, P < 0.01, 3 biological replicates, with 3 technical replicates each). c, Knocking down Rab27a in astrocytes inhibited exosome release. Forty-eight hours after Rab27a-targeting siRNAs were transfected, exosomes were collected from astrocyte-conditioned media and total proteins from exosomes were examined by BCA assay (normalized to total cell numbers) (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). d, Histogram showing relevant changes of Rab27a and Rab27b mRNA level in primary astrocytes infected with pLKO.shRab27a or pLKO.shRab27b virus (mean ± s.e.m., t-test, P < 0.001, 3 biological replicates, with 3 technical replicates each). e, Change of exosome protein level detected in the conditioned media from astrocytes infected by pLKO.shRab27a or pLKO.shRab27b virus by BCA assay (normalized to total cell numbers) (mean ± s.e.m., t-test, P < 0.001, 3 biological replicates, with 3 technical replicates each). f, g, IHC analysis showing the expression level of Rab27a and Rab27b (f) and exosome marker expression CD63 (g) in the brain tissue derived from mice injected with control lentivirus or Rab27a/b shRNA lentiviruses and subsequently intracranially injected with B16BL6 cells. h, i, IHC analysis showing the expression level of Rab27a and Rab27b (h) and exosome marker expression CD63 (i) in the brain tissue derived from mice injected with Rab27a/b shRNA lentiviruses and subsequently intracranially injected with B16BL6 cells and vehicle at the left side or B16BL6 cells and astrocyte-derived exosomes at the right side.

Extended Data Figure 6 Brain extravasation of MDA-MB-231 parental cells with or without induction of doxycycline-inducible PTEN shRNA knockdown, PTEN expression and CCL2 shRNA knockdown.

a, Western blot showing PTEN expression levels after treating MDA-MD-231 cells with doxycycline. MDA-MD-231 cells were stably infected with inducible shRNA expression vectors (pTRIPZ-control-shGFP as control and pTRIPZ-shRNA-RFP for PTEN shRNA). Doxycycline (1 μg ml−1) was added to induce shRNA expression for 5 days. As indicated, doxycycline was withdrawn in some samples for another 5 days before analysis. b, Schematics of in vivo extravasation assay. shControl-GFP and shPTEN-RFP cells were mixed at a 1:1 ratio. In total, 200,000 cells were ICA injected into mice, and doxycycline (50 μg kg−1) was given intraperitoneally daily. Brains were collected 5 days after ICA injection. c, Dot plot of extravasated cell counts 5 days after ICA injection of indicated MDA-MB-231 sublines. Tumour-bearing brains were collected and sectioned into 100 μm coronal slices. Extravasated tumour cells were counted under the fluorescence microscope (mean ± s.d., t-test). d, MDA-MB-231Br single cells were expanded into subclones (C12, C14, C18 and C19), which were transfected with doxycycline-inducible pTRIPZ-RFP or pTRIPZ-PTEN. 48 h after doxycycline (1 μg ml−1) treatment, PTEN induction was tested by western blotting. The C14 clone was used for further in vivo assays (see e, f and Fig. 4a). e, IHC staining of induced PTEN expression in brain metastases derived from mice injected with MDA-MB-231Br (231Br-RFP or 231Br-PTEN) cells. f, IHC analysis of PTEN downstream signalling pathway, including phosphorylated pAkt(T308), pAkt(S473) and pP70S6K(T389+T412) in brain metastases from mice injected with 231Br-RFP or 231Br-PTEN cells. Top, dot plot of IHC data quantification by IRS (mean ± s.d., t-test); bottom, representative IHC staining data. g, Histograms of PTEN and CCL2 mRNA levels (mean ± s.e.m., t-test) in indicated cancer cell lines 48 h after transfection with control or PTEN siRNAs (3 biological replicates, with 3 technical replicates each). h, Histogram showing the inducible CCL2 knockdown. MDA-MB-231Br cells were stably infected with pTRIPZ-inducible CCL2 shRNAs. 48 h after doxycycline (1 μg ml−1) treatment, CCL2 mRNA was examined by RT–PCR (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). i, Doxycycline-induced CCL2 knockdown in brain metastases. Mice were ICA injected with MDA-MB-231Br cells containing control or CCL2 shRNAs. Doxycycline (50 μg kg−1) was given to mice intraperitoneally daily after injection. IHC staining of CCL2 expression levels in brain metastases derived from MDA-MB-231Br cells. T, brain metastasis tumours at day 30 after ICA injection.

Extended Data Figure 7 PTEN-regulated CCL2 expression through the NF-κB pathway.

a, Heat-map showing differentially expressed protein markers of reverse-phase protein array analysis. MDA-MB-231Br cells were stably infected with pTRIPZ-RFP or pTRIPZ-PTEN (231Br-RFP or 231Br-PTEN) and induced by doxycycline (1 μg ml−1) for 48 h. b, Box chart showing the absolute intensity of PTEN and NF-κB p65(S536). c, Western blot analysis of NF-κB p65 nuclear translocation, after cells were treated with Akt inhibitor MK2206 (10 μg ml−1) 24 h before separation into cytosolic (Cyto) and nuclear (Nuc) fractions. d, Western blot analysis of NF-κB p65 nuclear translocation, after cells were treated with NF-κB inhibitor PDTC (0.2 mM) 16 h before separation into cytosolic and nuclear fractions. e, Relative CCL2 mRNA expression after NF-κB inhibitor PDTC treatment analysed by qRT–PCR (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). Cells were treated with PDTC (0.2 mM) for 16 h. f, Relative CCL2 protein expression after PDTC treatment analysed by ELISA (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each). Cells were treated as in e.

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Extended Data Figure 8 CCR2-mediated IBA1+ myeloid cell directional migration.

a, Co-expression of IBA1 and CCR2 on myeloid cells freshly isolated from mouse brain by CD11b beads. Representative immunofluorescence staining of IBA1 (left). FACS analysis of CD11b+ cells for CCR2 expression. b, Relative CCR2 expression in the BV2 microglia cell line compared with NIH3T3 fibroblasts. CCR2 mRNA level analysed by qRT–PCR (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each) (left) and protein expression analysed by FACS (right) c, Transwell migration assay examining the directional migration of BV2 cells towards CCL2. In total, 105 BV2 cells were seeded in the top chamber of the transwell units, and CCL2 or BSA (20 ng ml−1) was added into serum-free media in the bottom chamber. The migrated cell numbers were counted at 24 h. Next, CCR2 antagonists with different concentrations (10 μM, 1 μM and 0.1 μM) were added into the top chamber with BV2 cells, and CCL2 (20 ng ml−1) was added into serum-free media in the bottom chamber. The migrated cell numbers were counted at 24 h. d, Quantification of BV2 cell migration assay (mean ± s.e.m., t-test, 3 biological replicates, with 3 technical replicates each).

Extended Data Figure 9 The association between PTEN, CCL2 expression and recruitment of IBA1+ myeloid cells in patients’ brain metastases and matched primary breast tumours.

a, Summary histogram of CCL2 protein levels in primary breast tumours and matched brain metastases from 35 patients. Chi-square test was used to compare the IHC score in primary breast tumours versus matched brain metastases. P < 0.05 is defined as significantly different. b, Tables showing IHC scores of PTEN and CCL2 expression in primary breast tumours and matched brain metastases. c, Representative IHC staining of CCL2 proteins and IBA1+ myeloid cells in patients’ brain metastases, and the correlation plot showing the Pearson correlation between CCL2 and IBA1 staining in patients’ brain metastases (R = 0.371, P = 0.028).

Extended Data Figure 10 PTEN loss induced by astrocyte-derived exosomal microRNA primes brain metastasis outgrowth via functional cross-talk between disseminated tumour cells and brain metastatic microenvironment.

Top, disseminated tumour cells extravasate into the brain. a–-c, Exosomes secreted by astrocytes in the brain microenvironment transfer PTEN-targeting miRNA into extravasated brain metastatic tumour cells, leading to PTEN downregulation in tumour cells. c, d, PTEN loss in brain metastatic tumour cells increases their CCL2 secretion, facilitating the recruitment of IBA1+/CCR2+ myeloid cells at the micrometastasis site. d, e, The recruited IBA1+ myeloid cells enhance proliferation and inhibit apoptosis of metastatic tumour cells, and promote metastatic outgrowth.

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Zhang, L., Zhang, S., Yao, J. et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 527, 100–104 (2015). https://doi.org/10.1038/nature15376

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