Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism

Abstract

Breast cancer cells disseminate through TIE2/MENACalc/MENAINV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENAINV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENA gene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.

Conflict of interest statement

Competing interests: J.S.C. and M.H.O. are inventors on a patent application (#96700/2505) submitted by Albert Einstein College of Medicine that covers methods of detecting and reducing chemotherapy-induced prometastatic changes in breast tumors. All other authors declare that they have no competing interests

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1. Paclitaxel delays tumor growth and promotes infiltration of TIE2hi/VEGFhi macrophages and TMEM assembly

(A) Experimental design and chemotherapy scheme. i.v., intravenously. (B) Mouse models of breast carcinoma, estrogen receptor (ER) status in each model, and cohort sizes. (C) Tumor volume quantification on day 15 of the chemotherapy scheme shown in (A). Mann-Whitney U test. (D) TMEM score, assessed in 10 high-power fields (HPFs) by two pathologists, in mice treated as shown in (A). Mann-Whitney U test. (E) TMEM identification by triple-stain immunohistochemistry (IHC) and representative images for each mouse model. Scale bar, 50 μm. (F) Perivascular IBA1+ macrophages (Mφ) in 10 HPFs (absolute counts) in PyMT spontaneous and HT17 xenograft tumors, treated with paclitaxel or vehicle control. Mann-Whitney U test. (G) Perivascular TIE2hi/VEGFhi macrophages in 10 HPFs (absolute counts) quantified in PyMT spontaneous and HT17 xenograft tumors, treated with paclitaxel or vehicle control. Mann-Whitney U test. (H) Multichannel IF of IBA1, CD31, TIE2, VEGF, and 4′,6-diamidino-2-phenylindole (DAPI) in two sequential sections of an MMTV-PyMT breast tumor not treated with paclitaxel. Representative VEGFhi/TIE2hi macrophage (also coexpressing IBA1) is encircled with yellow dotted line. Scale bar, 10 μm. (I) Multichannel IF of IBA1, CD31, VEGF, and DAPI in an HT17 xenograft tumor, treated with paclitaxel, demonstrating one VEGFhi and one VEGFlo macrophage in a field. Scale bar, 15 μm. (J and K) Correlations of macrophage infiltration (IBA1+ macrophages or VEGFhi/TIE2hi macrophages) with TMEM score in the PyMT spontaneous (J) and HT17 xenograft (K) models. R2 = Pearson’s coefficient of determination; filled circles, control; open circles, paclitaxel.

Fig. 2. Paclitaxel induces vascular permeability exclusively at TMEM sites

(A) Experimental design and chemotherapy scheme. (B) Mouse models of breast carcinoma and cohort sizes. (C) Two examples of images taken from IVI of cfms-CFP mice grafted with MMTV-PyMT/Dendra2+ tumors. Left image: Bursting at TMEM (TMEM activity) identified by the presence of TMR-conjugated 155-kDa dextran in the extravascular space. Outline of the burst indicated by dotted yellow line. Right image: Absence of bursting at TMEM, as a control. Mϕ, macrophage; TC, tumor cell; EC, endothelial cell. Scale bar, 25 μm. (D) Left image: Region of interest (ROI) selection for calculation of Dendra2/TMR signal intensity over time. Right graph: The quantification of extravascular dextran (red lines) and Dendra2-labeled intravasating cancer cell (green lines) signal intensity in the selected TMEM-associated (solid lines) or non-TMEM (dotted lines) ROIs from the image shown on the left side. (E) Multichannel IF of endomucin (first column), dextran-TMR (second column), their merged image along with DAPI (third column), the corresponding thresholded blood vessel and ex-travascular dextran masks (fourth column), and the corresponding sequential section of TMEM IHC (fifth column) in MMTV-PyMT mice treated with paclitaxel. Top row: TMEM-associated vascular profile. Bottom row: Vascular profile away from TMEM. Scale bar, 20 μm. (F) Percentage of vascular profiles with extravascular dextran that have at least one TMEM site or no TMEM sites associated with them for vehicle-treated (top graph) or paclitaxel-treated (bottom graph) cases.

Fig. 3. Paclitaxel promotes TMEM-dependent vascular permeability, cancer cell dissemination, and metastasis in breast cancer

(A) Time-lapse images from videos S1 (top row) and S3 (bottom row). Time shown in minutes (t = 0 to 20 min). Top row: Arrowhead follows site of bursting from an active TMEM in a paclitaxel-treated mouse. Bottom row: Arrowhead marks the lack of bursting from an inactive TMEM in apaclitaxel-treated mouse. Scale bar, 50 μm. (B) Incidence of bursting (at least one complete event during ~4.5 hours of imaging per mouse) in paclitaxel-and vehicle-treated MMTV-PyMT/Dendra2 cfms-CFP mice. (C) Frequency of bursting in paclitaxel and control MMTV-PyMT/Dendra2 cfms-CFPmice. Mann-Whitney U test. (D) Representative blood vessel (endomucin) and extravascular dextran masks, as obtained by IF in mice treated with either vehicle or paclitaxel, showing TMEM-associated vascular permeability (yellow area). Scale bar, 100 μm. (E) Quantification of extra-vascular dextran area normalized to blood vessel area in mice treated with either vehicle or paclitaxel shown in (D). Mann-Whitney U test. (F) CTCs per milliliter of blood collected before sacrifice (day 15). Values normalized to the control group in each case to account for intercohort variability. Mann-Whitney U test. (G) Correlation between CTCs and TMEM. R2=Pearson’s coefficient of determination; filled circles, control; open circles, paclitaxel. (H) Detection of micrometastatic foci in the lungs of paclitaxel-treated mice. Two cases of histologically detectable metastases in lungs of PyMT transplants and HT17 xenografts, respectively, are shown. Scale bar, 100 μm. (I) Incidence of lung metastasis in mice treated with paclitaxel or vehicle control, χ2 test. (J) Quantification of histologically detectable lung metastases in mice treated with paclitaxel or vehicle control. Mann-Whitney U test. (K) Stereo-microscopy in extracted mouse lung. Blood vessels visualized via tail vein injection of rhodamine-labeled lectin 1 hour before sacrifice, and cancer cells identified through Dendra2 expression (arrow). Mann-Whitney U test. Scale bar, 20 μm. (L) Quantification of single cancer cell dissemination in lungs of PyMT transplants using fluorescence stereomicroscopy. Mann-Whitney U test.

Fig. 4. Paclitaxel promotes the expression of invasive isoforms of MENA in the primary breast cancer microenvironment

(A to D) Gene expression of MENA or MENA isoforms (real-time RT-PCR) after RNA extraction from FFPE tumors. Gene expression of Pan-Mena (A), Mena11a (B), MenaCalc (C), and MenaINV (D) indicated. Mann-Whitney U test. NS, not significant. (E and F) Correlations of MenaCalc with TMEM and MenaINV gene expression with TMEM in the PyMT spontaneous (E) and HT17 xenograft (F) tumors. R2 = Pearson’s coefficient of determination; filled circles, control; open circles, paclitaxel. (G) MENAINV protein expression visualized by MENAINV IF and DAPI in PyMT spontaneous and HT17 xenograft tumors, treated with paclitaxel or vehicle control. Scale bar, 100 μm. (H) Quantification of the MENAINV-positive area (%) in tumors shown in (G). Mann-Whitney U test. (I) Correlation of MENAINV-positive area (%) with TMEM score in the PyMT spontaneous (top plot) and HT17 xenograft (bottom plot) models. R2 = Pearson’s coefficient of determination; filled circles, control; open circles, paclitaxel.

Fig. 5. Paclitaxel promotes breast cancer cell dissemination and metastasis in a MENA-dependent manner

(A) Experimental design and chemotherapy scheme. (B) Mouse models of breast carcinoma and cohort sizes. (C) Multichannel IF of IBA1, CD31, VEGF, and DAPI in MENA−/−MMTV-PyMT-CFP transplanted tumors, treated with either vehicle control (top panel) or paclitaxel (bottom panel). Arrowheads: VEGFhi/IBA1+ macrophages. Scale bar, 25 μm. (D) Perivascular IBA1+ macrophages (Mφ) counted in 10 HPFs (absolute counts) in MENA−/− MMTV-PyMT-CFP transplanted tumors treated with paclitaxel or vehicle control. Mann-Whitney U test. (E and F) Perivascular TIE2hi/VEGFhi macrophages (Mφ) quantified in MENA−/− MMTV-PyMT-CFP transplanted tumors, treated with paclitaxel or vehicle control. Absolute counts (E) or percentages (F) among all perivascular IBA1+ macrophages (Mφ). Mann-Whitney U test. (G) CTCs per milliliter of blood collected from MENA+/+ and MENA−/− PyMT-CFP mice. Values normalized to the control group in each case to account for intercohort variability. Mann-Whitney U test. (H) Quantification of single cancer cell dissemination in the lungs of MENA+/+ and MENA−/− PyMT-CFP transplants, using fluorescence stereomicroscopy. Mann-Whitney U test.

Fig. 6. Nontaxane chemotherapies induce TMEM-dependent prometastatic changes in the breast cancer microenvironment

(A) Experimental design and chemotherapy scheme. (B) Mouse models of breast carcinoma and cohort sizes. (C) Representative histological [hematoxylin and eosin (H&E); left column] and TMEM IHC sections (right column) from MMTV-PyMT mice receiving doxorubicin/cyclophosphamide treatment or vehicle control, as shown in (A). Scale bar, 50 μm. (D) TMEM score in MMTV-PyMT mice treated with either vehicle control or doxorubicin/cyclophosphamide. Mann-Whitney U test. (E) CTCs per milliliter of blood collected before sacrifice (day 15). Values normalized to the control group in each case, to account for intercohort variability. Mann-Whitney U test. (F) Proportion of peri-vascular TIE2hi/VEGFhi macrophages among all IBA1+ macrophages, quantified in MMTV-PyMT mice treated with either doxorubicin/cyclophosphamide or vehicle control. Mann-Whitney U test.

Fig. 7. NAC in breast cancer patients promotes TMEM assembly and increased MENAINV expression

(A) Individual TMEM scores of 20 patients before and after receiving NAC, which included weekly paclitaxel (80 mg/m2 × 12 consecutive weeks) followed sequentially by dose-dense AC chemotherapy [doxorubicin (60 mg/m2) and cyclophosphamide (600 mg/m2) every 2 weeks × four cycles, plus pegfilgrastim (6 mg, subcutaneously) on day 2 of each cycle]. The patients did not receive tamoxifen. Red line: TMEM high-risk cutoff point according to (5). (B) Representative images of TMEM triple-stain IHC in patients #3 and #7 in the pre-NAC core biopsies (upper panels) and post-NAC resected tumors (lower panels). Scale bar, 50 μm. (C) Mean TMEM scores in the 20 human breast cancers shown in (A), before and after receiving NAC, Wilcoxon test. (D) Representative images of MENAINV protein expression, as visualized by MENAINV IF and DAPI in a patient receiving NAC. Scale bar, 100 μm. (E) Quantification of the MENAINV-positive area in pre- and post-NAC patient samples. Assay performed in only seven of the patients shown in (A) because of limited availability of pre-NAC biopsy material for the remaining 13 patients. Mann-Whitney U test. (F) MENAINV gene expression, as assessed by real-time RT-PCR, in FNA biopsies taken from five breast cancer patients before and after 2 weeks of receiving NAC with paclitaxel.

Fig. 8. TIE2 inhibitor rebastinib eliminates the prometastatic effects of paclitaxel

(A) Experimental design and chemotherapy scheme. (B) Mouse models of breast carcinoma and cohort sizes. (C and D) TMEM scores in the PyMT transplantation model (C) and the HT17 xenograft model (D), treated with vehicle control, rebastinib, paclitaxel, or a combination of rebastinib with paclitaxel. Mann-Whitney U test. (E and F) Perivascular TIE2hi/VEGFhi macrophages quantified in 10 HPFs in the PyMT transplantation model (E) or in the HT17 xenograft model (F), treated with vehicle control rebastinib, paclitaxel, or a combination of rebastinib with paclitaxel. Mann-Whitney U test. (G and H) CTCs per milliliter of blood collected before sacrifice (day 15) of mice [PyMT transplantation (G); HT17 xenograft (H)]. Values normalized to the control group in each case to account for intercohort variability. Mann-Whitney U test. (I) Incidence of bursting (at least one complete event during 4.5 hours of imaging per mouse) in paclitaxel-treated MMTV-PyMT/Dendra2 cfms-CFP mice that either received or did not receive rebastinib. (J) Frequency of bursting in paclitaxel-treated MMTV-PyMT/Dendra2 cfms-CFP mice that either received or did not receive rebastinib. Mann-Whitney U test. (K) Proposed model of chemotherapy-induced prometa-static changes and cancer cell dissemination. Chemotherapy treatment increases the density of TIE2hi/VEGFhi macrophages within the primary tumor. In addition to inducing angiogenesis, these macrophages assemble active TMEM structures. Chemotherapy treatment also increases expression of the actin-regulatory protein MENAINV isoform in tumor cells due to their contact with infiltrating macrophages, which makes these cancer cells highly migratory and invasive. Upon arriving at the blood vessels, some of the MENAINV-expressing cancer cells assemble active TMEM sites, which other MENAINV-expressing cells then use to intravasate. Together, chemotherapy-mediated MENAINV overexpression and TMEM assembly in breast cancer contribute to TMEM-dependent cancer cell dissemination and distant metastasis. Targeting the function of TMEM-associated macrophage subpopulation by a TIE2 inhibitor counteracts TMEM-mediated cancer cell dissemination induced by chemotherapy treatment.