NAC treatment increases metastatic burden in an EO771 experimental metastasis model.

<p><b>A)</b> Schematic of NAC treatment regimen for 4T1.2 primary-resection model. BALB/C mice were orthotopically injected with 4T1.2 breast tumor cells on day 0, and the primary tumor surgically resected at a volume of 35 mm³ (∼day 10). NAC was administered at 40 mM in the drinking water 7 days post-resection (green line) until metastatic endstage. <b>B)</b> Kaplan-Meier survival curve of mice treated as in A (n = 6 all groups; p = 0.57). p values calculated using Log-rank (Mantel-Cox) Test. <b>C)</b> Schematic of NAC treatment regimen for experimental metastasis model. NAC (40 mM) was administered in the drinking water from day -7 (red line) prior to EO771 tumor cell injection. Mice were intravenously injected with EO771 tumor cells through the tail vein on day 0, and lungs analyzed for metastatic burden at day 14. <b>D)</b> Representative histology images (H&E stained) of lungs from C, with metastatic foci indicated by black arrows. Scale bar represents 100 µm. <b>E)</b> Average number of metastatic foci per lung (average determined from 4 whole lung sections per mouse) from D (n = 9 per group). Mean ± SEM; *p<0.05; Student’s t-Test. <b>F)</b> Absolute frequency and percentage of mice with an average of between 0–5, 6–14, 15–50 and >50 metastatic foci per lung section from E.</p

NAC treatment does not alter the hypoxic phenotype of primary tumors.

<p>Tumors from NAC-treated or control mice were stained with Hif-1α (red), PIM (green) and DAPI (blue). Five sections per tumor were stained and imaged to obtain an average percentage threshold area of Hif-1α staining determined from 5 random images per section. <b>A)</b> Representative images of PyMT endstage tumors (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066388#pone-0066388-g003" target="_blank">Figure 3G</a>) with Hif-1α staining values calculated for individual tumors (<b>B)</b> and averaged <b>(C)</b> (control n = 5 tumors; NAC n = 7 tumors). <b>D)</b> Representative images of EO771 endstage tumors (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066388#pone-0066388-g003" target="_blank">Figure 3C–D</a>) with Hif-1α staining values calculated for individual tumors (<b>E)</b> and averaged <b>(F)</b> (n = 6 tumors for all groups). Mean ± SEM; n.s. indicates not significant. <b>G)</b> EO771 tumor lysates (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066388#pone-0066388-g003" target="_blank">Figure 3C–D</a>) were probed for Hif-1α and β-actin (loading control) by Western blot (control n = 3 tumors; NAC n = 4 tumors).</p

NAC treatment does not alter the angiogenic phenotype of primary tumors.

<p>Tumors from NAC-treated or control mice were stained with CD31 (red) and DAPI (blue). Five sections per tumor were stained and imaged to obtain an average percentage threshold area of CD31 staining determined from 5 random images per section. <b>A)</b> Representative images of PyMT tumors (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066388#pone-0066388-g003" target="_blank">Figure 3G</a>) with CD31 staining values calculated for individual tumors (<b>B)</b> and averaged <b>(C)</b> (n = 5 tumors for all groups). <b>D)</b> Representative images of EO771 tumors (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066388#pone-0066388-g003" target="_blank">Figure 3C–D</a>) with CD31 staining values calculated for individual tumors (<b>E)</b> and averaged <b>(F)</b> (n = 6 tumors for all groups). Scale bars represent 100 µm. Mean ± SEM; n.s. indicates not significant.</p

<i>In vitro</i> characterization of NAC treatment on the hypoxic response pathway.

<p><b>A)</b> Representative images of PyMT and EO771 cells exposed to normoxic (20% O₂) or hypoxic (2% O₂) conditions for 2 hours and treated with 0 (control) or 25 mM NAC. Cells were fixed with 10% NBF and stained for Hif-1α and DAB (positive cells are stained brown). Scale bars represent 100 µm. <b>B-C)</b> Cell lysates from PyMT and EO771 cells exposed to normoxic and hypoxic conditions for 2 hours and treated with 0 or 25 mM NAC, and probed for Hif-1α <b>(B)</b>, Hif-2α <b>(C)</b> and α–tubulin (loading control) by Western blot. <b>D)</b> Secreted VEGF-A (pg/mL) was measured by ELISA in the supernatant of PyMT and EO771 cells exposed to normoxic or hypoxic conditions for 8 hours with 0, 10 or 25 mM NAC. p value for EO771 is 0.174, n = 4. <b>E–F)</b> mRNA was extracted from PyMT and EO771 cells exposed to normoxic or hypoxic conditions for 8 hours and treated with 0, 10 or 25 mM NAC. Expression of hypoxic response genes VEGF <b>(E)</b> and LOX <b>(F)</b> was assessed using qRT-PCR and fold induction normalized to normoxic sample (broken line; n = 3 in triplicate). Mean ± SEM; n.s. indicates not significant; *p<0.05; ***p<0.001; Student’s t-Test.</p

The Antioxidant N-Acetylcysteine Prevents HIF-1 Stabilization under Hypoxia <i>In Vitro</i> but Does Not Affect Tumorigenesis in Multiple Breast Cancer Models <i>In Vivo</i>

<div><p>Intratumoral hypoxia is a poor prognostic factor associated with reduced disease-free survival in many cancer types, including breast cancer. Hypoxia encourages tumor cell proliferation, stimulates angiogenesis and lymphangiogenesis, and promotes epithelial-mesenchymal transition and metastasis. Tumor cells respond to a hypoxic state by stabilizing the Hif-1α subunit of the Hypoxia-Inducible Factor (HIF) transcription factor to promote expression of various tumor- and metastasis-promoting hypoxic response genes. The antioxidant N-acetylcysteine (NAC) was recently shown to prevent Hif-1α stabilization under hypoxia, and has been identified as a potential alternative method to target the hypoxic response in tumors. We utilized three orthotopic syngeneic murine models of breast cancer, the PyMT, EO771 and 4T1.2 models, to investigate the ability of NAC to modulate the hypoxic response <i>in vitro</i> and <i>in vivo</i>. While NAC prevented Hif-1α stabilization under hypoxia <i>in vitro</i> and increased levels of glutathione in the blood of mice <i>in vivo</i>, this did not translate to a difference in tumor growth or the hypoxic state of the tumor compared to untreated control mice. In addition, NAC treatment actually increased metastatic burden in an experimental metastasis model. This work raises questions regarding the validity of NAC as an anti-tumorigenic agent in breast cancer, and highlights the need to further investigate its properties <i>in vivo</i> in different cancer models.</p></div

NAC treatment does not prevent EMT under hypoxia <i>in vitro</i>.

<p><b>A)</b> Cell lysates from PyMT cells exposed to normoxic and hypoxic conditions for 2 hours and treated with 40 mM NAC and probed for E-cadherin and α–tubulin (loading control) by Western blot. <b>B–D)</b> mRNA was extracted from PyMT and EO771 cells exposed to normoxic or hypoxic conditions for 8 hours with 0, 10 or 25 mM NAC treatment. Expression of EMT target genes SNAIL <b>(B)</b>, SLUG <b>(C)</b> and TWIST <b>(D)</b> was quantified using qRT-PCR and fold induction normalized to normoxic sample (broken line; n = 3 in triplicate). Mean ± SEM; *p<0.05; Student’s t-Test.</p

NAC treatment does not significantly effect proliferation and apoptosis in EO771 metastatic tumors.

<p><b>A)</b> Representative images of lungs (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066388#pone-0066388-g006" target="_blank">Figure 6D–E</a>) stained with Ki67 (positive cells stained brown) from control and NAC-treated mice. Scale bars represent 100 µm. <b>B)</b> Percentage of Ki67 positive cells per metastatic tumor from A analyzed using the ImmunoRatio image analysis program (control n = 15 and NAC n = 21 tumors analyzed from 6 mice per group). Mean ± SEM; n.s. indicates not significant. <b>C)</b> Examples of 1+, 2+ or 3+ metastatic tumors regarding intensity of staining for apoptotic cells (positive cells stained brown) with tumors indicated by black arrows where necessary. Scale bars represent 100 µm. <b>D)</b> Percentage of metastatic tumors in control and NAC-treated mice (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066388#pone-0066388-g006" target="_blank">Figure 6D–E</a>) categorized as 1+, 2+ or 3+ for staining of apoptotic cells (control n = 16 and NAC n = 28 tumors analyzed from 7 mice per group).</p

Tracking the fate of adoptively transferred myeloid-derived suppressor cells in the primary breast tumor microenvironment

<div><p>Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that are expanded in cancer and act as potent suppressors of the anti-tumor immune response. MDSCs consist of two major subsets, namely monocytic (M-) MDSCs and granulocytic (G-) MDSCs that differ with respect to their phenotype, morphology and mechanisms of suppression. Here, we cultured bone marrow cells with IL-6 and GM-CSF <i>in vitro</i> to generate a population of bone marrow MDSCs (BM-MDSCs) similar to G-MDSCs from tumor-bearing mice in regards to phenotype, morphology and suppressive-function. Through fluorescent labeling of these BM-MDSCs and optical imaging, we could visualize the recruitment and localization of BM-MDSCs in breast tumor-bearing mice <i>in vivo</i>. Furthermore, we were able to demonstrate that BM-MDSCs home to primary and metastatic breast tumors, but have no significant effect on tumor growth or progression. <i>Ex vivo</i> flow cytometry characterization of BM-MDSCs after adoptive transfer demonstrated both organ-and tumor-specific effects on their phenotype and differentiation, demonstrating the importance of the local microenvironment on MDSC fate and function. In this study, we have developed a method to generate, visualize and detect BM-MDSCs <i>in vivo</i> and <i>ex vivo</i> through optical imaging and flow cytometry, in order to understand the organ-specific changes rendered to MDSCs in breast cancer.</p></div

Homing of adoptively transferred DiD-BM-MDSCs to established metastases.

<p><b>A</b>) Representative <i>in vivo</i> images of Luc-PyMT metastases (BL signal) 3 weeks after i.c. injection into C57Bl/6 mice. <b>B</b>) Representative e<i>x vivo</i> images of DiD-BM-MDSC (FL signal; top panel) localization to adrenal gland metastases (BL signal; bottom panel). DiD-BM-MDSCs were injected (i.v.) into mice from A, and images acquired 2 weeks later. Quantification of radiant efficiency (RE) of FL-signal for the adrenal gland shown on the right. <b>C</b>) Representative images of spleens (left) and RE quantification (right) from treatment groups described in <b>A and B</b>. Naïve C57Bl/6 mice were used as controls. Data represented as mean ± SEM; *p<0.05; ***p<0.001; n = 3–5 mice for all groups.</p

Fate of adoptively transferred DiD-BM-MDSC in the tumor and spleen of breast tumor-bearing mice.

<p><b>A</b>) Representative flow cytometry plots of the CD45.2/DiD-Cy5⁺ gated population in PyMT-WT tumors from control mice (primary tumor alone; left) and after adoptive transfer of DiD-BM-MDSCs (right) on day 24 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196040#pone.0196040.g005" target="_blank">Fig 5</a><b>B and</b> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196040#pone.0196040.g005" target="_blank">5</a><b>C</b>) Flow cytometry analysis of CD11b/Ly6C/Ly6G (B) and CD11b/Ly6C subpopulations (C) as a percentage of CD45/Cy5⁺ cells within the tumor. <b>D</b>) Flow cytometry analysis of mature myeloid populations including granulocytes (CD11b/Ly6G), macrophages and DCs as a percentage of CD45/Cy5⁺ cells within the tumor. <b>E</b>) Representative flow cytometry plots of the CD45.2/DiD-Cy5⁺ gated population within the spleen of PyMT-WT tumor-bearing mice (control; left) and after adoptive transfer of DiD-BM-MDSCs (right). <b>F,G</b>) Flow cytometry analysis of CD11b/Ly6C/Ly6G (F) and CD11b/Ly6C subpopulations (G) as a percentage of CD45/Cy5⁺ cells within the spleen. <b>H</b>) Flow cytometry analysis of mature myeloid populations including granulocytes, macrophages and DCs as a percentage of CD45/Cy5⁺ cells within the spleen. Data represented as mean ± SEM; n = 6 for all groups.</p